Use of fisetin for reducing the incidence of atrial fibrillation

Abstract

The present invention discloses a method for preventing or treating atrial fibrillation (AF) in a subject by administering a therapeutically effective amount of a flavonol compound (Formula 1 or a tautomer thereof), a pharmaceutically acceptable salt, hydrate, or solvate thereof. The method involves obtaining a subject in need of AF prevention or treatment, suspected to require such prevention or treatment, or a normal healthy subject, and administering the compound to the subject Formula 1 comprises specific atoms and substituents, including - H, -OCH3, -OH, -I, -Br, -F, or Cl. By utilizing the flavonol compound of Formula 1, the risk of AF development or the prevalence of AF in the subject is reduced, providing an effective approach for managing AF in individuals.

Description

Atty. Docket No.: 405002-549001WO USE OF FISETIN FOR REDUCING THE INCIDENCE OF ATRIAL FIBRILLATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent No.63 / 518,994, filed on 11-August-2023, the entirety of which is incorporated by reference as if fully reproduced and set forth herein in its entirety. FIELD OF THE INVENTION

[0002] This invention generally relates to methods for prevention and treatment of conditions often associated with high blood pressure and valvular heart disease, which are the most common modifiable risk factors for atrial fibrillation. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under R21 AG049608 awarded by the National Institutes of Health. The government has certain rights in the invention. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0004] This submission is accompanied by a “Sequence Listing XML” containing SEQ ID NOs: 1-5 (Table 2) and created on 05-August-2024, 6 KB, submitted with the filename: “405002-549001WO.xml”. No sequences contain less than 10 nucleotides and would be thus mandatorily marked as intentionally skipped sequences under WIPO Sequence software version 2.3.0. The Sequence Listing XML was generated using WIPO Sequence software version 2.3.0, in accordance with 37 CFR §§ 1.831 through 1.835, and is herewith submitted as an XML file, via the USPTO patent electronic filing system, 37 CFR § 1.835(a)(1). The Sequence Listing XML is incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION

[0005] Atrial fibrillation (AF) is a common cardiac arrhythmia characterized by rapid and irregular beating of the atria. AF is the most common serious abnormal heart rhythm and, as of 2020, affects more than 33 million people worldwide (e.g., see Centers for Disease Control, CDC, “About Atrial Fibrillation”). AF is associated with an increased risk of stroke, heart failure, and other cardiovascular complications. The prevalence of AF increases with age, and it is estimated that millions of people worldwide are affected by this condition. Current treatment options for AF include a variety of attempts to control heart rate and rhythm, anticoagulants to prevent stroke, and various interventional procedures such asAtty. Docket No.: 405002-549001WO catheter ablation. However, these treatments often come with significant side effects and may not be effective for all patients, highlighting the need for new therapeutic approaches. Unfortunately, AF can be associated with many other conditions and is the most common arrhythmia in the aging population, with an estimated lifetime risk of 25% after forty years of age. More than 5.6 million Americans are expected to be affected by atrial fibrillation in 2050.

[0006] Inflammation may have an important function in the pathogenesis of atrial fibrillation. See Harada, van Wagoner, & Nattel, Circ J., 79(3), 495-502 (2015)(1). The prevalence and prognosis of atrial fibrillation are associated with elevated inflammatory biomarkers in the serum, including the expression of inflammatory markers in cardiac tissues. Guo, Lip, & Apostolakis, J. Am. Coll. Cardiol., 60(22), 2263-70 (2012)(2); Ihara & Sasano, Front. Physiol., 13, 862164 (2022)(3). Anti-inflammatory drugs might have beneficial effects in atrial fibrillation model systems. See reviews by Harada, van Wagoner, & Nattel, Circ. J., 79(3), 495-502 (2015)(4).

[0007] Any mechanisms underlying the inflammatory process of AF remain to be determined. Furthermore, relationships between AF and inflammation due to metabolic conditions and disease may take many years to elucidate. There is an urgent need in the biomedical art for better transformational therapies for reducing the incidence of AF in the aging population. BRIEF SUMMARY OF THE INVENTION

[0008] The following presents a simplified summary of the innovation in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

[0009] The technology herein provides a new approach to solve a long-felt but unmet need, namely, to provide new solutions for atrial fibrillation (AF). The technology disclosed herein provides a central thesis that aging increases the number of senescent atrial myocytes (SAMs) and senescent atrial myofibroblasts (SAMFs) and that increased abundance of SAMs and SAMFs in the left and right atria predisposes the aging human and rabbit heart to atrial fibrillation by senescence-associated secretory phenotype (SASP)- mediated inflammatory response. See FIG.1. The arrhythmogenic effect can be driven either by directly changing the electrical phenotype of SAMs or by SASP-mediated pro- arrhythmic changes in the neighboring atrial myocytes (AMs). SASP-mediated inflammation triggers and accentuates atrial fibrosis and increases heterogeneity, providing a substrate for the maintenance of atrial fibrillation.Atty. Docket No.: 405002-549001WO

[0010] In some embodiments, the technology herein provides a senolytic therapy with direct, combined anti-inflammatory and anti-arrhythmic effects, reducing the incidence of spontaneous atrial fibrillation and induced atrial fibrillation in aging rabbit hearts. In some embodiments, the senolytic therapy is the administration of a small molecule plant flavonol (e.g., fisetin) to a person in need thereof.

[0011] The inventors found that the senolytic drug fisetin eliminated the senescence cells from aged rabbit atria. In a blinded prospective pilot assay of twenty-two rabbits in which eleven rabbits were treated with fisetin and eleven with the vehicle, two rabbits in the vehicle group developed spontaneous atrial fibrillation. None in the fisetin group developed spontaneous atrial fibrillation.

[0012] In one aspect, the invention provides that persistent senescent atrial myocytes and senescent atrial myofibroblasts are involved in an increased propensity for arrhythmogenesis with age.

[0013] In some embodiments, the invention provides for using senolytic drugs to eliminate senescent atrial myocytes and senescent atrial myofibroblasts in aged human or rabbit atria and mitigate atrial fibrillation and fibrosis.

[0014] In another aspect, atrial cell senescence causes “inflammaging” (inflammation + aging) underlies the inflammation and fibrosis in the aged atria. The invention thus provides the first molecular foundation for the pathogenesis of atrial inflammation and how it leads to atrial fibrillation. The invention enables the production of a detailed anatomical map of cellular senescence in the atrium and its proximity to ganglionic plexi and areas of fibrosis in the left atria.

[0015] In another aspect, the technology provides the discovery that aging is associated with the accumulation of senescent atrial myocytes and senescent atrial myofibroblasts in aging atria and with spontaneous atrial fibrillation enables mechanistic assays of how age, the major predisposing factor, affects the processes leading to atrial senescence, fibrosis, and atrial fibrillation.

[0016] For example, the inventors undertook a pilot assay of twenty-two rabbits, with eleven treated with fisetin and eleven vehicle controls. Two of the vehicle control rabbits developed atrial fibrillation. None of the fisetin-treated rabbits developed atrial fibrillation. Optical mapping showed that fisetin reduced reentry but increased automaticity.

[0017] Atrial fibrillation in aged rabbit hearts replicates important clinical features of atrial fibrillation in the elderly population such as automaticity, abnormal action potential duration, discontinuous conduction, and reentry formation. Screening of the aging rabbits’ colony (> four years) using surface electrocardiograms and echocardiograms detected spontaneous atrial fibrillation in four out of thirty-two rabbits (12.5%). This finding is important because spontaneous atrial fibrillation episodes in larger mammals are rare. The percentageAtty. Docket No.: 405002-549001WO of spontaneous atrial fibrillation in aged rabbits is similar to those in the aging human population. These exciting results make the aged rabbit an advantageous model system for mechanistic atrial fibrillation studies.

[0018] In some embodiments, the invention provides for using flavonols or senolytic drugs to prevent atrial fibrillation in an aged population, such as human subjects over 50-70 years old.

[0019] According to some aspects, the invention provides for using senolytic drugs to prevent atrial fibrillation in patients with paroxysmal atrial fibrillation.

[0020] In some embodiments, the invention provides for using senolytic drugs to prevent atrial fibrillation in patients after radiofrequency ablation.

[0021] According to some aspects, the invention provides for using senolytic drugs to treat paroxysmal atrial fibrillation, persistent atrial fibrillation, or chronic atrial fibrillation.

[0022] In another example embodiment, the invention provides for using senolytic drugs to prevent atrial fibrillation after open heart surgery.

[0023] In some embodiments, the invention provides for using senolytic drugs other than fisetin, such as the senolytics described by Zhang et al., The FEBS Journal, 290(5), 1362-1383 (2023)(5).

[0024] In an increasingly useful aspect, the invention provides for using senolytic drugs to prevent atrial fibrillation in obese human subjects.

[0025] In some embodiments, the invention provides for using senolytic drugs to prevent atrial fibrillation in diabetes mellitus patients.

[0026] According to some aspects, the invention provides for using senolytic drugs to prevent of atrial fibrillation in patients with hypertension and / or in patents with AF secondary to PH (pulmonary hypertension) and / or right heart failure.

[0027] In some embodiments, the invention provides for using senolytic drugs to prevent of atrial fibrillation in patients with ischemic heart disease, myocardial infarction, or heart failure.

[0028] To further describe a brief summary of some embodiments, the technology disclosed herein can be discussed by a discussion including the following list of features:

[0029] Feature 1: A method for preventing or treating atrial fibrillation (AF) in a subject, the method comprising the steps of: (1) obtaining a subject in need of a prevention and / or a treating of AF, a subject suspected to be in need of a prevention and / or a treating of AF, or a normal healthy subject without a suspected need for the prevention and / or treating of AF; (2) administering a therapeutically effective amount of a compound of formula 1, a pharmaceutically acceptable salt of formula 1, and / or a pharmaceutically acceptable hydrate and / or solvate of formula 1 to the subject:Atty. Docket No.: 405002-549001WO, Formula 1;

[0030] wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms as indicated in Formula 1; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl; whereby a risk of the subject acquiring AF or a prevalence of AF in the subject is lowered.

[0031] Feature 2: The method of feature 1, wherein Formula 1 further comprises a tautomer and / or an isomer thereof wherein atom 9 is a tautomeric -OH or atom 10 is a tautomeric =O as is represented by Formula 1 Tautomer below:, Formula 1 Tautomer;Atty. Docket No.: 405002-549001WO

[0032] wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent independently comprises: -H, -OCH3, -OH; -I, - Br, -F, or Cl.

[0033] Feature 3: The method of feature 1, wherein Formula 1 comprises one of the following compounds:,

[0034] 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one);,

[0035] Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one);,

[0036] Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0037] Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one);,

[0038] Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one);,

[0039] Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one);Atty. Docket No.: 405002-549001WO,

[0040] Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one);,

[0041] Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4- one);,

[0042] Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one);Atty. Docket No.: 405002-549001WO,

[0043] Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one);,

[0044] Natsudaidain (2-(3,4-dimethoxyphenyl)-3-hydroxy-5,6,7,8- tetramethoxychromen-4-one);,

[0045] Pachypodol (5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7- dimethoxychromen-4-one);Atty. Docket No.: 405002-549001WO,

[0046] Quercetin (3,3′,4′,5,7-pentahydroxy-2-phenylchromen-4-one);,

[0047] Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7- methoxychromen-4-one);,

[0048] Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxychromen-4- one); or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide,Atty. Docket No.: 405002-549001WO Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, or Rhamnetin.

[0049] Feature 4: The method of feature 1, further comprising atom 10 is replaced by a glycoside.

[0050] Feature 5: The method of feature 1, further comprising Formula 1 comprises Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin.

[0051] Feature 6: The method of feature 1, wherein the risk of the subject acquiring AF or a prevalence of AF in the subject is lowered is measured by a symptom reported by the subject; by a sign perceived by a healthcare provider; by a measurement of a device in contact with the subject and / or implanted with the subject; by an iWatch®; by a FitBit®; or by a remote measurement device.

[0052] Feature 7: The method of feature 1, whereby a vulnerability of the subject acquiring AF or a prevalence of AF in the subject is lowered.

[0053] Feature 8: The method of feature 1, wherein Formula 1 is administered in a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis; as measured by the mass of the non-salt and / or non- hydrate / solvate form and the mass of the subject in kg; or in a daily dose of about 100 mg; in a dose administered by a device in contact with the subject; or in a monthly dose.

[0054] Feature 9: The method of feature 1, wherein the administering is executed for a time period of longer than one year.

[0055] Feature 10: The method of feature 1, wherein the administration decreases a number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in an atrium of the subject.

[0056] Feature 11: The method of feature 10, wherein one or more persistent SAMs and / or SAMFs increase an arrhythmic propensity with an aging of the subject, and wherein the decreases of feature 10 is measured as a comparison to the number of the SAMs and / or SAMFs that would be measured in the same subject without the administering during the aging of the subject.

[0057] Feature 12: The method of feature 1, further comprising a spatial transcriptomic measurement of a cell composition in the subject, whereby the measurement elucidates the role of different cell types in modulating atrial senescence in the context of human atrial-like engineered heart tissues (a-EHTs).

[0058] Feature 13: The method of feature 1, whereby the method prevents an accumulation of senescent cells in the atria.

[0059] Feature 14: The method of feature 1, wherein the method is executed including a monthly dose of Formula 1 to the subject.Atty. Docket No.: 405002-549001WO

[0060] Feature 15: The method of feature 1, whereby the method reduces an incidence and / or a duration of spontaneous AF episodes.

[0061] Feature 16: The method of feature 1, further comprising an optical mapping (OM) of a tissue from the subject, whereby the OM is utilized to record and visualize one or more electrophysiological properties in -myocardial preparations including Langendorff- perfused isolated hearts, coronary-perfused wedge preparations, or cell culture monolayers, and wherein the OM is capable to reveal a marked reduction in a re-entrant wave.

[0062] Feature 17: The method of feature 1, further comprising the method is executed for a time period of longer than five years, and a fibrosis of the atrium is reduced compared to the same atrium of the same subject without the method of feature 1.

[0063] Feature 18: The method of feature 1, wherein the method prevents a disease of aging, an atrial senescence of a myofibroblast, and atrial senescence of a myocyte, an inflammation, or a fibrosis.

[0064] Feature 19: The method of feature 1, wherein the administration is a senolytic therapy that is an administration of Formula 1 to a person in need thereof.

[0065] Feature 20: The method of feature 19, wherein the method is utilized to prevent AF in an aged population ≥ 50 years old, wherein the method is to prevent atrial fibrillation in subjects with paroxysmal atrial fibrillation; wherein the method is to prevent atrial fibrillation in subjects after radiofrequency ablation; wherein the method is to treat paroxysmal atrial fibrillation, persistent atrial fibrillation, or chronic atrial fibrillation; wherein the method is to prevent atrial fibrillation after an open heart surgery; wherein the method is to prevent AF in obese and / or overweight human subjects; wherein the method is executed to prevent atrial fibrillation in diabetes mellitus subjects; wherein the method is executed to treat and / or prevent AF related to obesity or AF risk due to obesity; wherein the method is executed to prevent a complication of persistent AF like stroke; wherein the method is executed to prevent AF in subjects with hypertension; or wherein the method is executed to prevent AF in subjects with ischemic heart disease, myocardial infarction, or heart failure.

[0066] Feature 21: The method of feature 1, wherein the method is executed as a combination therapy with one or more therapeutic agents administered simultaneously with Formula 1 or before / after an administration of Formula 1.

[0067] Feature 22: The method of feature 1, wherein the method is utilized for treating AF in an aging population.

[0068] Feature 23: A drug screening platform for measuring one or more effects of a proposed therapeutic agent (candidate) on AF, the platform comprising a heart cell, tissue, or a human atrial-like engineered heart tissue (a-EHTs) and a compound of formula 1, a pharmaceutically acceptable salt of formula 1, and / or a pharmaceutically acceptable hydrate and / or solvate of formula 1:Atty. Docket No.: 405002-549001WO, Formula 1;

[0069] wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms as indicated in Formula 1; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl; whereby a screening of the candidate for a risk of AF or a prevalence of AF in the cell or tissue in the platform is enabled.

[0070] Feature 24: The drug screening platform of feature 23, further comprising a control unit including the heart cell or the a-EHTs without the presence of Formula 1; whereby the control unit enables a testing of the candidate on the same cell or a-EHTs without the presence of Formula 1.

[0071] Feature 25: The drug screening platform of feature 23, wherein Formula 1 further comprises a tautomer and / or an isomer thereof wherein atom 9 is a tautomeric -OH or atom 10 is a tautomeric =O as is represented by Formula 1 Tautomer below:Atty. Docket No.: 405002-549001WO, Formula 1 Tautomer;

[0072] wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent independently comprises: -H, -OCH3, -OH; -I, - Br, -F, or Cl.

[0073] Feature 26: The drug screening platform of feature 23, wherein Formula 1 comprises one of the following compounds:,

[0074] 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0075] Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one);,

[0076] Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one);,

[0077] Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0078] Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one);,

[0079] Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one);,

[0080] Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0081] Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4- one);,

[0082] Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one);,

[0083] Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0084] Natsudaidain (2-(3,4-dimethoxyphenyl)-3-hydroxy-5,6,7,8- tetramethoxychromen-4-one);,

[0085] Pachypodol (5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7- dimethoxychromen-4-one);,

[0086] Quercetin (3,3′,4′,5,7-pentahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0087] Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7- methoxychromen-4-one);,

[0088] Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxychromen-4- one); or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, or Rhamnetin.

[0089] Feature 27: The drug screening platform of feature 23, further comprising atom 10 is replaced by a glycoside.

[0090] Feature 28: The drug screening platform of feature 23, further comprising Formula 1 comprises Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin.

[0091] Feature 29: The drug screening platform of feature 23, wherein the risk an AF or a prevalence of AF in the cell, tissue, or a-EHTs is measured by one or more analytical methods performed on the platform.Atty. Docket No.: 405002-549001WO

[0092] Feature 30: The drug screening platform of feature 23, wherein the candidate decreases a number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in a cell, tissue, or a-EHTs of the platform.

[0093] Feature 31: The drug screening platform of feature 23, further comprising a spatial transcriptomic measurement of a cell composition in the subject, whereby the measurement elucidates the role of different cell types in modulating atrial senescence in the context of the cell, tissue, or human atrial-like engineered heart tissues (a-EHTs).

[0094] Feature 32: The drug screening platform of feature 23, further comprising an optical mapping (OM) of a tissue from the subject, whereby the OM is utilized to record and visualize the electrophysiological properties in a variety of myocardial preparations such as Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, and cell culture monolayers will reveal marked reduction in reentrant waves.

[0095] Feature 33: The drug screening platform of feature 23, wherein the screening is executed as a combination therapy with one or more therapeutic agents administered simultaneously with Formula 1 or before / after an administration of Formula 1.

[0096] Feature 34: A supplement for preventing or treating atrial fibrillation (AF) in a subject, the supplement comprising: a therapeutically effective amount of a compound of formula 1, a pharmaceutically acceptable salt of formula 1, and / or a pharmaceutically acceptable hydrate and / or solvate of formula 1 to the subject:, Formula 1;

[0097] wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms as indicated in Formula 1; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent 5, 6, 7, 8, 2*, 3*, 4*,Atty. Docket No.: 405002-549001WO 5*, and 6* independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl; wherein the supplement is provided in a pharmaceutical formulation.

[0098] Feature 35: The supplement of feature 34, wherein Formula 1 further comprises a tautomer and / or an isomer thereof wherein atom 9 is a tautomeric -OH or atom 10 is a tautomeric =O as is represented by Formula 1 Tautomer below: ,

[0099] wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent independently comprises: -H, -OCH3, -OH; -I, - Br, -F, or Cl.

[0100] Feature 36: The supplement of feature 34, wherein Formula 1 comprises one of the following compounds:,

[0101] 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0102] Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one);,

[0103] Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one);,

[0104] Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0105] Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one);,

[0106] Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one);,

[0107] Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0108] Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4- one);,

[0109] Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one);,

[0110] Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0111] Natsudaidain (2-(3,4-dimethoxyphenyl)-3-hydroxy-5,6,7,8- tetramethoxychromen-4-one);,

[0112] Pachypodol (5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7- dimethoxychromen-4-one);,

[0113] Quercetin (3,3′,4′,5,7-pentahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO,

[0114] Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7- methoxychromen-4-one);,

[0115] Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxychromen-4- one); or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, or Rhamnetin.

[0116] Feature 37: The supplement of feature 34, further comprising atom 10 is replaced by a glycoside.

[0117] Feature 38: The supplement of feature 34, further comprising Formula 1 comprises Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin.

[0118] In yet other embodiments, a method of making a supplement is provided comprising a composition discussed in any of the features above and optionally adding one or more pharmaceutically acceptable excipients. Many of the compositions areAtty. Docket No.: 405002-549001WO pharmaceutically effective as a composition or “drug in capsule”, without needing excipients. As will be discussed in more detail below, the technology disclosed herein is surprisingly effective and solves many of the larger problems introduced above.

[0119] Other implementations are also described and recited herein. These and other features and advantages will be apparent from a reading of the following detailed description and a review of the associated drawings. It is to be understood that both the foregoing general description and the following detailed description are explanatory only and are not restrictive of aspects as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Solely for the purpose of illustration, certain embodiments of the present invention are explained using examples in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and configurations shown. In the figures:

[0121] FIG.1 is a schematic showing senescence leading to atrial fibrillation. The hypothesis is that aging increases the number of senescent atrial myocytes (SAMs) and senescent atrial myofibroblasts (SAMFs). Increased abundance of SAMs and SAMFs in the left atria and right atria predisposes the aging human and rabbit heart to atrial fibrillation by SASP-mediated inflammatory response.

[0122] FIG.2A shows a two-part chart showing the characterization of atrial action potential and mapping of reentry during atrial fibrillation in aged rabbit heart. The chart shows that focal activation (a, b) and (c) maintain atrial fibrillation. Multiple atrial fibrillations occurred, lasting from a few seconds to more than ten minutes which terminated spontaneously in this rabbit. FIG.2B, FIG.2C, and FIG.2D show summary data of CV, APD, and APD dispersion from young and aged rabbit hearts. FIG.2E and FIG.2F show historic data of young and old rabbit optical mapping show that 23% of aged rabbit atria showed spontaneous AF (FIG.2E) and 61% showed AF induction under S1S2 protocol (FIG.2F). FIG.2G, FIG.2H, and FIG.2I show characterization of AF in aged rabbit hearts.

[0123] FIG.3A and FIG.3B show Action Potential Recordings of Freshly Isolated Young and Aged Atrial Myocytes: Electrophysiological experiments with acutely isolated aged and young rabbit atrial cardiomyocytes were performed to compare their electrophysiological features. The set of charts is showing APD90at a 1.25 Hz stimulation rate of atrial myocytes from young and aged rabbits. (A) Representative action potential (AP) waveforms of atrial myocytes from young rabbits and aged rabbits. (B) Bar graph showing cumulative action potential duration (APD) data from forty-nine cells of four young and four aged rabbit hearts (mean±SD, p = 0.35). In the young rabbit group, ADP90was 97±62Atty. Docket No.: 405002-549001WO milliseconds (n=26). In the aged rabbit group, ADP90was equal to 113±58 milliseconds (n=23, p=0.35).

[0124] FIG.4A and FIG.4B show results of use of a SPiDER SA-β-Gal kit to identify senescent atrial cardiomyocytes. The set of representative images of aged and young live atrial myocytes stained with SPiDER SA-β-Gal and imaged in the green channel. The first row (A) shows two representative epifluorescence images of freshly isolated aged rabbit atrial myocytes. The bottom row (B) shows a representative brightfield image and a representative epifluorescence image of freshly isolated young rabbit atrial myocytes. SA-β- Gal-positive cells show intense green fluorescence. Regular cardiomyocytes exhibit faint autofluorescence. Aged cardiomyocytes appear to have more lipofuscin, a pigmented molecule that is autofluorescent and accumulates over time in lysosomes of postmitotic cells, compared to young cardiomyocytes. Scale bars are 100 μm.

[0125] FIG.5A a set of images and FIG.5B is a pair of charts showing senescence in the atria of aged and young rabbits. The images (A) show SA-β-gal-stained images of aged (four years six months) and young animals (eleven months) (the 40x images are from the dotted areas of the 2x images). The scale bar of aged animal’s 2x image is 6 mm. The scale bar of young animal’s 2x image is 4 mm. Scale bars of 40x images are 60 µm. Blue color represents SA-β-gal positivity. The scatter plots (mean ± SEM) show the measurement of the percent SA-β-gal+cells from atria. At least five randomly selected images at 40x were used for measurement. Bars=SEM, **p<0.01, Mann-Whitney U test (n=5 aged and n=7 young rabbits).

[0126] FIG.6A depicts representative images of an aged (top panel) and a young atrium (bottom panel) stained for the myocyte marker Desmin and senescence marker γH2AX. FIG.6B depicts aged rabbit atria stained for the myofibroblast marker αSMA and senescence marker γH2AX (a marker for DNA damage repair as a surrogate for senescence). In the set of images and a set of charts showing senescence in the atria of aged and young rabbits. The images (A) in the first two rows show immunofluorescence confocal images showing staining for γH2AX and desmin from both an aged animal and a young animal. No γH2AX+nuclei were detected in the region depicted in the young animal. The top row shows images from an aged atrium. The second row shows images from a young atrium. No γH2AX+ nuclei were detected in the region depicted from the young atrium. The immunofluorescent confocal images (B) in the third row show staining for γH2AX and αSMA in an aged atrium. Panels A and B: Left images: DAPI in blue and γH2AX in red; center images: DAPI in blue and desmin in white or αSMA in green; right images include all channels together. Scale bars of confocal images are 10μm. The large yellow rectangles are magnified regions indicated by the smaller yellow rectangles. Yellow arrows indicate cells that are positive for both γH2AX and the cell type marker. The first three scatter plots (C) inAtty. Docket No.: 405002-549001WO the third row shows the results of the quantification of total percentage of desmin+, αSMA+, and γH2AX+ nuclei in aged and young atria. The fourth scatter plot (D) shows the results of the quantification of percentage ofγH2AX+ nuclei that are either desmin+ or αSMA+ in aged animals. Left and right atria were analyzed separately but no significant differences in the staining ofγH2AX and desmin / αSMA were observed. n=5 for aged, n=5 for young animals. Scatter plots= mean±SEM, *p<0.05, **p<0.01, Mann-Whitney U test. The images (E) in the bottom row show representative immunofluorescence confocal images of atria showing staining for γH2AX and F4 / 80. No γH2AX+ nuclei that were also F4 / 80+ were detected in the region depicted). Scale bars are 5.50 μm.

[0127] FIG.6C, FIG.6D, and FIG.6E illustrate identification of the types of senescent cells in rabbit atria with identification of the types of senescent cells in rabbit atria. FIG.6E shows representative IF confocal images of atria showing staining for γH2AX F4 / 80 (note: no γH2AX+ nuclei that were also F4 / 80+ were detected in the region depicted).

[0128] FIG.7A, FIG.7B, and FIG.7C show bulk RNAseq analysis of aged and young rabbit right atrial samples. The set of charts show the bulk RNAseq analysis of aged and young rabbit right atrial samples. The top left chart (A) provides results showing the SenMayo gene set enrichment analysis. GSEA shows that the SenMayo gene set is significantly enriched in aged vs young rabbit right atria (NES: normalized enrichment score). The volcano plot (B) shows the genes that up and downregulated in the aged rabbits compared to young rabbits. The bottom left scatter plots show RNA level expression differences of senescence genes p16 (CDKN2A) and p21(CDKN1A) in aged rabbit atria versus young rabbit atria (***p<0.001).

[0129] FIG.8A and FIG.8B show a senescence assessment of human RAA and LAA samples with and without AF. The set of images and a bar graph show a senescence assessment of human right atrial appendages (RAA) and left atrial appendages (LAA) samples with and without atrial fibrillation. The images show SA-β-gal -stained images of human RAA and LAA samples either with atrial fibrillation or sinus rhythm (SR) counterstained with NFR. The bar graph shows the measurement results of the percent SA- β-gal+cells. At least five randomly selected images at 40x were used for measurement. Bars=SEM. One-way ANOVA with post-hoc Tukey test.

[0130] FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E show the IF and IHC data from human right atrial specimens with and without AF. The set of images and the set of bar graphs show immunofluorescence data and immunohistochemistry data from human right atrial specimens with and without atrial fibrillation. The images in the top row (A) are representative immunofluorescence confocal images at 60x of atria with atrial fibrillation showing staining for γH2AX and desmin. The graph on the right: quantification of percent desmin+ cells. The images in the top row (B) are representative immunofluorescenceAtty. Docket No.: 405002-549001WO confocal images of atria with atrial fibrillation showing staining for γH2AX and αSMA. The graph on the right: quantification of percent αSMA+ cells. The scatter plot in the third row (C) show the measurement of the percent desmin+ cells. The images show immunofluorescence confocal images at 60x of atria with atrial fibrillation showing staining for γH2AX and desmin. The bar graph in the second row shows a measurement of the percent αSMA+ cells. The images show immunofluorescence confocal images of atria with atrial fibrillation showing staining for γH2AX and αSMA. The bar graphs in the third row show measurement of the total % γH2AX+nuclei and % γH2AX+nuclei that are desmin+or αSMA+. The bar graph in the bottom row shows the staining results for γH2AX and CD68, a macrophage marker. The images in the fourth row (D) show representative immunofluorescence confocal images of atria with atrial fibrillation showing staining for γH2AX and CD68. Panels A, B and D: Left images: DAPI in blue and γH2AX in red; center images: DAPI in blue and desmin in white, αSMA in green, or CD68 in yellow; right images include all channels together. Bigger yellow rectangles show focused areas outlined by the smaller rectangles. Yellow arrows indicate cells that are positive for both γH2AX and the cell lineage marker. Scale bars of confocal images are 20 μm. Scale bars of focused nuclear images are 10 μm. n=5 for AF, n=5 for SR. Age average of atrial fibrillation RAA is 65.8 years. Age average of SR RAA is 68.3 years. Error bars denote SEM, *p<0.05, **p<0.01, 2- tailed Exact test. The images in the bottom row (E) show representative images of female (F) and male (M) human atrial samples stained for p16 antibody. Counterstaining was done with nuclear fast red. Black rectangles indicate the positive areas for the staining. Scale bars are 10 μm. FIG.9E shows representative images of female (F) and male (M) human atrial samples stained for p16 antibody.

[0131] FIG.10A (and FIG.10B) illustrate fisetin application protocols. FIG.10A is a schematic showing fisetin application protocols. In protocol A, rabbits received fisetin (20 mg / kg) for two days throughout the protocol. In protocol B, rabbits received either fisetin (20 mg / kg) or vehicle for two days per week and for two weekly cycles throughout the protocol. See green and blue arrows. This protocol is used in the pilot assay. FIG.10B shows Fisetin 3-week treatment protocols.

[0132] FIG.11A and FIG.11B shows the senescence assessment of the Fisetin treatments on aged rabbits. The set of images and scatter plots show senescence assessment of the fisetin treatments on aged rabbits. The images (A) show representative SA-β-gal-stained images, counterstained with nuclear fast red. Blue color and blue arrows indicate areas with SA-β-gal positive cells. Small scale bars are 10μm. Big scale bar is 100μm. The scatter plot (B) shows the measurement results of the percent SA-β-gal+cells. LA: Left atrium, RA: Right atrium, Veh: Vehicle, 1-Cyc: 2 days-1 week of treatment, 2-Cyc: 2Atty. Docket No.: 405002-549001WO days-2 weeks of treatment. n=3 for all groups. The age average per group is 4.4 years. Scatter plot: Mean ± SEM, *p<0.05.One-way ANOVA with post-hoc Tukey test.

[0133] FIG.12A, FIG.12B, FIG.12C, FIG.12D, FIG.12E, FIG.12F, and FIG.12G show characterization of atrial action potential and AF in hearts of fisetin-treated aged rabbits. FIG.12H and FIG.12I show results of RT-qPCR and Western Blot experiments. The scatter plots (Mean ± SEM) in the top row (FIG.12H) show relative RNA expression levels of senescence genes p16 and p21 in the right and left atria of aged and young rabbits. The images (FIG.12I) on the left show representative right atrium blot images of three young and three aged rabbits. Y: young, A: aged, RA: right atrium, LA: left atrium. The scatter plots (mean ± SEM) on the right show relative protein expression levels of senescence genes p21 and p53 in the right and left atria of aged and young rabbits* p<0.05, **p<0.01, Mann- Whitney U test (n=5 aged rabbits and n=5 young rabbits).

[0134] FIG.13 shows a proposed fisetin study design.

[0135] FIG.14 shows an example of cellular senescence in atrial cells leading to AF (example created with BioRender).

[0136] FIG.15 shows examples of triggers, substrates and modulators that play role in the development and maintenance of AF.

[0137] FIG.16 shows a presentation of AF (bottom) compared to sinus rhythm on ECG (top).

[0138] FIG.17 shows an example chemical structure of fisetin (not taking into account salts, solvates, or tautomers).

[0139] FIG.18A and FIG.18B show representative ECG recordings from aged rabbits with sinus rhythm (FIG.18A) and AF (FIG.18B).

[0140] FIG.19A and FIG.19B illustrate fibrosis (stained blue) in atria of aged and young rabbits.

[0141] FIG.20 shows senescent cell islands (stained darker or blue) in human atrial samples.

[0142] FIG.21A and FIG.21B illustrate fibrosis in human RAA and LAA samples.

[0143] FIG.22A illustrates that in the Fisetin group 4 out 11 rabbits developed PACs (Premature Atrial Contractions) that did not progress to AF. FIG.22B shows an analysis of pre- and post-treatment ECGs revealed no significant difference between Fisetin and control group in PR, QRS, QT and RR intervals.

[0144] FIG.23A and FIG.23B show Optical mapping results of the pilot Fisetin study.

[0145] FIG.24 shows an accumulation of senescent cells and the SASP-mediated inflammatory response predisposes the atria to AF.Atty. Docket No.: 405002-549001WO

[0146] FIG.25A and FIG.25B illustrate, in a flowchart, a method for preventing or treating atrial fibrillation (AF) in various subjects.

[0147] FIG.26A and FIG.26B illustrate, in a flowchart, the administration of various doses of Formula 1 or Fisetin derivatives.

[0148] Any trademarks mentioned or shown herein are used in fair use. It should be understood that while different depictions and / or numbers / numbering are / is sometimes used in some of the figures above to describe different embodiments and different aspects of the technology, any depiction and / or number from any figure can be inter-combined with a depiction or numbered aspect from any other figures. All trademarks, images, likenesses, words, and depictions in the drawings and the disclosure are plainly in fair use and are provided solely for the purposes of illustration of the invention in view of an urgent need to treat subjects as further discussed in detail below. DETAILED DESCRIPTION OF THE INVENTION Industrial applicability

[0149] Previous clinical, animal, and in vitro results showed that cytokine-mediated ion channel remodeling in cardiomyocytes can prolong action potential duration (APD) and QT interval and increase risk for atrial and ventricular arrhythmias and sudden cardiac death (SCD). See Mitrokhin, Mladenov, & Kamkin, Immunobiology, ;220(9), 1107-12 (2015); Empana et al., Arterioscler. Thromb. Vasc. Biol., 30(10), 2047-52 (2010)(6); Hussein et al., Heart Rhythm., 10(10), 1425-32 (2013); Issac, Dokainish, & Lakkis, J. Am. Coll. Cardiol., 50(21), 2021-8 (2007)(8); and Parekh et al., Kidney Int., 74(10), 1335-42 (2008)(9). Higher IL-6 levels were associated with a greater risk of atrial fibrillation in the general population and atrial fibrillation recurrence after cardioversion or catheter ablation. Wu et al., Int. J. Cardiol., 169(1):62-72 (2013)(10). Both IL-1β and IL-6 increased L-type calcium current, increasing myocytes' calcium load. Francis Stuart, De Jesus, Lindsey, & Ripplinger, J. Mol. Cell Cardiol., 91, 114-22 (2016)(12); Aromolaran et al., PLoS One, 13(12), e0208321 (2018)(13). Increased calcium load in atrial cells is associated with atrial fibrillation. De Jesus et al., Heart Rhythm.2017;14(5):727-36(14); Ali, Boutjdir, & Aromolaran, Front Physiol., 9, 1866 (2018)(15); and Qi et al., Circ Res., 103(8), 845-54 (2008)(16). Senescence-associated secretory phenotype (SASP) also causes secretion of TGF-β, an inducer of atrial fibrosis. Nattel, JACC Clin. Electrophysiol., 3(5), 425-35 (2017)(17). SASP could induce both trigger and substrate for atrial fibrillation.

[0150] Targeting senescence to suppress senescence-associated secretory phenotype should have an anti-arrhythmic effect. Recent reports indicate that senescent cells can be targeted by drugs and selectively removed from tissues. The approach is founded on observations that senescent cells resist apoptosis, downregulate proapoptoticAtty. Docket No.: 405002-549001WO genes, and upregulate anti-apoptotic genes. Chang et al., Nature Med., 22(1), 78-83 (2016)(18). Results

[0151] The inventors showed that accumulations of multiple cell types were positively stained for senescence-associated β-galactosidase (SA-β-gal) and the senesce- associated DNA damage marker γH2AX in aging mouse atria, in rabbit atria, in human atria of explanted hearts from patients with atrial fibrillation, and in atrial appendages biopsies of patients who underwent open-heart surgery. The inventors showed that most of these cells are senescent atrial myocytes (SAMs). A small fraction are senescent atrial myofibroblasts (SAMFs). These stained cells are absent from young mouse, rabbit, and human atria. RNAseq of aged rabbits’ atria showed a significant elevation of the SenMayo gene set and elevated markers of inflammation. Aged rabbits have a low threshold for induction of atrial arrhythmias.

[0152] The inventors documented spontaneous atrial fibrillation in four of thirty-two aged rabbits. By pacing or stretching the left atria, the inventors documented induced atrial fibrillation in four of five aged rabbits. Young rabbits (N=4) were not induced. Definitions

[0153] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are listed below. Unless stated otherwise or implicit from context, these terms and phrases shall have the meanings below. These definitions aid in describing particular embodiments but are not intended to limit the claimed invention. Unless otherwise defined, the technical and scientific terms have the same meaning as commonly understood by a person having ordinary skill in the biomedical art. A term's meaning provided in this specification shall prevail if any apparent discrepancy arises between the meaning of a definition provided in this specification and the term's use in the biomedical art.

[0154] Aging has the biomedical art-recognized meaning. Aging is the time-related deterioration (characteristic of a species) of the physiological functions necessary for survival and reproduction. The phenotypic changes of senescence (which affect all members of the species) are not the same as diseases of senescence, such as cancer and heart disease (which affect individuals).

[0155] Senescent cardiomyocytes have the biomedical art-recognized meaning.

[0156] Atrial fibrillation (AF) has the biomedical art-recognized meaning.

[0157] Cellular senescence has the biomedical art-recognized meaning of an irreversible proliferative arrest of normal somatic cells triggered by many repetitions of cell division. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res.1961;25:585-621(19). The underlying cause of this phenomenon is telomereAtty. Docket No.: 405002-549001WO shortening. Many intrinsic and extrinsic stimuli can also trigger cellular senescence, stresses such as oncogene activation, exposure to reactive oxygen species (ROS), or injury. Rodier & Campisi, J. Cell Biol., 192(4), 547-56 (2011)(20); Sedivy, Munoz-Najar, Jeyapalan, & Campisi, In: Guarente & Partridge, editors. The Molecular Biology of Aging (Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2007)(21).

[0158] Cluster of Differentiation 68 (CD68) has the biomedical art-recognized meaning as a macrophage marker.

[0159] Comprises and comprising refer to elements, components, or steps in a non- exclusive manner, indicating that the referenced elements, components, or steps may be present, used, or combined with other elements, components, or steps. The singular terms "a," "an," and "the" include plural referents unless context indicates otherwise. The inclusive term "or" should cover the term "and" unless the context indicates otherwise. The abbreviation "e.g." means a non-limiting example and is synonymous with the term “for example.”

[0160] Desmin has the biomedical art-recognized meaning of the muscle-specific, type III intermediate filament that integrates the sarcolemma, Z disk, and nuclear membrane in sarcomeres and regulates sarcomere architecture. Desmin is a myocyte marker.

[0161] ECG and EKG have the biomedical art-recognized meaning of electrocardiogram.

[0162] γH2AX has the biomedical art-recognized meaning of the phosphorylated histone variant H2AX, which is a sensitive marker for DNA damage.

[0163] Fisetin has the biomedical art-recognized meaning of the flavonol 3,7,30,40- tetrahydroxyflavone (IUPAC name), 2-(3,4-Dihydroxyphenyl)-3,7-dihydroxy-4H-1- benzopyran-4-one (systematic IUPAC name), Systematic IUPAC name, CAS Number 528- 48-3, which is widely found as glycosides or as aglycone in various edible fruits and vegetables. Fisetin is an effective “senolytic” drug, effectively removing most senescent cells and prolonging lifespan in different models. See Yousefzadeh et al., eBioMedicine, (2018)(22), reviewed by Grynkiewicz et al., Front. Chem. (2019)(23) and Elsallabi et al., Molecules (2022)(24). Fisetin functions by suppressing BCL-2 family members such as BCL- xL and HIF-α and other parts of the senescent cells' anti-apoptotic pathways (SCAPs) network. See Yousefzadeh et al., eBioMedicine, (2018)(22). Fisetin is also a topoisomerase inhibitor, which may also contribute to its anti-cancer activity. Salerno et al., Current Medicinal Chemistry (2010)(25). Fisetin may directly inhibit cytokines like TNFα and IL6. See Gupta et al., Archives of Biochemistry and Biophysics (2014)(26). A 20 mg / kg dose is like the one used in human clinical trials.

[0164] Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for understanding high-level functions of a biological system from molecular-levelAtty. Docket No.: 405002-549001WO information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput technologies. KEGG pathway has the biomedical art-recognized meaning of a molecular interaction / reaction network diagram represented in terms of the KEGG Orthology (KO) groups so that evidence in specific organisms can be generalized to other organisms through genomic information.

[0165] Left atrial appendages (LAA) have the biomedical art-recognized meaning of small ear-shaped outpouchings of the muscular wall of the left atrium, lying anteriorly in the atrioventricular sulcus, close to the left circumflex artery, phrenic nerve, and pulmonary veins.

[0166] Normal sinus rhythm (NSR) has the biomedical art-recognized meaning of the rhythm that originates from the sinus node and describes the typical rhythm of the healthy human heart.

[0167] Right atrial appendages (RAA) have the biomedical art-recognized meaning of right auricles (TA), trapezoidal pouches forming the anterosuperior part of the right atrium.

[0168] Senescence-associated secretory phenotype (SASP) has the biomedical art- recognized meaning. The senescence-associated secretory phenotype is characterized by the increased expression and secretion of pro-inflammatory cytokines and chemokines, some growth factors, and tissue-remodeling metalloproteinases (MMPs). van Deursen, Nature, 509(7501), 439-46 (2014)(27). SASP is the most important physiological manifestation of senescence. Coppe et al., PLoS Biol., 6(12), 2853-68 (2008)(28). SASP is characterized by increased expression and secretion of pro-inflammatory cytokines and chemokines, some growth factors, and tissue-remodeling metalloproteinases. Coppe et al., PLoS Biol., 6(12), 2853-68 (2008)(28). Accumulation of senescent cells during aging is now viewed as a pro-inflammatory process, and, in mouse models, whole-body genetic or pharmacologic ablation of these cells has clear health benefits, including the extension of lifespan. Baker et al., Nature, 530(7589), 184-9 (2016)(29); Baker et al., Nature, 479(7372), 232-6 (2011)(30).

[0169] Senescent atrial myocytes (SAMs) have the biomedical art-recognized meaning.

[0170] Senescent atrial myofibroblasts (SAMFs) have the biomedical art-recognized meaning.

[0171] Senolytic therapy has the biomedical art-recognized meaning. Several senolytic drugs are known in the biomedical art, such as the combination of dasatinib and quercetin. See review by Zhang et al., The FEBS Journal, 290(5), 1362-1383 (2023)(5), for a list of senolytics.

[0172] Smooth muscle alpha-actin (αSMA) has the biomedical art-recognized meaning of the marker for the activated cardiac fibroblast population known as theAtty. Docket No.: 405002-549001WO myofibroblasts. αSMA is a myofibroblast marker.

[0173] Troponin T has the biomedical art-recognized meaning of a cardiomyocyte marker.

[0174] von Willebrand Factor (vWF) has the biomedical art-recognized meaning of the glycoprotein crucial to primary hemostasis through platelet and subendothelial collagen adhesion, and the intrinsic coagulation cascade, through Factor VIII stabilization. vWF is an endothelial cell marker.

[0175] Unless otherwise defined, scientific and technical terms used with this application shall have the meanings commonly understood by persons having ordinary skill in the biomedical art. This invention is not limited to the method, protocols, reagents, etc., described herein and can vary.

[0176] This specification does not concern a process for cloning humans, methods for modifying the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or procedures for modifying the genetic identity of animals likely to cause them suffering with no substantial medical benefit to humans or animals resulting from such processes. Guidance from materials and methods

[0177] Persons having ordinary skill in the biomedical art can use these materials and methods as guidance to predictable results when making and using the invention:

[0178] Aged female (retired breeders) rabbits model system for assaying the aging heart: The inventors’ published results showed that aged rabbit hearts effectively recapitulate the human aged heart, with increased interstitial fibrosis and slowing of conduction in the Purkinje system. Cooper, Am J. Physiol. Heart Circ. Physiol. (2012)(31). The inventors also showed that aging increased the production of mitochondrial ROS in ventricular cells, which led to abnormal calcium dynamics and membrane oscillations through oxidation of the ryanodine receptor. Cooper et al., J. Physiol., 591(Pt 23), 5895-911 (2013)(31). The inventors further showed that stimulating autophagy in aged ventricular myocytes reduced mito-ROS production and the arrhythmogenic phenotype. Murphy, Front. Physiol. (2019)(32). Preliminary results from using aged atrial tissues showed accumulation of multiple cell types positively stained for SA-β-gal and nuclear γH2AX in the aging mouse atria, rabbit atria, human atria, and in biopsies of atrial appendages of patients who underwent open-heart surgery. A scientific publication documented SA-β-gal positive cells in atrial appendages of atrial fibrillation patients. Jesel, J. Clin. Med. (2019)(33). Although gaps remain in the characterization of senescent cells, detailed lineage assays enabled by this invention can bridge this gap.

[0179] Aged New Zealand white rabbit (Oryctolagus cuniculus) as an animal model for atrial fibrillation: The aging rabbit recapitulates age-associated cardiac dysfunctions seenAtty. Docket No.: 405002-549001WO in people. Cooper et al., Am. J. Physiol. Heart Circ. Physiol., 302(8), H1625-35 (2012)(34). These dysfunctions include aortic stiffening, increased interstitial fibrosis leading to decreased compliance of the ventricular myocardium, decreased complexity of the His- Purkinje network leading to abnormal conduction, and lower thresholds for inducible ventricular arrhythmias. Cooper et al., Am. J. Physiol. Heart Circ. Physiol., 302(8), H1625-35 (2012)(34). Optical mapping showed shortened activation time and expansion of the QRS complex without significant changes in action potential duration. Cooper et al., Am. J. Physiol. Heart Circ. Physiol., 302(8), H1625-35 (2012)(34). Studies have shown that a greater proportion of individuals show a prolonged QT interval as people age, with the duration of the QT interval increasing with age. Aging increased the production of mitochondrial ROS in ventricular cells, which led to abnormal calcium dynamics and membrane oscillations through oxidation of the ryanodine receptor. Cooper et al., J. Physiol., 591(Pt 23), 5895-911 (2013)(31). These studies also showed that stimulating autophagy in aged ventricular myocytes reduced mito-ROS production and the arrhythmogenic phenotype. Murphy et al., Front. Physiol., 10, 1277 (2019)(32). See also Baggett et al., eLife (May 19, 2023)(35) (myofibroblast senescence in the infarct border zone is proarrhythmogenic).

[0180] Atrial fibrillation induction in rodents is difficult due to the small size of the atrial tissue and species-dependent action potential characteristics. Larger animal models of atrial fibrillation were developed in sheep or dogs using tachy-pacing of atria to trigger atrial fibrillation artificially. Nattel et al., Prog Cardiovasc Dis., 48(1), 9-28 (2005)(17).

[0181] Atrial fibrillation in aged rabbit hearts replicates important clinical features of atrial fibrillation in the elderly population such as automaticity, abnormal action potential duration, discontinuous conduction, and reentry formation. Screening of the aging rabbits’ colony (> four years) using surface ECG and echocardiogram detected spontaneous atrial fibrillation in four out of thirty-two rabbits (12.5%).

[0182] This finding is important because spontaneous atrial fibrillation episodes in larger mammals are rare. Schuttler et al., Circ. Res., 127(1), 91-110 (2020)(36). The percentage of spontaneous atrial fibrillation in aged rabbits is similar to those in the aging human population. See Institutional Animal Care and Use Committee (IACUC) protocol number 5034-21. These exciting results make the aged rabbit a good model system for mechanistic atrial fibrillation studies.

[0183] Animal ethical statement. The assays in this disclosure conformed with the current Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication, Revised 2011) and the standards recently delineated by the American Physiological Society (“Guiding Principles for Research Involving Animals and Human Beings”) and was approved by the Institutional Animal Care and Use Committee ofAtty. Docket No.: 405002-549001WO Rhode Island Hospital. Young (≤1 year old) and aged (≥4 years old) female New Zealand White rabbits acquired from Robinson Services Inc. (RSI, NC, USA) were anesthetized.

[0184] Echocardiogram. Echo was performed by a PGY-6 cardiology fellow undergoing level 3 echocardiography certification. Rabbits were sedated. Excess fur was removed before imaging. Echo was performed before and after treatment with fisetin. Left atrial strain and left atrial volumes were compared pre-treatment and post-treatment. Echocardiographic measurements: Mitral valve E and A waves, e' septal and e' lateral, E / e' (mean), Left atrial volume (Simpson's biplane), Left ventricular ejection fraction by Simpson's biplane, Isovolumic relaxation time, Left atrial global longitudinal strain (reservoir strain), TAPSE, RV S', Right ventricle global longitudinal strain, Tricuspid regurgitation maximum velocity. The assay was powered to detect a difference of 25% between the fisetin and vehicle groups requiring a population of twenty-five rabbits. Left atrial strain analysis was done using TomTec software and speckle tracking. The cardiac cycle of interest was selected using the R-R intervals. The left atrium was traced in systole and diastole, using automated tracing within the software, followed by manual adjustment as needed. Speckles are tracked on a frame-by-frame basis throughout the cardiac cycle. The measured change in distance between the different speckles was used to calculate left atrial movement. The left atrial strain was measured twice on two images from an apical four-chamber view with a frame rate between forty-sixty frames per second. Left atrial volumes in systole and diastole were recorded from tracing and speckle tracking. Left atrial reservoir strain was recorded.

[0185] Tissues. Rabbits were anesthetized with ketamine (60 mg / kg), xylazine (15 mg / kg IM), buprenorphine (0.03 mg / kg SQ), and sodium pentobarbital (150 mg / kg IV). Hearts were excised and washed in phosphate-buffered saline. Gross anatomy photos of the left and right atria were taken right after the heart harvest. Heart tissue for histological analysis was embedded in optimal cutting temperature (OCT) compound solution (Fisher Scientific, Waltham, MA, USA) and rapidly frozen on liquid nitrogen. Heart tissue for biochemical analysis was snap-frozen in liquid nitrogen and stored immediately at -80°C. Cryosectioning was performed at 5 µm thickness for immunofluorescence (IF), 6 µm for immunohistochemistry (IHC), and 10µm for Senescence-associated β-galactosidase (SA-β- gal) and Masson's Trichrome staining using a Leica CM1950. Slides were immediately fixed in cold 4% paraformaldehyde (PFA) in phosphate-buffered saline for ten minutes, followed by a wash in phosphate-buffered saline for ten minutes for IF, IHC, and Masson's Trichrome. For SA-β-gal, slides were fixed in 0.5% glutaraldehyde, followed by a wash in phosphate- buffered saline for ten minutes.

[0186] RNA purification and sequencing. In an example, total RNA can be purified from cultured cells using the Qiagen RNAeasy kit. RNA sequencing can be performed by Genewiz®, Inc. Our total RNA isolation was from tissues using TRIzol reagent (Invitrogen,Atty. Docket No.: 405002-549001WO 15596018) according to the manufacturer’s instructions and sequencing was performed by the company now called Azenta®. Qiagen kit can be used for the same purpose as well.

[0187] Senescence-associated beta-galactosidase (SA-β-gal) assay is one of the most common methods for detecting senescence, taking advantage of increased lysosomal β-galactosidase expression in senescent cells. In normal cells, the activity of lysosomal β- galactosidase enzyme is detectable at a pH of about 4. However, the activity of the senescence-associated enzyme, which is highly overexpressed in senescent cells, is detectable at pH 6.0 and used as a marker for senescent cells through a histological staining assay. Itahana, Itahana, & Dimri, Methods Mol. Biol., 965, 143–156 (2013)(37); Debacq- Chainiaux, Erusalimsky, Campisi, & Toussaint, Nature Protocols, 4, 1798–1806 (November 19, 2009)(38).

[0188] Persistent DNA damage markers ɣH2AX, ATM, ATR foci, and telomere dysfunction-induced foci (TIFs) are commonly used to identify senescent cells. ɣ-H2AX is a histone variant protein rapidly phosphorylated after receiving a DNA damage signal.

[0189] Senescent cells are typically identified by the upregulation of senescence- associated gene expression pathways, such as the p53-p21CIP1 (also called p21) and the p16INK4a (hereafter called p16)-Retinoblastoma (Rb) pathways. Other senescence biomarkers include an absence of DNA proliferation using EdU and decreased lamin B1 levels. Hernandez-Segura, Nehme, & Demaria, Trends Cell. Biol., 28(6), 436-53 (2018).

[0190] Adult rabbit cardiac myocyte isolation. The heart was removed from anesthetized young or aged New Zealand White rabbits and perfused for five minutes (20 mL / min) with a basic buffer (130 mM NaCl, 4.5 mM KCl, 3.5 mM MgCl2, 0.4 mM NaH2PO4, 5 mM HEPES, and 10 mM glucose), to which 0.75 mM CaCl2was added. After eight minutes, 0.1 mM EGTA was added to the buffer. Then the heart was perfused with enzyme buffer (basic buffer with 216 or 432 U / mL collagenase I (Worthington Biochemical), 0.52 or 1.08 U / mL protease XIV, and 240 or 480 µM CaCl2). The left atria and right atria were cut off from the heart, minced into fine pieces, and then transferred into a small beaker that contained 2.0 mL of enzyme buffer and 2% Bovine Serum Albumin (BSA) (Sigma, Cat# A7638-25G). The beaker was then incubated in a 37°C water bath shaker at 137 revolutions per minute. A drop of the supernatant was checked under the microscope every three-four minutes until an amount of single atrial cells appeared. The supernatant was collected by filtering through a 100 µm cell strainer into a 50 mL tube that contained 1.5 mL of potassium buffer (KB) (85 mM KCl, 5 mM MgSO4, 30 mM KH2PO4, 7 mM H2O, 5 mM Na2ATP, 5 mM pyruvate Na, 5 mM DL-ß-hydroxybutyric acid sodium salt, 5 mM phosphocreatine di(tris) salt, 20 mM Taurine, 0.56 mM EGTA, 50 g / L PVP 40, 0.1 mM CaCl2, and 20 mM glucose), to which 3% bovine serum albumin was added. The tissue was rinsed from the strainer into a beaker with 1.5 mL of fresh enzyme buffer and 2% bovine serum albumin to continue incubation. TheAtty. Docket No.: 405002-549001WO collection was repeated every five minutes until sufficient. All fractions were combined and filtered again into a 50 mL tube. The tube was spun down at 80 g for four minutes, and the pellet was resuspended in a 15 mL tube with 4-5 mL of potassium buffer. Filtration was done again if needed. The 15 mL tube was spun down at 80g for two minutes. The pellet was again resuspended in 7-8 mL of potassium buffer. Myocytes sat in the buffer with a loose cap for 30-45 minutes to recover before use.

[0191] Optical mapping, stimulation of epicardial surface, and data analysis. See FIG.2A. Young (n=4) and aged rabbits (n=5) were euthanized with buprenorphine (0.03 mg / kg IM), acepromazine (0.5 mg / kg IM), xylazine (15 mg. / g IM), ketamine (60 mg / kg IM), pentothal (35 mg / kg IV), and heparin (200 U / kg). Hearts were excised from the chest and perfused in a Langendorff perfusion apparatus (Radnoti LLC, Covina, CA, USA). Blebbistatin (10 µmol / L) was perfused to reduce movement artifacts (Fedorov et al., 2007). Hearts were stained with the voltage-sensitive dye di-4-ANEPPS, and ECG and perfusion pressure were constantly monitored (PowerLab, AD Instruments, Sydney, Australia). A dual camera system (Ultima-L, Scimedia, Japan) was used to simultaneously record the right and left atria. The pacing electrode was placed on the SA nodal region to test vulnerability to atrial fibrillation under stimulation protocol. The hearts underwent two stimulation protocols: S1S1 basic cycle length (CL=350 milliseconds) followed by a single premature S2 beat to reduce S2 cycle length (CL) by ten milliseconds for action potential duration restitution and tissue refractory period or atrial tachycardia (AT) / AF induction. The Journal of Physiology, 587(19), 4661-4680 (October 1, 2009)(39). S1S1 basic cycle length (CL=350 milliseconds) followed by five premature S2 beats to reduce the cycle length of S2 beat by ten milliseconds until it reaches refractoriness or AT / atrial fibrillation induction. The data were analyzed with a custom-built software program developed in Interactive Data Language (Harris Geospatial Solutions). Action potential durations and APD maps were calculated via the difference between activation and repolarization timepoints, determined from fluorescence (F) signals by calculating (dF / dt)max and (d2F / dt2)max. Data were filtered using a temporal polynomial filter (3rdorder, thirteen points). Salerno et al., Curr. Med. Chem., 17(35), 4270-90 (2010)(25). Action potential conduction velocity was calculated using the spatial gradient of activation time (11x11 pixels) and spatial resolution of the acquired image (200 µm / pixel).

[0192] Electrophysiology recording via patch clamping. Whole-cell patch clamp recordings from freshly isolated rabbit myocytes. Assays were conducted in the whole-cell configuration at 35–37°C with Axopatch-200B, Digidata 1440A, and pClamp 10 software (Molecular Devices). After the cell membrane was broken by suction, the cell membrane capacitance and series resistance were compensated by 70–80% in the voltage clamp mode. Tyrode solution (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.33 mM NaH2PO4, 7.5 mM glucose, and 5 mM HEPES, pH 7.4 adjusted with NaOH) was a standardAtty. Docket No.: 405002-549001WO bath solution. The pipette solution contained 90 mM potassium aspartate, 50 mM KCl, 5 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 5 mM MgATP, and 0.1 mM GTP Tris (pH 7.2 adjusted with KOH). Action potential was activated in the current clamp mode by a three-millisecond injection of a depolarizing current, which was 20% higher than the threshold of action potential activation. The action potential stimulation rate was 1.25 Hz or 0.25 Hz. The voltage output was filtered by a low-pass filter with a cutoff frequency of 10 kHz and sampled at 20 kHz. The data are presented as mean±SEM.

[0193] SPiDER-ß-gal assay. Wells were covered with laminin (Santa Cruz Biotechnology, Inc. sc-20912) and placed in the incubator at 37°C until the myocytes were isolated and ready for seeding. Myocytes were seeded and allowed to adhere for forty-five minutes. The culture medium was discarded, and the cells were washed with 2 mL of HBSS. Bafilomycin A1 working solution was prepared as directed by the manufacturer, Dojindo Molecular Technologies Inc. Then, 1 mL of bafilomycin A1 working solution was added. The cells were incubated at 37°C for one hour in a 5% CO2incubator. Next, 1 mL of the SPiDER- β-gal working solution was added. The cells were incubated at 37°C for one hour in a 5% CO2incubator. After removing the supernatant, the cells were washed twice with 2 mL of HBSS. The cells were then imaged using a Nikon Eclipse TE2000U Epifluorescence Microscope.

[0194] Senescence-associated ß-galactosidase assay. SA-β-gal staining was performed with modifications as described by Debacq-Chainiaux, Erusalimsky, Campisi, & Toussaint, Nature Protoc., 4(12), 1798-806 (2009)(38); Dimri et al., Proc. Natl. Acad. Sci. U. S. A., 92(20), 9363-7 (1995)(40). Right after cryosectioning, tissue sections were immersed in 0.5% glutaraldehyde (in 1x phosphate-buffered saline) for ten minutes and then placed in fresh 1x phosphate-buffered saline for ten minutes. Sections were kept at -20°C. For staining, sections were incubated with fresh β-galactosidase staining solution (40 mM citric acid / Na phosphate buffer, 5 mM K4[Fe(CN)6]·3H2O, 5 mM K3[Fe(CN)6], 150 mM sodium chloride, 2 mM magnesium chloride, and 1 mg / ml X-gal) at 37°C with no CO2for sixteen hours. Sections were counterstained with Nuclear Fast Red (Vector Labs), and slides were imaged using an Aperio ScanScope (Leica Biosystems, Buffalo Grove, IL, USA) with 2x-40x objective. Images were analyzed using Aperio ImageScope v12 software (Leica Biosystems, Buffalo Grove, IL, USA). Randomly selected five representative images at 40x magnification were used for measurement. The two atrial chambers were analyzed separately. The percentage of SA-β-gal-positive cells was calculated by hand counting the number of SA-β- gal-positive and SA-β-gal-negative cells using the counter tool in ImageJ.

[0195] Immunofluorescence (IF) staining.5 µm-thick frozen sections were acquired and fixed as described above. Steps were performed in a humidified chamber. The tissue was encircled with a PAP pen and washed in phosphate-buffered saline three times. ForAtty. Docket No.: 405002-549001WO intracellular targets, frozen sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for thirty minutes. Slides were then blocked with normal donkey or goat serum in phosphate-buffered saline for one hour. Slides were incubated with a primary antibody (γH2AX, αSMA, desmin, CD31, or CD68) diluted in blocking buffer overnight at 4°C in the dark, then washed in phosphate-buffered saline three times for five minutes each, then incubated in secondary antibody (Alexa 594 goat anti-mouse or Alex 647 donkey anti-goat) in blocking buffer for one hour and thirty minutes in the dark. Slides were again washed in phosphate-buffered saline three times for five minutes each. When slides were being stained for multiple intracellular targets, the slides were placed in blocking buffer again. The steps of adding primary and secondary antibodies were repeated.

[0196] After the antibodies had been added, incubated, and washed off, slides were mounted with Prolong Gold Reagent with DAPI. Antibodies used in the assays were validated with rabbit atrial samples before starting any assays and are listed in Table 1. The slides were imaged with a Nikon Ti2 confocal microscope with an A1R scanner. Randomly selected five representative Z-stack images at 60x magnification were used for measurement. Images were analyzed via Nikon NIS Element AR software. The number and percentage of cells with positive signals were derived from maximum projection images by hand using the counter tool in ImageJ. Nuclei with small but more than three γH2AX foci and with big γH2AX foci were counted as positive. Mann-Whitney U or 2-tailed Exact test was used for statistical analyses. Table 1 (further below) shows examples of antibodies used in histology assays.

[0197] Immunohistochemistry (IHC) staining. Frozen sections were stained with immunohistochemistry.6 µm sections were obtained and fixed as described above. The steps were performed in a humidified chamber. The tissue was encircled with a PAP pen, then washed in phosphate-buffered saline three times. For intracellular targets, the slides were permeabilized using 0.1% Triton X-100 in phosphate-buffered saline for fifteen minutes. Slides were incubated in BLOXALL Blocking Solution (VECTOR Laboratories: VECTASTAIN Elite ABC Kit, peroxidase [mouse IgG]) for ten minutes to quench endogenous peroxidase activity. The slides were then washed in phosphate-buffered saline for five minutes. Next, the slides were incubated in 1.5% blocking serum (VECTOR Laboratories: VECTASTAIN Elite ABC Kit, peroxidase [mouse IgG]) for 30 minutes at room temperature. After aspirating the blocking serum, the slides were incubated with a primary antibody (p16, Ventana, Lot# J03988) diluted as desired in 2.5% normal animal serum for thirty minutes, then washed in phosphate-buffered saline for five minutes and incubated with a secondary antibody (VECTOR Laboratories: VECTASTAIN Elite ABC Kit, peroxidase [mouse IgG]) diluted in 1.5% normal animal serum for 30 minutes at room temperature. After another 5-minute phosphate-buffered saline wash, the slides were incubated in ABC solutionAtty. Docket No.: 405002-549001WO (VECTOR Laboratories kit: 100 µL from reagent A.100 µL from reagent B per 5 mL phosphate-buffered saline), prepared at least fifteen-thirty minutes beforehand, for thirty minutes at room temperature. The slides were washed in phosphate-buffered saline for five minutes. Then they incubated in DAB solution (Vector Laboratories DAB Substrate Kit, peroxidase for five-eight minutes before the slides were rinsed in tap water to stop the reaction and then rinsed in distilled water. Finally, the slides were counterstained with Nuclear Fast Red, dehydrated through a graded series of ethanol and xylene (1x 70% ethanol, 2x 100% ethanol, 3x Xylene), and mounted with Micromount media (Leica). The slides were dried overnight at room temperature and stored in a slide box. Slides were imaged using a Nikon Eclipse TE2000U epifluorescence microscope using a 60x objective.

[0198] Masson’s trichrome staining. Frozen sections were stained with Masson's Trichrome for fibrosis assessments.10 µm sections were obtained and fixed as described above. Slides were kept at -20°C until staining was performed. Bouin's fixative (Electron Microscopy Sciences) was preheated to 56°C in a water bath. After briefly rinsing the slides in distilled water, the slides were placed in heated Bouin’s fixative for one hour at 56°C in the water bath. The slides were washed until the yellow color of the fixative disappeared and then rinsed in distilled water before being stained in Weigert’s Iron Hematoxylin Working Solution (equal parts Weigert’s iron hematoxylin A and hematoxylin B, from Electron Microscopy Sciences) for five minutes. The slides were washed in running tap water for ten minutes, rinsed in distilled water, and then stained in Biebrich scarlet-acid fuchsin (Electron Microscopy Sciences) for fifteen minutes. After rinsing in distilled water, the slides were stained in phosphomolybdic acid-phosphotungstic acid (Electron Microscopy Sciences) for fifteen minutes. The slides were then stained in Aniline Blue Solution (Electron Microscopy Sciences) for fifteen minutes and rinsed in distilled water afterward. Next, the slides were differentiated in 1% acetic acid (Electron Microscopy Sciences) for four minutes and then dehydrated through a graded series of ethanol and xylene (1x 70% ethanol, 2x 100% ethanol, 3x xylene) before being mounted with Micromount Media (Leica). Slides were imaged using an Aperio ScanScope (Leica Biosystems, Buffalo Grove, IL, USA) with a 2x- 40x objective. Randomly selected five representative images at 40x magnification were used for measurement, and the two atrial chambers were analyzed separately. Computerized assessment via Image J software and a script developed by Eric Mi in the Gideon Koren laboratory at Brown University was used to calculate the percentage of fibrotic areas. Images with less than 25% empty (white) areas were used for measurement and analysis purposes. The analyses were done blinded. Mann-Whitney U test was used for statistical analysis.

[0199] Fisetin preparation. Fisetin (MedChemExpress, Cat#: HY-N01892) was added at a concentration of 10 mg / mL to a solvent composed of 45% PEG300, 5% Tween-Atty. Docket No.: 405002-549001WO 80, and 50% Saline. The solution was placed in a sonicator for one hour or until the fisetin was dissolved.

[0200] Introduction and Aims: Atrial fibrillation (AF) is the most prevalent arrhythmia among the elderly population, with a lifetime risk of 25% after the age of 40 years. According to recent projections, by 2050, there will be more than likely 12 million people living with AF in the US (42, 43). The significance of AF is underscored by its increasing prevalence and the potential health complications associated with it. AF is associated with an increased incidence of stroke, heart failure, and death. The economic burden of AF is substantial due to the high healthcare costs of hospitalizations related to its complications and the procedures to treat AF (44).

[0201] A key risk factor for the development of AF is aging, and AF prevalence increases with age. (45). Cellular senescence has come to light as a potential contributing component to the complex and multifaceted mechanisms underpinning the link between aging and AF. (46). Cellular senescence is a state of permanent growth arrest that occurs in response to numerous stressors, including chronological aging (19). Senescent cells secrete a range of bioactive molecules, collectively known as the senescence-associated secretory phenotype (SASP), which can promote inflammation, fibrosis, and oxidative stress, all of which are implicated in the pathophysiology of AF (27).

[0202] The relationship between cellular senescence and inflammation is particularly relevant in the context of AF, as many studies now suggest that inflammation plays a key role in the pathogenesis of AF (1). Elevated levels of inflammatory biomarkers have been associated with the prevalence and prognosis of AF, and AF has been linked to the expression of inflammatory markers in cardiac tissues (2, 47). Importantly, higher IL-6 levels were associated with greater risk of AF in the general population and AF recurrence after cardioversion or catheter ablation (48). Both IL-1β and IL-6 increase L-type calcium current, which in turn increases calcium load in myocytes (11, 49), which in atrial cells is associated with arrhythmogenesis (50-52). Moreover, a recent study demonstrated that senescent ventricular cardiomyocytes produced from human induced pluripotent stem cells replicate the phenotype of aged cardiomyocytes in terms of senescence markers, electrical and Ca2+handling characteristics (e.g., prolonged multicellular QTc and single cell Action Potential Duration (APD), with prolonged APD variability and increase frequency of delayed afterdepolarizations (DADs) incidence), and metabolic features (53).

[0203] Despite significant advances in the understanding of the mechanisms underlying AF, current therapies remain suboptimal. Anti-inflammatory drugs have been shown to have beneficial effects in experimental AF, and some studies have reported positive outcomes in clinical trials (1, 54). However, the efficacy of anti-inflammatory therapy in AF remains controversial, likely due to the incomplete understanding of the inflammatoryAtty. Docket No.: 405002-549001WO processes that underlie AF. Anticoagulation, rate control, and rhythm control strategies have limited efficacy and are associated with various side effects (55). Therefore, there is a pressing need for new therapeutic approaches and the understanding of the complex interplay between cellular senescence, inflammation and the pathogenesis of AF is critical for the development of effective therapeutic strategies for this debilitating condition. FIG.14 shows an example of cellular senescence in atrial cells leading to AF (example created with BioRender).

[0204] A central hypothesis of this work is that the aging process leads to the accumulation of senescent atrial myocytes (SAMs) and senescent atrial myofibroblasts (SAMFs) in the left and right atria. The accumulation of these cells predisposes the heart (herein rabbit heart) to AF through the senescence-associated secretory phenotype (SASP)- mediated inflammatory reaction. Specifically, cellular senescence could promote arrhythmias through two possible mechanisms: 1) By directly modifying the electrical properties of SAMs, or through the pro-arrhythmic effects of SASP on the adjacent atrial myocytes (AMs) (FIG. 14).2) SASP-induced inflammation may initiate and exacerbate atrial fibrosis, promoting electrical heterogeneity and conduction block thereby forming the substrate that can maintain reentrant wavelets that underlie AF. We posit that a therapy with a senolytic drug such as Fisetin (if started early) will eliminate senescent cells and will have a direct anti- inflammatory and thereby anti-arrhythmic effect, diminishing the incidence of spontaneous and inducible AF in aging rabbit hearts and will potentially open up an avenue to a novel pharmacological therapy for AF in clinical practice.

[0205] Overview and aims: To investigate the technology herein, the following three specific aims are proposed; Aim 1: To develop a unique larger-animal model in which to investigate the role of cellular senescence in the development of AF by comparing the atrial mechanical and electrical properties of young (<11 months old) and aged (>4 year old) rabbit atria via surface Electrocardiogram (ECG), Echocardiography (Echo) recordings and optical mapping (OM) experiments. In addition to these, to characterize the cellular electrical features of atrial CMs (ACMs) that are freshly isolated from aged and young rabbits via patch clamp recordings. To define and characterize the type and quantity of senescent cells in the atria of young and aged rabbits using histochemical and biochemical techniques including tissue level assays (SA-β-Gal staining, immunofluorescence, Masson’s Trichrome staining for fibrosis assessment), RT-qPCR, Western Blot, and bulk RNA-sequencing (RNAseq).

[0206] Aim 2: To identify the senescence profile of human atria with and without AF and define the types of senescent cells by conducting histological and biochemical experiments.Atty. Docket No.: 405002-549001WO

[0207] Aim 3: To investigate the effect of senolysis on the aged rabbit atria by treating them with a promising senolytic drug, Fisetin, and assessing outcomes of the treatment via SA-β-Gal staining, ECG, Echo recordings and OM experiments.

[0208] This work provides compelling evidence that both aged human and aged rabbit atria have senescent cells, including cardiomyocytes and myofibroblasts, aged rabbits develop spontaneous AF and inflammatory pathways are upregulated in aged rabbits compared to young rabbits. Furthermore, the thesis demonstrates that senolytic treatment reduces the senescence burden in aged rabbit atria suggesting that targeting cellular senescence may represent a novel strategy for curbing inflammation and possibly early- stage fibrosis in the context of AF. All together these findings may have important clinical implications, as they suggest that preventive senolytic therapy may represent a safe and effective treatment for reducing the incidence of AF in the aging population. Additional background information

[0209] Atrial Fibrillation; Cardiac Anatomy and Electrophysiology; composed of 4 chambers, the heart’s main function is to act as a pump to help circulate blood throughout the body. While the right side of the heart pumps blood to the lungs for oxygenation via the pulmonary circulation, the left side of the heart pumps blood to the rest of the organs and tissues of the body via the aorta and systemic circulation. The upper two heart chambers, known as the right and left atria, are in charge of drawing blood from the systemic and pulmonary veins and pumping it into the right and left ventricles through the tricuspid and mitral valves, respectively. The right and left ventricles act as the main pumping force of the body, with the right ventricle pumping blood through the pulmonary valve into the pulmonary circulation and the left ventricle pumping blood through the aortic valve into the systemic circulation. The superior and inferior vena cava, which drain blood from the upper and lower regions of the body, respectively, deliver deoxygenated blood from the rest of the body to the right atrium. The pulmonary veins, which carry blood from the lungs, deliver oxygenated blood to the left atrium. As they play a more minor role in the pumping action of the heart, the walls of the atria are thinner than that of the ventricles. The wall of the heart is composed of multiple layers: the endocardium, the myocardium, and the epicardium. The endocardium and epicardium are made of epithelial cells and connective tissue while the myocardium is mainly composed of cardiac myocytes along with blood vessels, nerve cells, fibroblasts, and adipose cells.

[0210] The atrium's unique structure plays a crucial role in performing its electrophysiological function. The sinoatrial (SA) node, a group of specialized muscle fibers located in the superior posterolateral wall of the right atrium, is in charge of producing the electrical impulses that control the heart rate. Directly connected to the atrial muscle fibers, the electrical impulses travel throughout the atria, with small bundles of specialized atrialAtty. Docket No.: 405002-549001WO fibers called intratrial and internodal pathways helping to speed the impulse to the left atrium as well as the atrioventricular (AV) node. The AV node acts as a conduit for electrical impulses to travel from the atria to the ventricles and serves to delay the conduction to the ventricles. The purpose of the delay is to allow the atria to empty their blood into the ventricles prior to the contraction of the ventricles, maintaining the efficient pumping of blood. Located in the posterior wall of the right atrium behind the tricuspid valve, the cells of the AV node have fewer gap junction between neighboring cells, increasing the resistance to conduction. The location and function make it prone to damage and damaging the AV node leads to atrioventricular block and slowing of the heart rate (56, 57). Once past the AV node, the electrical impulses travel into the ventricles via the Bundle of His. The signals are then split into the left and right bundle branches before continuing into the Purkinje Fibers.The path of the electrical impulse in the ventricles travels down the center of the heart before spreading to the sides of the heart and up the ventricle walls. Unlike the AV node, the fibers of the Bundle of His and Purkinje Fibers are very large and able to transmit the electrical impulses very quickly alllowing almost instantanous transmission to the ventricles.

[0211] The creation and transmission of electrical signals in the atria are orchestrated by a complex interplay of ion channels, transporters, and exchangers which work together to regulate membrane potential and action potential duration (APD). Voltage- gated sodium (Na+), potassium (K+), and calcium (Ca2+) channels are three of the most important ion channels in atrial electrophysiology. In atrial cells the action potential is initiated by sodium channels, which allow a fast inward current of sodium ions, depolarizing the resting membrane potential. The action potential upstroke is followed by a fast repolarization phase driven by K+channels. The transient outward current (Ito) and the rapid delayed rectifier current (IKr) are key K+channels involved in early and late action potential repolarization, respectively (58). The L-type Ca2+channels are responsible for the Ca2+ion inflow that causes atrial myocyte contraction, whereas T-type Ca2+channels are involved in the creation of pacemaker activity in the atria. The funny current (If) channels play an important role in controlling the pacemaker activity in the sinoatrial node and the atrial muscle. In addition, the inward rectifier potassium (Kir) channels control the resting membrane potential and the duration of the action potential, and the G-protein-gated K+channel (IKACh), which is responsible for regulating pacemaker activity and recovering resting heart rate after sympathetic stimulation, are additional ion channels that contribute to atrial electrophysiology (58, 59).

[0212] The heart is also under the control of both the parasympathetic and sympathetic nervous systems. Stimulation through sympathetic nerves cause excitation of the heart, increasing the heart rate from a normal resting heart rate between 60-100 beats per minute to 180-200 beats per minute. This stimulation can even double the pumpingAtty. Docket No.: 405002-549001WO force, increasing the amount of blood being circulated throughout the body. Overall, the increased heart rate and volume of blood increases the cardiac output of the heart. Parasympathetic stimulation through the vagus nerve reduces heart rate and contraction strength. Mainly distributed to the atria, vagal nerve stimulation plays a larger role in decreasing heart rate than it does in decreasing contraction strength (60)

[0213] Introduction to Atrial Fibrillation (AF) and Its History: the first description of AF dates back to the early 18thcentury when an Irish physician, William Stokes, described an irregular pulse and palpitations in patients with valvular heart disease. However, it was not until the advent of electrocardiography in the early 20thcentury that the diagnosis of AF could be accurately made. In 1909, Einthoven, a Dutch physician, first demonstrated the presence of rapid irregular atrial activity in patients with AF using a string galvanometer. Over the years, more refined electrocardiographic techniques were developed that enabled more accurate diagnosis and classification of AF (61). AF presents with a narrow QRS complex with an irregularly irregular pattern and no visibly distinguishable P-waves on an ECG. Fibrillatory waves, which are small oscillations around the amplitude of the baseline of an ECG, may or may not be present. The ventricular rate of an ECG typically ranges from 80 to 180 per minute (62).

[0214] AF is the most prevalent cardiac arrhythmia encountered in clinical practice (63, 64). It is an irregular and often rapid heart rhythm originating in the atria. During AF, the atria contract in a disorganized manner, leading to ineffective blood flow from the atria to the ventricles. As a result, blood clots may form within the atria, increasing the risk of stroke and other thromboembolic complications. The severity of symptoms and clinical outcomes associated with AF can vary widely, ranging from asymptomatic to severe and life- threatening complications (63, 64). The increasing prevalence and its related complications of AF represent a significant public health concern. AF is associated with an increased risk of morbidity and mortality, including stroke and exacerbation of heart failure. Despite the availability of multiple therapeutic options, including rate control, rhythm control, and anticoagulation, the management of AF remains challenging, and in many patients, the healthcare system fails to achieve satisfactory outcomes such as conversion to sinus rhythm (44). Therefore, there is an urgent need to identify novel therapeutic targets and treatment strategies for AF, with the ultimate goal of reducing the incidence of AF, improving patient outcomes and reducing the economic burden of this disease on the healthcare systems.

[0215] The treatment of AF has evolved over time as new therapeutic options have become available. In the early 20thcentury, the mainstay of treatment was digitalis, which helped to control the ventricular response rate in AF. The introduction of antiarrhythmic drugs (AADs) in the 1960s and 1970s allowed for the direct suppression of AF, but these drugs were often associated with proarrhythmic effects and other side effects. In the 1980s,Atty. Docket No.: 405002-549001WO the class III AAD amiodarone was introduced, which was effective at suppressing AF but also had significant side effects. The development of catheter ablation techniques in the 1990s provided a new option for the treatment of AF. Initially, catheter ablation was only used in patients who had failed AAD therapy, but as the techniques improved and the success rates increased, catheter ablation became a first-line therapy for many patients with AF. More recently, the development of newer technologies such as cryoballoon ablation and contact force sensing catheters have further improved the success rates of catheter ablation (65). In addition to these treatments, anticoagulation therapy is also an important component of AF management. The use of vitamin K antagonists such as warfarin has been the standard of care for many years, but newer direct oral anticoagulants (DOACs) have now become available and are increasingly being used in clinical practice. Overall, the treatment of AF requires a multimodal approach, with individualized treatment plans tailored to the patient’s specific clinical characteristics and risk factors (65).

[0216] Incidence and Prevalence of AF; AF is the most prevalent cardiac arrhythmia. The incidence of AF is increasing, with an estimated 3-6 million new cases diagnosed each year worldwide (44). The estimated global prevalence of AF is 33.5 million. Because of an aging population and an increase in risk factors such as obesity and hypertension, this number is anticipated to rise to over 76 million by 2050 (43). In the United States, AF affects approximately 2.7-6.1 million people, with the number expected to double by 2035 (42). The prevalence of AF is 1-2% in the general population. Its prevalence increases to 5-10% in the population older than 65, and more than 10% in humans older than 80. Additionally, the prevalence of AF is higher in men than in women, as well as in people with specific concomitant conditions such as diabetes, obesity, heart failure, chronic renal disease, and hypertension (66). Classification of AF

[0217] AF is commonly classified based on its duration and underlying etiology (67, 68). In terms of duration, AF can be classified as follows:

[0218] Paroxysmal AF: on and off AF self-terminating within 7 days.

[0219] Persistent AF: lasting longer than 7 days and requiring intervention to terminate.

[0220] Long-standing Persistent AF: continuous episodes of persistent AF longer than 1 year.

[0221] Permanent AF: ongoing and unable to be terminated (67).

[0222] AF can also be classified as non-valvular (i.e. without structural heart disease) or valvular (i.e. with structural heart disease or valvular abnormalities). In addition, the presence or absence of underlying cardiovascular risk factors like hypertension, diabetes, and obesity can also be used to further define AF.Atty. Docket No.: 405002-549001WO

[0223] Pathophysiology and Mechanisms of AF; AF is characterized by rapid heart rate caused by disorganized atrial activity. The underlying mechanisms that cause AF are complex and there are multiple factors contributing to its origin but they can be briefly divided into 3 groups: Triggers, substrates and modulators (FIG.15) (69).

[0224] FIG.15 shows examples of triggers, substrates and modulators that play role in the development and maintenance of AF. During AF the fibrillatory pattern of electricity in the baseline is caused by reentrant circuits that are originated from ectopic foci in the atria (64). Early and delayed afterdepolarizations (EADs and DADs, respectively) initiate the ectopic activity. Ectopic activity can initiate reentry in a substrate that is vulnerable and displays structural and electric remodeling, in which electrical signals circulate around the atria in a self-sustaining manner, resulting in irregular and chaotic atrial depolarizations or can sustain AF as a driver when it occurs repeatedly (64, 70). Described as oscillatory changes in the cell membrane voltage, EADs and DADs are known to play a role in a variety of cardiac arrythmias. EADs can occur during the repolarization (phase 2 and 3 EADs) of the action potential and are caused mostly by a transitory inward current carried by calcium ions (ICa,L). These currents have the potential to increase the membrane potential, although the increase may be small due to the high resistivity of the cell membrane, and produce an early depolarization of the cell. Cells having longer action potential duration, such as those impacted by specific medications, electrolyte imbalances, or genetic abnormalities, are more prone to developing EADs. Prolongation of action potentials can occur due to the loss of repolarizing potassium currents or because of an excessive late component of noninactivating sodium current. Normally, the calcium and voltage-dependent inactivation of the L-type calcium channels occurs; however, when the action potential is prolonged, the channels are able to recover and cause the inward calcium current. Contrarily, DADs, which occur after action potential repolarization during diastole, are brought on by an increase in intracellular calcium concentration, which activates the sodium-calcium exchanger (NCX) and creates a net inward current that depolarizes the membrane. DADs are more likely to occur in cells with calcium handling abnormalities like high calcium load, which can occur in those affected by heart failure or ischemia. Abnormal sarcoplasmic reticulum (SR) calcium leaks and diastolic SR calcium release events (SCaEs) are the most common triggers for DADs. It is the diastolic calcium release that activates NCX (68). Early or delayed afterdepolarization-induced triggered activity (EAD-TA or DAD-TA), which can start ectopic beats and arrhythmias, can be generated by both EADs and DADs. Both EADs and DADs can result in sufficient increase in the membrane potential of the cell to activate the inward sodium current (INa) to induce the activity (71).

[0225] As one of the substrates, structural remodeling includes atrial dilatation and slow / heterogeneous conduction caused by fibrosis. Atrial fibrosis is a major feature of AF-Atty. Docket No.: 405002-549001WO related remodeling and acts as an important substrate for arrhythmias. Tissue fibrosis can cause physical separation of muscle fibers, resulting in atrial conduction delays or conduction blocks (72). Fibrosis also predisposes the tissue to reentry during AF. One molecule implicated in the development of atrial fibrosis is Angiotensin II in various cardiac diseases. The binding of Angiotensin II to the Angiotensin type I (AT1) receptor is generally assosicated with fibrotic effects, stimulating various actions such as fibroblast stimulation, cardiomyocyte hypertrophy, and apoptosis (72, 73). The Shc / Grb2 / SOS adapter protein activates Ras which in turn intiates MAPK cascades. Downstream, these actions activate ERK1 / 2, p38 and JNK to activate various transciption factors to change gene expression. Additionally, the binding of Angiotensin II to AT1 also activates the Phospholipase C (PLC) pathway (74). In addition to activating its own pro-fibrotic pathway, Angiotensin II also plays a role in increasing the activity of various other pro-fibrotic molecules such as TGF-β1which stimulates fibroblast activation and collagen deposition. TGF-β1is stimulated by Angiotensin II which consequently through the stimulation of fibroblast activity enhances the production of Angiotensin II creating a positive feedback loop (75, 76). Both Angiotensin II and TGF-β are implicated as SASP factors, a hallmark of senescence.

[0226] The group of modulators, for example, includes atrial stretch, calcium load, genetic predisposition, and increased vagal tone. The first 2 modulators can also play roles by assisting triggers and substrates. Vagal discharge decreases APD, stabilizes reentrant rotors, and increases acetylcholine-dependent K+ current (IKACh) (64). In addition, genetic predisposition has a significant impact in the development of AF. It is responsible for familial occurrences of AF with early onset, regardless of other cardiovascular problems (69). Certain ion channel dysfunction might lead to atrial arrhythmias like AF. For instance, loss- of-function mutations in the Kv1.5 K+channel gene (KCNA5) have been linked to familial AF. Similar to this, AF has also been associated with mutations in the genes for the L-type Ca2+channel CACNA1C and the Na+channel SCN5A (70, 77).

[0227] The molecular and cellular mechanisms underlying AF are complex and often interact with each other in intricate ways. Understanding how different mechasims interact and contribute to the initiation, maintenance, and progression of AF is critical for finding more effective treatments for this condition.

[0228] Clinical Presentation of AF; there are different approaches to diagnose AF including electrocardiogram (ECG), implantable loop recorder, and Holter monitor. The ECG is the primary diagnostic technique for AF since it can detect irregular heartbeats and provide information on the heart's rhythm and pace. A portable device called Holter monitor tracks the electrical activity of the heart over the course of 24 to 48 hours, whereas an event recorder can be used for up to 30 days. An implantable loop recorder is a small deviceAtty. Docket No.: 405002-549001WO placed beneath the skin that constantly monitors the electrical activity of the heart for up to three years (78).

[0229] AF can present with a range of symptoms or can be asymptomatic. Palpitations, shortness of breath, chest discomfort, dizziness, weariness, and weakness are the most prevalent symptoms. Anxiety, perspiration, and a quick heartbeat may also occur in patients. In rare circumstances, patients may have no symptoms, and the AF may be discovered incidentally during a routine examination or ECG (FIG.16) (63, 79). AF on an ECG exhibits distinctive patterns. FIG.16 shows a presentation of AF (bottom) compared to sinus rhythm on ECG (top). AF displays the absence of prominent P waves, which signify atrial depolarization. Instead of P waves, disorganized and chaotic irregular fibrillatory or "f" waves are seen. The f waves can have different morphologies and amplitudes, and they frequently have an oscillating look. A distinguishing characteristic of AF on ECG is the irregularity of the R-R intervals, which signify ventricular depolarization and produce an irregular rhythm. In addition to these, the absence of consistent AV conduction intervals, such as PR intervals, could provide supportive evidence to the diagnosis of AF. Depending on the underlying conditions and the degree of AV block present, an irregular ventricular response in AF patients is seen as exemplified by ST segment abnormalities or T wave alterations on the ECG (80).

[0230] Risk Factors for AF; AF is a complicated arrhythmia which can be induced by several modifiable and non-modifiable risk factors. Age is the most prevalent non-modifiable risk factor for AF. The incidence of AF increases with age, with a dramatic increase found in persons over the age of 60. The accumulation of comorbidities, structural and functional changes in the atria, and changes in cellular signaling pathways are thought to contribute to this age-related risk.

[0231] Aging is the most significant risk factor for AF (81). The increased risk of AF in older persons is assumed to be due to age-related changes in atrial tissue, such as fibrosis, inflammation, and oxidative stress, which can disturb normal atrial electrophysiology. Oxidative stress can lead to cell damage and dysfunction, while inflammation can promote fibrosis (scarring) of the atrial tissue, which can further disrupt the electrical activity of the heart (70). Furthermore, older persons are more likely to have comorbidities such as hypertension, heart failure after myocardial infarction (ischemic cardiomyopathy) and dilated cardiomyopathy, which all can increase the risk of AF even further. As a result, interventions to prevent AF in older persons should emphasize comorbidity treatment as well as lifestyle changes, such as weight control and regular exercise, to lower the risk of AF and its associated consequences (44).

[0232] Another commonly cited risk factor for the development of AF is the dilation of the left atrium. Several studies have shown that there is strong association betweenAtty. Docket No.: 405002-549001WO increased dilation and increased development of AF (82). Enlargement of the left atrium can result from a variety of conditions, whether congenital or acquired, many of which are associated with aging. It is most commonly caused by pressure and / or volume overload. Pressure overload can result from the left ventricle dysfunction where the non-compliance of the left ventricle results in increased pressure in the left atrium. Mitral valve regurgitation can cause volume overload of the left atrium and also result in enlargement (83).

[0233] Male sex, genetic susceptibility, and underlying cardiac diseases such as valvular heart disease, congenital heart disease, and heart failure are additional non- modifiable risk factors for AF (70). Modifiable risk factors for AF include lifestyle choices such as alcohol consumption, obesity, and smoking. Excessive alcohol intake has been associated with an increased risk of AF. Obesity is also a significant risk factor for AF, as it has been linked to metabolic changes, such as insulin resistance and inflammation. Other modifiable risk factors for AF include hypertension, diabetes, sleep apnea, chronic renal disease, physical inactivity, and poor diet. These conditions can contribute to the development of atrial fibrosis and remodeling, as well as other cardiovascular complications, which can increase the risk of AF (44).

[0234] Current Treatments and Management Strategies for AF: There are several approaches to the management of AF, including pharmacological, interventional, and lifestyle modifications. The choice of treatment depends on the type and severity of AF, as well as the underlying comorbidities and individual patient characteristics (78). The goal of pharmacological management of AF is to control the heart rate and rhythm while lowering the risk of thromboembolic events (65). Several classes of drugs are used for the treatment of AF such as antiarrhythmic agents, rate-control agents, and anticoagulants.

[0235] Antiarrhythmic medications are used for maintaining sinus rhythm or to convert AF to sinus rhythm. These drugs function by inhibiting the activity of certain ion channels or receptors involved in cardiac conduction, such as potassium, sodium, and calcium channels. Antiarrhythmic medications commonly used include amiodarone, flecainide, propafenone, and sotalol. These medications, however, can have serious side effects, including proarrhythmic effects, hypotension, and liver and lung toxicity. Antiarrhythmic medicines' efficiency in maintaining sinus rhythm varies, and the risk of recurrence following successful cardioversion is significant (84). As a result, these drugs are frequently used in in combination with other therapies, such as ablation or rate control medications.

[0236] In individuals with persistent AF, rate-controlling medications are utilized to regulate ventricular rate (61). These medications function by reducing the atrioventricular node's ability to conduct electrical impulses. Digoxin, calcium channel blockers, and beta- blockers are all frequently used rate-controlling medications. Although these medications areAtty. Docket No.: 405002-549001WO typically well tolerated, they can have side effects such as bradycardia, hypotension, and fatigue. A resting heart rate of under 80 beats per minute is the target for rate management. According to the patient's comorbidities and symptoms, a more liberal heart rate control aim may be suitable in some circumstances.

[0237] As AF is linked to an increased risk of thromboembolic events, such as stroke, anticoagulation is a crucial part of managing AF. The CHA2DS2-VASc score, which accounts for age, sex, comorbidities, and a patient's prior history of stroke or transient ischemic attack, is used to determine the patient's risk of stroke. Direct oral anticoagulants (DOACs), such as edoxaban, rivaroxaban, and apixaban as well as vitamin K antagonists like warfarin, are often prescribed anticoagulants (85). There are fewer bleeding complications with DOACs compared to warfarin, and they have been demonstrated to be at least as effective in preventing stroke. However, they need to be dose-adjusted in individuals with mild renal impairment and are contraindicated in those with severe renal or hepatic impairment.

[0238] Electrical cardioversion, catheter ablation, and surgical ablation are the main non-pharmacological therapies utilized for AF (69). Electrical cardioversion is most successful when AF has only been present for less than 48 hours and includes shocking the heart to reestablish sinus rhythm. During ablation a catheter is introduced into the left atrium and radiofrequency energy is delivered to small patches of tissue that are identified as the source of the aberrant electrical signals, usually around the pulmonary veins and the posterior wall of the left atrium. This technique has a success rate of about 70% and is particularly useful for younger patients with paroxysmal AF. A more invasive method to stop the aberrant electrical signals is surgical ablation, which includes making scars on the atria (maze procedure) (85). Patients who have tried various treatments without success or who are undergoing concurrent heart surgery are often the only ones eligible for this therapy.

[0239] In addition to pharmacological and interventional approaches, lifestyle modifications can also be helpful to effectively manage AF (70). These include regular exercise and weight control, as well as dietary adjustments including cutting back on salt and alcohol intake and upping fruit and vegetable consumption. Additionally, patients with AF should be counseled to abstain from stimulants like caffeine and nicotine and to reduce stress in their lives. Treatment of underlying comorbidities including diabetes and hypertension is also crucial in the management of AF.

[0240] The Aged New Zealand White Rabbit (Oryctolagus cuniculus) as an Animal Model for AF: A popular animal model for cardiac electrophysiology is the New Zealand White rabbit. The Koren laboratory has previously demonstrated that the aging rabbit recapitulates age-associated cardiac dysfunctions seen in people (86). These dysfunctions include aortic stiffening, increased interstitial fibrosis leading to decreasedAtty. Docket No.: 405002-549001WO compliance of the ventricular myocardium, decreased complexity of the His-Purkinje network leading to abnormal conduction, and lower thresholds for inducible ventricular arrhythmias (86). Optical mapping showed that shortened activation time and expansion of the QRS complex without significant changes in APD (86). Studies have shown that a greater proportion of individuals exhibit a prolonged QT interval as people age, with the duration of the QT interval increasing with age. Additionally, the Koren Lab showed that aging increased production of mitochondrial reactive oxygen species (ROS) in ventricular cells, which led to abnormal calcium dynamics and membrane oscillations through oxidation of the ryanodine receptor (87). These studies also revealed that stimulating autophagy in aged ventricular myocytes reduced mito-ROS production and the arrhythmogenic phenotype (88).

[0241] One of the major difficulties in studying AF mechanisms in the field is the lack of a clinically relevant animal model with spontaneous AF. AF induction in rodents is difficult due to the small size of the atrial tissue and species-dependent unique action potential characteristics. Larger animal models of AF were developed in sheep or dogs using tachy- pacing of atria to artificially trigger AF (17). Recent studies in the Koren and Choi Labs showed that AF in aged rabbit hearts replicates important clinical features of AF in the elderly population such as automaticity, abnormal APD, discontinuous conduction, and reentry formation (unpublished data). Screening of the aging rabbits’ colony (>4 years) using surface ECG and echocardiogram detected spontaneous AF in four out 32 rabbits (12.5%). This is a major finding as spontaneous AF episodes in larger mammals are rare (36) and the percentage of spontaneous AF in aged rabbits is similar to of those in the aging human population. These exciting results make the aged rabbit a unique model system for mechanistic AF studies.

[0242] Aging and Cellular Senescence, an Introduction to Cellular Senescence: Cellular senescence was first described in 1961 by Leonard Hayflick and Paul Moorhead as a phenomenon of limited replicative capacity in normal human cells, giving rise to the concept of a "cellular clock" that determines the lifespan of cells (19). Senescence is a stress response which brings about replicative arrest and can be triggered by various stimuli, such as telomere shortening, DNA damage, oxidative stress, and oncogene activation (89). Extensive research has been conducted since then to comprehend the molecular mechanisms underlying this process and its function in aging and age-related diseases such as cancer, cardiovascular disease, and neurodegenerative disorders.

[0243] The induction of senescence in cells comes from a variety of factors and can occur in diverse cell types, including post-mitotic cells. Accordingly, senescent cells have a wide and variable range of characteristics. However, all senescent cells exhibit a stable, often irreversible, cell cycle arrest, resistance to apoptosis mediated by upregulation of anti- apoptotic genes, persistent DNA damage signaling, and senescence-associated secretoryAtty. Docket No.: 405002-549001WO phenotype (SASP), which is now widely regarded as the most important physiological manifestation of senescence. SASP involves the secretion of a wide range of pro- inflammatory chemokines, cytokines, growth factors, and proteases that have been shown to contribute to chronic inflammation, tissue remodeling, fibrosis, and the development of age- related diseases (90, 91). Through paracrine signaling, senescent cells can potentially interact with nearby cells and tissues and impact the structure and function of those tissues (92). Additionally, with aging, senescent cells have been found to accumulate in a variety of tissues and organs of humans increasing the risk of developing age-related diseases.

[0244] Cellular Features of Senescence and Its Detection: Several important characteristics define cellular senescence and some of them are shared throughtout different senescence and cell types (93). These changes include alterations in cell morphology, metabolism, and gene expression. When a cell becomes senescent, it undergoes a dramatic change in its morphology, characterized by a flattened and enlarged shape. The morphological changes are associated with the impairment of autophagy and metabolic dysfunction including a shift towards glycolysis (94, 95). Additionally, gene expression undergoes substantial changes, including the downregulation of genes involved in cell cycle progression and the upregulation of genes associated with inflammation and tissue remodeling.

[0245] Various techniques can be used to identify cellular senescence. The senescence-associated beta-galactosidase (SA-β-Gal) assay is one of the most common methods for detecting senescence, taking advantage of increased lysosomal β- galactosidase expression in senescent cells. In normal cells, the activity of lysosomal β- galactosidase enzyme is detectable at a pH of approximately 4. However, the activity of senescence-associated enzyme, which is highly overexpressed in senescent cells, is detectable at pH 6.0 and therefore used as a marker for senescent cells through a histological staining assay (96, 97). Persistent DNA damage markers ɣH2AX, ATM, ATR foci, and telomere dysfunction-induced foci (TIFs) are also commonly used to identify senescent cells. H2AX is a histone variant protein which is phosphorylated upon receiving a DNA damage signal, in so becoming ɣ-H2AX. Senescent cells are also typically identified by the activity of the cyclin-dependent kinase inhibitors (CDKIs) p21CIP1(hereafter referred to as p21) and the p16INK4a(hereafter referred to as p16). Additional senescence biomarkers include absence of DNA proliferation, as measured by EdU labelling assays, and decreased lamin B1 levels (98).

[0246] The development of the SASP is one of the most prominent features of cellular senescence. The SASP, which varies depending on cell type, duration of senescence, and senescence induction method, is usually a complex combination of chemokines, cytokines, growth factors, tissue remodeling metalloproteinases and otherAtty. Docket No.: 405002-549001WO signaling molecules (99). This highly inflammatory secretome can promote tissue injury and dysfunction, as well as the onset of age-related diseases such as cancer, cardiovascular and neurodegenerative diseases (100). Through paracrine and juxtacrine signaling mechanisms, the SASP may also play a role in the propagation of senescence to neighboring cells. Various methods are used to detect SASP factors including bulk RNA sequencing (RNAseq) and Western blotting or immunohistochemistry techniques. Of note, a gene set purporting to constitute a ‘senescence profile’ developed by a consortium of researchers led by scientists from the Mayo Clinic (101). This gene set, termed ‘SenMayo’ is comprised of a panel of 125 genes that were manually curated through literature review according to their informative value as markers of senescence and is now widely used to analyze RNAseq results across different species and tissues.

[0247] Mechanisms and Triggers of Cellular Senescence: Cellular senescence is a complex biological process that can be triggered by various intrinsic and extrinsic factors (89). The triggers including telomere attrition, ionizing radiation, DNA damage, oncogene activation, viral infection, oxidative stress. These triggers activate specific pathways leading to initiation of different types of senescence, each with unique molecular and cellular characteristics. Replicative senescence is the most well-known and studied type of cellular senescence, and is a natural consequence of aging. As telomeres shorten with each cell division, they reach a critical length that initiates senescence. Stress-induced premature senescence (SIPS), oncogene-induced senescence (OIS), and DNA damage-induced senescence (DIS) are other well-known forms of senescence (101, 102).

[0248] Multiple molecular pathways regulate the complex mechanism of cellular senescence. The p53-p21 and p16-Rb tumor suppressor pathways act by inhibiting cyclin- dependent kinases (CDKs) and in turn promoting retinoblastoma (Rb) protein inhibition of E2F targets. Studies have suggested that while the p53-p21 pathway is important for the induction of senescence, the p16-Rb pathway plays a larger role in the maintenance of senescence (103). Ionizing radiation and DNA damaging agents have been shown to activate the p16-Rb pathway. p38 also plays a role in the initiation of the p16-Rb pathway in response to ROS-mediated oxidative stress, UV light, heat, and osmotic stress. After activation, p38 phosphorylates transcription factor ESE-3 which directly increases p16 expression by binding to its promoter (104). The upregulation of p16 inhibits CDK4 / 6 and results in the hypophosphorylation and activation of Rb. Rb remains bound to the E2F family of transcription factors preventing the transcription of the genes necessary for the G1 to S phase progression, causing cell cycle arrest and changes in chromatin structure, including the formation of senescence-associated heterochromatic foci (SAHF) and the downregulation of histone H1, which causes the activation of the expression of senescence-Atty. Docket No.: 405002-549001WO specific genes (105, 106). The activated p16-Rb pathway induces ROS formation leading to a positive feed back loop that eventually causes irreversible cell cycle arrest (104).

[0249] Several upstream signaling pathways, including the DNA damage response (DDR), the telomere attrition response (TAR), and the OIS response, activate the p53-p21 pathway in the cells. In response to DNA damage, the DDR pathway entails the activation of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases, which results in phosphorylation and activation of p53 and its downstream effector p21. In the absence of stress, p53 is constitutively produced and degraded as a result of its ubiquitination by the E3 ubiquitin ligase MDM2 which leads to its degradation by the proteasomes pathway. Phosphorylation of p53 by ATM, Chk1 / 2, HIPK2 prevents its degradation by MDM2 enabling nuclear translocation and transciription of genes involved in DNA repair, cell cycle arrest, apoptosis, and senescence. Specifically, p53 transcriptionally activates CDKI p21, which then inhibits Cdk2- and Cdk4- mediated phosphorylation allowing for the activation of retinoblastoma protein Rb, thereby inducing cell cycle arrest (107).

[0250] Physiological and Pathological Roles of Cellular Senescence: Cellular senescence plays a significant role in a number of physiological processes (102, 108, 109). In response to tissue injury, senescent cells can be formed at the site of injury and secrete factors that stimulate the growth and differentiation of adjacent stem cells, which can aid in tissue regeneration and in the clearance of the damaged. Studies have also shown that transient senescence prevents excessive fibrosis during tissue regeneration, allowing for proper healing (110). It has also been shown that senescence plays an important function in embryonic development (111). The proliferation and differentiation of various cell types during development is believed to be regulated by senescence, and the process is essential for tissue patterning and function. Studies have demonstrated that senescent cells contribute to the development of the placenta, the formation of heart valves, and the differentiation of numerous neural cell types (112).

[0251] In neoplastic cells, induction of senescence, along with apoptosis, is one of two modes aimed at preventing growth through cell cycle arrest. Indeed, most tumor cells exhibit mutations in the p53 or p16 pathway, highlighting the selective pressure aimed at disabling these mediators of senescence / apoptosis. However, despite, the possible positive effects of senescence, the environment fostered by the presence of senescence cells, particularly the pro-inflammatory aspect resulting from the secreted SASP factors, can instead play a role in the development and progression of cancer by promoting proliferation of pre-neoplastic cells, angiogenesis, de-differentiation of nearby cells, and the immune evasion of tumorigenic cells (113, 114).

[0252] Besides cancer, senescence, and in particular SASP factors, also play a role in aging and age-related diseases. For example, accumulation of senescent cells in arterialAtty. Docket No.: 405002-549001WO walls and the secretion of SASP factors that promote inflammation and fibrosis could lead to the formation of atherosclerotic plaques. In neurodegenerative disorders, senescence has been linked to neuronal degeneration and mortality. Senescent neurons have been observed in the brains of patients with Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders contributing to the pathogenesis of these conditions by secreting factors that promote inflammation and scarring (102). Cellular senescence was also implicated in diseases such as type 2 diabetes, non-alcoholic fatty liver dieases, pulmonary fibrosis, osteoporosis, and osteoartritis (100).

[0253] Therapeutic Interventions Targeting Cellular Senescence: Several therapeutic approaches are currently being investigated to target and modify cellular senescence (89, 115). It has been demonstrated that lifestyle modifications can impact cellular senescence. Both caloric restriction and physical activity have been shown to reduce the number of senescent cells and delay the onset of age-related diseases. Currently, various types of pharmaceutical approaches like senolytics and senomorphics are also being studied. Senomorphics are drugs that inhibit the SASP without inducing apoptosis. This strategy has the potential to mitigate the negative effects of senescent cells while maintaining their positive effects. JAK inhibitors and metformin are two of the senomorphics being investigated. Senolytics are drugs that eliminate senescent cells by inducing their pro- apoptotic pathways (116). Dasatinib, quercetin, and navitoclax have been used in pre-clinical trials.

[0254] A combination of dasatinib and quercetin is perhaps the most thoroughly researched senolytic therapy. Dasatinib is a pan-tyrosine kinase inhibitor, whereas quercetin is a natural flavonoid that inhibits BCL-2 proteins, the PI3K / AKT signaling pathway, and p53. FIG.17 shows an example chemical structure of fisetin (not taking into account salts, solvates, or tautomers). These proteins play a role in inhibiting the anti-apoptotic pathways of senescent cells and promoting apoptosis of senescent cells. The combination of the two medications was more effective than either drug alone in mitigating a variety of age-related diseases in mice, including musculoskeletal, renal, liver, cardiovascular (specifically atherosclerosis), and neurological diseases (117).

[0255] Fisetin (3.3’.4’.7-tetrahydroxyflavone) is a flavonol available in fruits and vegetables like apples, onions, cucumbers, and strawberries. Like all other flavonoids, Fisetin, is composed of a general 15-carbon skeleton, with two phenyl rings and a heterocyclic ring (FIG.17). While soluble in polar organic compounds, Fisetin is basically insoluble in water. The bioavailability of Fisetin has been studied in rats and mice (118). A 223mg / kg IP dose of Fisetin in mice resulted in a plasma half-life of 0.09 hours and a terminal half-life of 3.1 hours, suggesting a quick clearance of the drug. Fisetin is observed to undergo conjugation metabolism in the liver, mainly via sulfation. Another study reportedAtty. Docket No.: 405002-549001WO highest concentrations of Fisetin and its metabolites in liver, intestines, and kidneys of mice 30 minutes after IP injection of 233mg / kg Fisetin (62). For example, glucuronide metabolite M1 concentrations were highest in the plasma and kidneys (119). No side effects of relatively high doses of Fisetin have been reported in in-vivo studies. Additional studies with Fisetin, have shown that it exhibits anti-inflammatory and antioxidant effects (120, 121).

[0256] Fisetin has been shown to have senolytic effects, eliminating senescent cells and ameliorating aging in mice as well as in vitro with human adipose cells (22). It has also been found to eliminate senescent cells but not proliferating human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. A related study showed that Fisetin treatment in rats alleviates atrial inflammation, remodeling, and vulnerability to AF after myocardial infarction (122). Fisetin has gained a lot of attention within the last years and to date, clinical studies utilizing it have been conducted in the context of different pathological conditions. As a senolytic, Fisetin functions in a similar manner to quercetin by suppressing BCL-2 family members such as BCL-xL, and other components of the senescent cell anti-apoptotic pathways (SCAPs) (22). As a topoisomerase inhibitor Fisetin has other effects which may also contribute to its anti-cancer activity (123) and may directly inhibit cytokines like TNFα and IL-6 (124).

[0257] Fisetin has been shown to have cardioprotective effects. In rats, after an acute myocardial infarction, Fisetin greatly reduced the damage that ischemia / reperfusion (I / R) caused to the cardiac tissue, reduced oxidative stress, and restored the structure and function of the mitochondria (125). Another study done in mice demonstrated that Fisetin inhibited pressure overload-induced cardiac hypertrophy, improved cardiac function in vivo and decreased phenylephrine (PE)-induced cardiomyocyte hypertrophy (126). An in vivo study with mice on Fisetin’s effects on high-glucose-induced vascular inflammation revealed that Fisetin inhibits monocyte adhesion, expressions of cell adhesion molecules (CAMs), formation of ROS, and activation of NF-κB (127).

[0258] A recent study has shown that Fisetin decreases mortality in old mice infected with β-coronavirus which experienced increased senescence and inflammation (128). Fisetin is also being used in an ongoing clinical trial to combat frailty and inflammation in aged adults (ClinicalTrials.gov Identifier NCT03675724, 20mg / kg / day orally for 2 consecutive days), as well as to combat osteoarthritis-related articular cartilage degeneration (ClinicalTrials.gov Identifier NCT0421098620mg / kg / day orally for 2 consecutive days for 2 cycles with 28 days between cycles) and to combat complications due to COVID-19 (ClinicalTrials.gov Identifier NCT0447695320mg / kg / day orally for 2 consecutive days).

[0259] In conclusion, preclinical studies indicate that therapeutic interventions targeting cellular senescence have the potential to alleviate age-related diseases. However, additional research is required to ascertain their safety and effectiveness in humans. TheAtty. Docket No.: 405002-549001WO creation of effective interventions that target cellular senescence may open up an entirely novel path for the prevention and treatment of age-related diseases.

[0260] Atrial Fibrillation and Cellular Senescence: Several studies have documented the presence of senescent cells in the atrium of patients with AF including cardiomyocytes, and fibroblasts (129, 130). A recent study in diabetic mice suggested that advanced glycation end products (AGEs), which accumulate in diabetes mellitus (DM), can accelerate atrial electrical remodeling and cellular senescence, thereby activating the p16- Rb pathway and increasing programmed electrical stimulated (PES) AF susceptibility (131). The study documented no spontaneous episodes of AF in the diabetic mouse models. Currently, the field of clinical electrophysiology is skeptical whether PES in mice is a proper methodology to study AF vulnerability.

[0261] In one study, the relationship between AF and senescence was examined by comparing the expression of senescence markers with prothrombotic and inflammatory proteins in the right atrial appendages (RAAs) of patients with AF and sinus rhythm (SR). Based on the findings, AF progression was associated with cell senescence burden, as determined by p53 and p16 expression, and senescent cells induce inflammation through their SASP (33). Another study compared 26 patients with AF undergoing open-heart surgery to 12 patients with sinus rhythm (controls). Left atrial appendages (LAAs) with AF were found to have a substantially increased positive area of cellular senescence, as well as upregulated expression of p16, p21, and p53 (129). Although the aforementioned studies established a link between senescence and AF, it remains to be shown whether AF drives senescence or senescence causes AF.

[0262] Introduction to Studies in the Thesis and Rationale: To the best of our knowledge, there are no studies in the literature yet that use a more clinically relevant larger animal model to shed light on the molecular and cellular mechanisms and features of atrial senescence or provide data on preclinical senolytic trials using such a larger animal model. Unfortunately, significant gaps remain in the characterization of senescent cells in the aged atria. In addititon to that, the findings in the current literature were not studied in the context of the origin or maintenance of AF in humans and did not offer detailed maps of cellular senescence and its relationship to fibrosis and arrhythmogenesis. More importantly, the question that could reshape the treatment and prevention of AF in the near future has not been answered yet: does cellular senescence that comes with aging create the necessary niche for AF or does AF bring the senescence burden to the atria and cause the detrimental changes? The data provided with the studies presented in this thesis are aimed to bring detailed and rational explanation to this question and elucidate the relationship between AF and cellular senescence that may potentially bring about a novel therapy for AF in the clinics.Atty. Docket No.: 405002-549001WO

[0263] Based on our preliminary data and the information from the current literature, we hypothesize that the aging process leads to the accumulation of senescent atrial myocytes (SAMs) and senescent atrial myofibroblasts (SAMFs) in the left and right atria. Accumulation of these cells predisposes the rabbit heart to AF through the senescence- associated secretory phenotype (SASP)-mediated inflammatory reaction. Specifically, the development of arrhythmia could be caused by two factors: by directly modifying the electrical properties of SAMs or through the pro-arrhythmic effects of SASP on the adjacent atrial myocytes (AMs). Furthermore, SASP-induced inflammation may initiate and exacerbate atrial fibrosis, promoting heterogeneity in conduction thereby forming the substrate that can maintain reentrant wavelets that underlie and maintain AF. The following aims are being proposed to test the hypothesis.

[0264] Rationale for Aim 1: An ever-growing number of studies implicated senescence as a major cause of age-related disease [reviewed in (132)]. For example, genetic or pharmacological ablation of senescent cells improved health and increased life expectancy in different animal models. Our central hypothesis is somewhat supported by recent studies that documented the presence of SA-β-Gal-positive cells and increased levels of p16 and p53 in atrial appendages of AF patients (33, 133). Previous studies in the Koren lab showed that the aged rabbit heart closely recapitulates the aged human heart as explained under model introduction section. Thus, the aged rabbit will serve as a larger- animal model that enables us to elucidate the arrhythmogenic consequences of having cells that continuously secrete proinflammatory molecules into the atrium. We propose histochemical (SA-β-Gal staining for the detection of senescent cells, Masson’s Trichrome staining for fibrosis assessment, immunofluorescence / immunohistochemistry to detect senescence markers and determine the cellular identity of senescent atrial cells) and molecular studies (RT-qPCR, Western blotting and bulk RNA sequencing to assess expression levels of senescence markers or SASP factors) that will quantify senescence and fibrosis in the left and right atria of young and aged rabbits. We will correlate these findings with optical mappings (to study Action Potential Durations [APDs], Conduction Velocities [CVs] as well as to map automaticity, APD dispersions, CV dispersions, and heterogeneity in the rabbit atria), ECG, and Echo recordings (to assess cardiac function and incidence of AF). These studies will be complemented with electrophysiological experiments (patch clamp recordings to measure APD) of (senescent) atrial myocytes derived from young and aged atria.

[0265] Rationale for Aim 2: We posit that cellular senescence in the aged human atria underlies fibrosis as well as vulnerability to develop AF. Besides, a pathologic accumulation of senescent cells in atria will likely accelerate AF progression and increase the LA diameter, a known risk factor of AF, in humans. Thus, we will assess the type andAtty. Docket No.: 405002-549001WO quantity of senescent cells and fibrosis in atria from aged patients in normal sinus rhythm (SR) or AF. This allows us to correlate the findings with the pathogenesis of AF in humans. Here, we will conduct histochemical (SA-β-Gal staining for the detection of senescent cells, Masson’s Trichrome staining for fibrosis assessment, immunofluorescence / -histochemistry to detect senescence markers and to determine the cellular identity of senescent atrial cells) to quantify senescence and fibrosis in the human atria. Analysis of RNAseq data obtained from patients’ LAAs will allow us to correlate the LA diameter with expression levels of SASP factors.

[0266] Rationale for Aim 3: We posit that the elimination of senescent cells in the atria of aged rabbits will attenuate the SASP-mediated inflammatory response and fibrosis and reduce the atrial fibrillation burden or lower the chance to develop AF in these rabbits. We also argue that hearts from Fisetin-treated animals will be less inducible for atrial tachycardia (AT) ex vivo. Here, we will test our hypothesis by treating aged rabbits with Fisetin. As we have no experience with administering senolytics to rabbits, we will first use different protocols, based on reported Fisetin treatments of other species and clinical trials, to evaluate the safety and efficacy of oral 20 mg / kg Fisetin in rabbits. SA-β-Gal staining will be performed on harvested atria to assess the degree of senescence elimination at the tissue level after Fisetin treatment.

[0267] Once satisfactory safety and efficacy of the treatment are achieved, we will conduct a pilot study with Fisetin on aged female rabbits. ECGs and Echocardiograms will be recorded before and after the treatment to assess the effects of Fisetin on the overall cardiac, particularly atrial function. The efficacy of Fisetin treatment will be evaluated using SA-β-Gal staining of senescent cells. Lastly, we will use optical mapping to map cardiac electrophysiology and inducibility for AT of hearts from control and Fisetin-treated animals.

[0268] The identification of cellular senescence as a potential contributing factor to the development of AF opens new avenues for the development of effective therapeutic strategies. This doctoral thesis provides compelling evidence that both aged human and aged rabbit atria have senescent cells, including cardiomyocytes and myofibroblasts, aged rabbits develop spontaneous AF, and inflammatory pathways are upregulated in aged rabbits compared to young rabbits. Furthermore, our observations show that senolytic treatment reduces the senescent burden in aged rabbit atria suggesting that targeting cellular senescence may present a novel strategy for modulating inflammation and possibly early-stage fibrosis in the context of AF. All together these findings have important clinical implications, as they suggest that senolytic therapy may have potential as a safe and effective treatment for AF in the aging population.

[0269] Material and Methods; Animal Ethical Statement: This study was approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital and compliedAtty. Docket No.: 405002-549001WO with the most recent guidelines established by the American Physiological Society (Guiding Principles for Research Involving Animals and Human Beings) as well as the National Institutes of Health's current guide for the Care and Use of Laboratory Animals (NIH Publication, Revised 2011). Young (≤1 year old) and aged (≥4 years old) female New Zealand White rabbits (Oryctolagus cuniculus) were acquired from Robinson Services Inc. (RSI, NC, USA).

[0270] Human Ethical Statement: The Institutional Review Board (IRB) of Rhode Island Hospital has reviewed and approved Dr. Gideon Koren's human studies (project 1362230-4 renewal on August 9, 2023). He is exempt from the secondary uses of identifiable private information or identifiable biospecimens.

[0271] Tissues: Rabbits were anesthetized with ketamine (60 mg / kg), xylazine (15 mg / kg IM), buprenorphine (0.03 mg / kg SQ), and sodium pentobarbital (150 mg / kg IV). Hearts were removed as previously described (134) and washed in cold phosphate buffered saline (PBS). Gross anatomy photos of the whole heart were taken before any dissection. Cardiac tissues for histology experiments were embedded in optimal cutting temperature (OCT) compound solution (Fisher Scientific, Waltham, MA, USA) and rapidly frozen on liquid nitrogen. Cardiac tissue for biochemical experiments were snap frozen in liquid nitrogen and stored immediately at -80°C. Cryosectioning was performed at 5 µm thickness for immunofluorescence (IF), 6 µm for immunohistochemistry (IHC), and 10µm for Senescence- associated β-galactosidase (SA-β-Gal) and Masson’s Trichrome staining using a Leica CM1950. Slides were immediately fixed in cold 4% paraformaldehyde (PFA) in PBS for 10 minutes followed by a wash in PBS for 10 minutes for IF, IHC, and Masson’s Trichrome. For SA-β-Gal, slides were fixed in 0.5% glutaraldehyde for 10 minutes followed by a wash in PBS for 10 minutes.

[0272] 3.4. Echocardiogram: Echo was performed by a PGY-6 cardiology fellow undergoing level 3 echocardiography certification. Rabbits were sedated and excess fur removed prior to imaging. Echo was performed before and after treatment with Fisetin. Left atrial strain and left atrial volumes were compared pre- and post-treatment. Echocardiographic measurements: Mitral valve E and A waves, e’ septal and e’ lateral, E / e’ (mean), Left atrial volume (Simpson’s biplane), Left ventricular ejection fraction by Simpson’s biplane, Isovolumic relaxation time, Left atrial global longitudinal strain (reservoir strain), TAPSE, RV S’, Right ventricle global longitudinal strain, Tricuspid regurgitation maximum velocity. The study was powered to detect a difference of 25% between the Fisetin and vehicle groups requiring a population of 25 rabbits. Left atrial strain analysis was done using TomTec software and speckle tracking. Cardiac cycle of interest was selected using the R-R intervals. The left atrium was traced in systole and diastole, using automated tracing within the software followed by manual adjustment as needed. Speckles were tracked on frame-by-Atty. Docket No.: 405002-549001WO frame basis throughout the cardiac cycle. The measured change in distance between the different speckles was used to calculate left atrial movement. The left atrial strain was measured twice on two different images from an apical 4 chamber view with a frame rate between 40-60 frames per second. Left atrial volumes in systole and diastole were recorded from tracing and speckle tracking. Left atrial reservoir strain was recorded.

[0273] Adult Rabbit Cardiac Myocyte Isolation: The heart was removed from anesthetized young or aged rabbits and perfused for 5 minutes (20 mL / min) with a basic buffer (130mM NaCl, 4.5 mM KCl, 3.5 mM MgCl2, 0.4 mM NaH2PO4, 5 mM HEPES, and 10 mM Glucose), to which 0.75 mM CaCl2was added. After 8 minutes, 0.1 mM EGTA was added to the buffer. Subsequently the heart was perfused with enzyme buffer (basic buffer with 216 or 432 U / mL Collagenase I (Worthington Biochemical), 0.52 or 1.08 U / mL Protease XIV, and 240 or 480 µM CaCl2). The left and right atria were cut off from the heart, minced into fine pieces, and then transferred into a small beaker that contained 2.0 mL of enzyme buffer and 2% Bovine Serum Albumin (BSA) (Sigma, Cat# A7638-25G). The beaker was then incubated in a 37°C water bath shaker at 137 revolutions per minute. A drop of the supernatant was checked under the microscope every 3-4 minutes until single atrial cells appeared. The supernatant was collected by filtering through a 100 µm cell strainer into a 50mL tube that contained 1.5 mL of Potassium Buffer (KB) (85 mM KCl, 5 mM MgSO4, 30 mMKH2PO4, 7 mM H2O, 5 mM Na2ATP, 5 mM Pyruvate Na, 5 mM DL-ß-Hydroxybutyric acid sodium salt, 5 mM Phosphocreatine di(tris) salt, 20 mM Taurine, 0.56 mM EGTA, 50 g / L PVP 40, 0.1 mM CaCl2, and 20 mM Glucose), to which 3% BSA was added. The tissue was rinsed off from the strainer into a beaker with 1.5 mL of fresh enzyme buffer and 2% BSA to continue incubation. The collection was repeated every 5 minutes until sufficient number of single, rod shape myocytes were acquired. All fractions were combined and filtered again into a 50 mL tube. The tube was spun down at 80g for 4 minutes, and the pellet was resuspended in a 15 mL tube with 4-5 mL of KB. Filtration was done again if needed. The 15 mL tube was spun down at 80g for two minutes and the pellet was again resuspended in 7-8 mL of KB. Myocytes were let sit in the buffer with a loose cap for 30-45 min to recover before use.

[0274] Optical Mapping Procedure, and Data Analysis: Young (n=4) and aged rabbits (n=5) were euthanized with buprenorphine (0.03 mg / kg IM), acepromazine (0.5 mg / kg IM), xylazine (15 mg. / g IM), ketamine (60 mg / kg IM), pentothal (35 mg / kg IV), and heparin (200 U / kg). Hearts were excised from the chest and perfused in a Langendorff perfusion apparatus (Radnoti LLC, Covina, CA). Blebbistatin (10 µmol / L) was perfused to reduce movement artifacts (135). Hearts were stained with the voltage-sensitive dye di-4- ANEPPS, and ECG and perfusion pressure were constantly monitored (PowerLab, AD Instruments, Sydney, Australia). A dual camera system (Ultima-L, Scimedia, Japan) wasAtty. Docket No.: 405002-549001WO used to record RA and LA simultaneously. The pacing electrode was placed on the SA nodal region to test vulnerability to AF under stimulation protocol. The hearts underwent two stimulation protocols: 1) S1S1 basic cycle length (CL=350 ms) followed by a single premature S2 beat to reduce S2 cycle length (CL) by 10 ms for APD restitution and tissue refractory period or atrial tachycardia (AT) / AF induction (39), S1S1 basic cycle length (CL=350 ms) followed by five premature S2 beats to reduce CL of S2 beat by 10 ms until it reaches refractoriness or AT / AF induction. The data were analyzed with a custom-built software program developed in Interactive Data Language (Harris Geospatial Solutions). Action potential durations and APD maps were calculated via the difference between activation and repolarization time points, determined from fluorescence (F) signals by calculating (dF / dt)max and 75% depolarization (9x9 pixels). Data were filtered using a temporal polynomial filter (3rd order, 13 points). Action potential conduction velocity was calculated using the spatial gradient of activation time (11x11 pixels) and spatial resolution of acquired image (200 µm / pixel).

[0275] Electrophysiology Recording via Patch Clamping: Whole-cell patch clamp recordings from freshly isolated rabbit myocytes. All experiments were conducted in the whole-cell configuration at 35–37°C with Axopatch-200B, Digidata 1440A, and pClamp 10 software (Molecular Devices). After the cell membrane was broken by suction, the cell membrane capacitance and series resistance were compensated by 70–80% in the voltage clamp-mode. Tyrode solution (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.33 mM NaH2PO4, 7.5 mM Glucose, and 5 mM HEPES, pH 7.4 adjusted with NaOH) was used as a standard bath solution. The pipette solution contained 90 mM potassium aspartate, 50 mM KCl, 5 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 5 mM MgATP, and 0.1 mM GTP Tris (pH 7.2 adjusted with KOH). AP was activated in the current clamp mode by a 3 ms injection of a depolarizing current, which was 20% higher than the threshold of AP activation. The AP stimulation rate was 1.25 Hz or 0.25 Hz. The voltage output was filtered by a low-pass filter with a cut-off frequency of 10 kHz and sampled at 20 kHz.

[0276] SPiDER-ß-Gal Assay: Wells were covered with laminin (Santa Cruz Biotechnology, Inc. sc-20912) and placed in the incubator at 37°C until the myocytes were isolated and ready for seeding. Myocytes were seeded and allowed to adhere for 45 minutes. The culture medium was discarded, and the cells were washed with 2 mL of HBSS. 1 mL of Bafilomycin A1 working solution (prepared as directed by the manufacturer, Dojindo Molecular Technologies Inc.) was added and the cells were incubated at 37°C for 1 hour in a 5% CO2incubator. Next, 1 mL of the SPiDER-β-Gal working solution was added, and the cells were incubated at 37°C for 1 hour in a 5% CO2incubator. After removing the supernatant, the cells were washed with 2 mL of HBSS twice. The cells were then imaged using a Nikon Eclipse TE2000U Epifluorescence Microscope.Atty. Docket No.: 405002-549001WO

[0277] Senescence-associated ß-Galactosidase Assay: The procedure for SA-β- Gal staining was modified from that previously described (97, 136). Immediately after cryosectioning, frozen sections of rabbit or human atrial tissues were immersed in 0.5% glutaraldehyde (in 1x PBS) for 10 minutes and then placed in fresh 1x PBS for 10 minutes. Sections were kept at -20°C for the same day staining. For staining, sections were incubated with fresh β-galactosidase staining solution (40 mM citric acid / Na phosphate buffer, 5 mM K4[Fe(CN)6]·3H2O, 5 mM K3[Fe(CN)6], 150 mM sodium chloride, 2 mM magnesium chloride, and 20 mg / ml X-gal in dimethylformamide) at 37°C with no CO2for 16 hours. Nuclear Fast Red (Vector Laboratories, H-3403) was used for the counterstaining of the sections, and slides were imaged using an Aperio ScanScope (Leica Biosystems, Buffalo Grove, IL, USA) with 2x - 40x objective. Images were analyzed using Aperio ImageScope v12 software (Leica Biosystems, Buffalo Grove, IL, USA). Randomly selected 5 representative images at 40x magnification were used for quantification, and the 2 atrial chambers were analyzed separately. The percentage of SA-β-Gal-positive cells was calculated by hand counting the number of SA-β-Gal-positive and SA-β-Gal-negative cells using the counter tool in ImageJ.

[0278] Immunofluorescence (IF) staining: 5 µm-thick frozen sections of rabbit or human atrial tissues were acquired and fixed as described above. All steps were performed in a humidified chamber. The tissue was encircled with a PAP pen and washed in PBS 3 times. For intracellular targets, frozen sections were permeabilized with 0.1% Triton X-100 in PBS for 30 min. Slides were blocked with 10% normal donkey or goat serum including Triton X-100 in PBS for 1 hr at room temperature. Slides were incubated with primary antibodies diluted in antibody dilution buffer prepared with BSA overnight at 4°C in the dark, then washed in PBS 3 times for 5 minutes each and incubated in suitable secondary antibodies diluted in antibody dilution buffer prepared with BSA for 1 hr and 30 minutes in the dark at room temperature. Slides were again washed in PBS 3 times for 5 minutes each. When slides were being stained for multiple targets, the slides were placed in blocking buffer again and the steps of adding primary and secondary antibodies were repeated. Once all antibodies had been added, incubated, and washed off, slides were mounted with Prolong Gold Reagent with DAPI (Invitrogen, P36931). All antibodies used in the experiments were validated with rabbit atrial samples before initiating any experiments and are listed in Table 1.

[0279] The slides were imaged with a Nikon Ti2 confocal microscope with an A1R scanner. Randomly selected 5 representative Z-stack images at 60x magnification were used for quantification. Images were analyzed via Nikon NIS Element AR software and the number and percentage of cells with positive signal were derived from maximum projection images by hand using the counter tool in ImageJ. Nuclei with 3 or more γH2AX foci or with big 1 or 2 γH2AX foci were counted as positive. Mann-Whitney U or 2-tailed Exact test wasAtty. Docket No.: 405002-549001WO used for statistical analyses. Table 1 (also discussed above) shows examples of antibodies used in histology experiments. Table 1: Antibodies used in histology experiments. Target Host Species Company Product Number

[0280] Immunohistochemistry (IHC) staining: 6 µm sections of rabbit or human atrial tissues were obtained and fixed as described above. All steps were performed in a humidified chamber. The tissue was encircled with a PAP pen and then washed in PBS 3 times. For intracellular targets, the slides were permeabilized using 0.1% Triton X-100 in PBS for 15 minutes. Slides were incubated in BLOXALL Blocking Solution (VECTOR Laboratories, SP-6000) for 10 minutes to quench endogenous peroxidase activity. The slides were then washed in PBS for 5 minutes. VECTASTAIN Elite ABC Kit was used for the assay (VECTOR Laboratories, VECTASTAIN Elite ABC Kit, Peroxidase (Mouse IgG), PK-6102). The slides were incubated in 1.5% blocking serum for 30 minutes at room temperature. After aspirating the blocking serum, the slides were incubated with a primary antibody (p16, Ventana, 705-4793) diluted as desired in 2.5% normal animal serum for 30 minutes, then washed in PBS for 5 minutes, and incubated with the secondary antibody diluted in 1.5% normal animal serum for 30 minutes at room temperature. After another 5-minute PBS wash, the slides were incubated in ABC solution (VECTOR Laboratories kit: 100 µL from reagentAtty. Docket No.: 405002-549001WO A.100 µL from reagent B per 5mL PBS), prepared at least 15-30 minutes beforehand, for 30 minutes at room temperature. The slides were washed in PBS for 5 minutes and then incubated in DAB solution (VECTOR Laboratories, DAB Substrate Kit, Peroxidase (with Nickel), SK-4100) for 5-8 minutes before the slides were rinsed in tap water to stop the reaction and then rinsed in distilled water. Finally, the slides were counterstained with Nuclear Fast Red, dehydrated through a graded series of ethanol and xylene (1x 70% ethanol, 2x 100% ethanol, 3x Xylene), and mounted with Micromount medium (Leica). The slides were let dry overnight at room temperature and stored in a slide box. Slides were imaged using a Nikon Eclipse TE2000U Epifluorescence Microscope using a 60x Objective.

[0281] Masson’s Trichrome Staining: For fibrosis assessments, frozen sections of rabbit or human atrial tissues were stained with Masson’s Trichrome and reagents from Electron Microscopy Sciences (catalog # 26367-series) were used throughout the protocol. 10 µm sections were obtained and fixed as described above. Bouin’s Fixative was preheated to 56°C in a water bath. After briefly rinsing the slides in distilled water, the slides were placed in heated Bouin’s Fixative for 1 hour at 56°C in the water bath. The slides were washed until the yellow color of the fixative disappeared and then rinsed in distilled water before being stained in Weigert’s Iron Hematoxylin Working Solution (equal parts Weigert’s Iron Hematoxylin A and Hematoxylin B) for 5 minutes. The slides were washed in running tap water for 10 minutes, rinsed in distilled water, and then stained in Biebrich Scarlet-Acid Fuchsin for 15 minutes. After rinsing in distilled water, the slides were stained in Phosphomolybdic Acid-Phosphotungstic Acid for 15 minutes. The slides were then stained in Aniline Blue Solution for 15 minutes and rinsed in distilled water afterwards. Next, the slides were differentiated in 1% Acetic Acid for 4 minutes and then dehydrated through a graded series of ethanol and xylene (1x 70% ethanol, 2x 100% ethanol, 3x Xylene) before being mounted with Micromount medium (Leica).

[0282] Slides were imaged using an Aperio ScanScope (Leica Biosystems, Buffalo Grove, IL, USA) with a 2x - 40x objective. Randomly selected 5 representative images at 40x magnification were used for quantification, and the 2 atrial chambers were analyzed separately. Computerized assessment via Image J software and a script developed by Eric Mi (Koren Lab) was used to calculate the percentage of fibrotic areas. Images for quantification and analysis purposes were used that had less than 25% empty (white) areas. All analyses were done blinded.

[0283] Fisetin Preparation: Fisetin (MedChemExpress, HY-N01892) was added at a concentration of 10 mg / mL to a solvent composed of 45% Polyethylene glycol 300 (PEG300) (MedChemExpress, HY-Y0873), 5% Tween-80 (MedChemExpress, HY-Y1891), and 50% Saline. The solution was placed in a sonicator for 1 hour or until all the Fisetin was completely dissolved.Atty. Docket No.: 405002-549001WO

[0284] Aged Rabbit Fisetin Treatment Protocol: Fisetin treatment was conducted on aged rabbits. The experimental group was given Fisetin in doses of 20 mg / kg, while the control group was given a vehicle (45% PEG300, 5% Tween-80, and 50% Saline). Both Fisetin and vehicle were mixed with apple sauce and given to the rabbits orally via a syringe. Two different protocols were used to treat the rabbits: Protocol A consisted of one cycle of two days, each day with a daily dose of 20 mg / kg Fisetin followed by observation for 3 weeks, whereas Protocol B consisted of two cycles (one cycle per week) of two days, each day with a daily dose of 20 mg / kg Fisetin followed by observation for a week. The control group followed Protocol B. The protocols were completed by the end of the 22nd day of treatment. All rabbits were assessed pre- and post-treatment via ECG and Echo recordings, and blood samples were collected prior to treatment, 8-12 hours after the first dose of each cycle, and prior to euthanizing the rabbits for beating heart harvest (BHH).

[0285] RNA Isolation: Total RNA was isolated from rabbit LA and RA tissues. Samples were lysed and homogenized in TRIzol Reagent (Invitrogen), with 1 mL of TRIzol used per 50-100 mg of tissue, using a PRO Scientific Bio-Gen PRO200 homogenizer. Samples were then centrifuged at 4°C for 10 minutes at 12,000g before the supernatant was transferred to a new tube. After being incubated for 5 minutes, 0.2 mL chloroform per 1 mL TRIzol reagent used was added to each tube, and the tubes incubated for another 2-3 minutes. The samples were then centrifuged at 4°C for 15 minutes at 12,000g. The upper colorless aqueous phase containing the RNA was transferred into a new tube, and the samples were again centrifuged at 4°C for 15 minutes at 12,000g. The aqueous phase containing the RNA was again transferred to a new tube and 0.5 mL of isopropanol per 1 mL of TRIzol Reagent used was added before the tubes were shaken lightly. After 10 minutes of incubation, the tubes were centrifuged at 4°C for 10 minutes at 12,000g. The supernatant was discarded, and the pellet was resuspended in 1 mL of 75% ethanol per 1 mL TRIzol Reagent used. After briefly vortexing and centrifuging at 4°C for 5 minutes at 12,000g, the supernatant was removed, and the pellet was again resuspended in 75% ethanol. The steps of vortexing, centrifuging, and removing the supernatant were repeated one more time before the pellet was allowed to air dry for 5-10 minutes. The pellets were finally resuspended in 20 µL of RNase-free water. Nucleic acid concentration of the RNA was determined using a Nanodrop 2000c Spectrophotometer (Thermo Fisher, Waltham, MA, USA). Each sample was treated with the TURBO DNA-free Kit (Invitrogen, AM1907) according to the manual instructions. RNA integrity was checked using an Agilent 2100 Bioanalyzer and Agilent (formerly AATI) Fragment Analyzer prior to use. Samples with ≥8.5 RIN were used for downstream applications.

[0286] RT-qPCR: iScript Reverse Transcription kit (BioRad, Hercules, CA, USA) and a C1000 Thermocycler (BioRad, Hercules, CA, USA) were used for the reverse transcription.Atty. Docket No.: 405002-549001WO For each gene of interest as well as endogenous regulation, primer sets were created. Each gene's accession number was obtained from the National Center for Biotechnology Information gene database (ncbi.nlm.nih.gov / gene), and multiple primer sets were generated for each gene using the Primer Blast tool (ncbi.nlm.nih.gov / tools / primer-blast / ). For further testing, suitable primer sets were chosen for each gene of interest. For the purpose of accurately calculating normalized change gene expression (ΔCT), the efficiencies of the chosen primer set for each gene were determined (Table 2). In Table 2, SEQ ID NO: 1 is 5’- TTCCAGAAATGCCGGTGTC-3; SEQ ID NO: 2 is 5’-GCTTCGATGCTGGACTTCTCA-3; SEQ ID NO: 3 is 5’-GCCGAAGGCGGTAACCA-3’; SEQ ID NO: 4 is 5’- CGCTCCCAGGCGAAGTT-3’; SEQ ID NO: 5 is 5’-GAGACTGCGACGCACTCATG-3’; (Table 2). SYBR Green reagent (BioRad) and a ViiA 7 Real Time System (ThermoFisher, Waltham, MA, USA) were used to conduct RT-qPCR.100 ng, 10 ng, 1 ng, 0.1 ng, and 0.01 ng of cDNA from rabbit atrial tissue were used to calculate the effectiveness of each primer set. SRP14 (internal control) and no template were used as positive and negative controls, respectively. All experiments were run in triplicate for each primer set. Standard curves, melt curves, and primer efficiency were reported by the ViiA7 QuantStudio software. Ta y

[0287] Western Blots: The 20-30mg of rabbit atrial tissue was rinsed in PBS, centrifuged, and lysed in a volume-appropriate amount of RIPA buffer (Boston BioProducts) containing a Protease / Phosphatase Inhibitor Cocktail (Cell Signaling Technology). Sonication was followed by 90 minutes of rotation at 4°C, centrifugation, and the transfer of supernatants to new tubes. Following protein quantification, 40–80 g of heat-denatured (15 min at 60°C) protein were loaded into each lane of an SDS-PAGE in 4x Laemmli Sample Buffer (Bio-Rad; final concentration: 2x Sample Buffer; 100 mM DTT). Protein wasAtty. Docket No.: 405002-549001WO transferred to PVDF membranes, which were afterward blocked in 3% BSA. Membranes were incubated with the primary antibodies overnight (Table 3). Suitable secondary HRP- conjugated antibodies at 1:10,000 titer (Thermo Fisher) were used. SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and the ChemiDoc MP Imaging System (Bio-Rad) were used to detect signals. Protein band quantification was done with ImageLab software (Bio-Rad). Respective total (and phosphorylated) protein expressions were normalized to GAPDH levels. Table 3 shows examples of antibodies used in Western Blot (WB) experiments. Table 3: Antibodies used in WB experiments

[0288] RNA-sequencing: High quality RNA was isolated and sent to Azenta Life Sciences US, Inc. (Burlington, MA, USA) for RNA sequencing and library creation. Analysis of the raw data was performed in-house.

[0289] Statistical Analysis: Unless otherwise stated, all data are represented as Mean ± SD in the text. To compare the two groups, the two-tailed Exact test or Mann- Whitney U test was used. To compare more than two groups, the one-way ANOVA with post-hoc Tukey test was applied. GraphPad Prism 9.5.1 was used to perform all statistics and create all graphs. Scatter plots of the graphs show Mean ± SEM.

[0290] Results: Aged Rabbit Atria Are More Prone to Atrial Arrhythmias; in order to study the electrophysiological characteristics of the atria of aged rabbits, we conducted OM experiments on both atria using voltage-sensitive dyes. The aim of these experiments was to to detect AF in aged rabbit hearts and verify that it replicates important clinical features of AF in the aged human population. The picture of a representative aged rabbit left atrium mapped by Dr. Choi’s laboratory is shown in FIG.2A. Respective activation APD, and conduction velocity (CV) maps are shown in FIG.2A demonstrating large APD dispersion between posterior and anterior regions of the left and right atria.

[0291] The CV map also shows heterogeneity near the pulmonary vein (right top quadrant of CV map). The bottom panels show ECG tracing and OM of a spontaneous AFAtty. Docket No.: 405002-549001WO episode. Activation maps show that both multiple focal activity (maps ‘a’ and ‘b’) and rotors (map ‘c’) maintain AF (FIG.2A). In summary, our experiments showed large heterogenenity in CV and APD resulting in greater APD dispersion in the aged group, and large fibrotic areas were also visible. Trends in slow CV, shorter APD, and greater APD dispersion in the aged group were also detected. Of note, 4 out of 5 aged rabbits were inducible for AF by either pacing or stretch suggesting that aged rabbit atria have a low threshold for induction of atrial arrhythmias. Importantly, none of the four young rabbits was inducible by pacing or stretch. Our optical mapping studies did not reveal significant differences between the APD of aged vs young atria (data not shown).

[0292] We have also conducted ECG and Echo recordings on all rabbits in our colony to create a database and verify the spontaneous AF incidence in the aged rabbit (FIG.18A, FIG.18B). We have found spontaneous AF developing in 4 out of 32 rabbits. Interestingly, this 12.5% AF incidence in our aged rabbit cohort is is very similar to the AF incidence found in the aged human population.

[0293] Action Potential Recordings of Freshly Isolated Young and Aged Atrial Myocytes: We performed electrophysiological experiments with acutely isolated aged and young rabbit atrial cardiomyocytes to compare their electrophysiological features (FIG.3A, FIG.3B). Representative action potential (AP) traces from young and aged cardiomyocytes recorded during 1.25 Hz stimulation are shown in FIG.3A and FIG.3B. In FIG.3B, the average values of AP duration at 90% repolarization level (APD90) are shown for atrial cardiomyocytes isolated from 4 young and 4 aged rabbits. We found no significant difference between the APD90values in the two groups (FIG.3B). In the young group, ADP90was 97±62 ms (n=26), while in the aged group ADP90was equal to 113±58 ms (n=23, p=0.35). We also did not see any significant difference in APD50(AP duration at 50% repolarization level) values between these two groups at a 1.25 Hz stimulation rate (data not shown). Furthermore, we measured APD50and APD90values at a non-physiologically low stimulation at 0.25 Hz. At that rate, all cardiomyocyte ion channels are recovered from inactivation. Again, we observed no significant differences in APD50and APD90between the groups (data not shown).

[0294] For a few years, the SPiDER SA-β-Gal kit developed by Dojindo Molecular Technologies has been available. This kit stains live or fixed cells for SA-β-Gal activity using a fluorogenic β-galactosidase substrate (SPiDER β-Gal). First bafilomycin A1 (an antibiotic that blocks the lysosomal proton pump resulting in an increased pH to inhibit endogenous β- Gal) is added to the media, followed by the addition of the fluoregenic SPiDER β-Gal stain (137), which is converted to a fluorescent end product by SA-β-Gal. In a matter of hours, live senescent cells are readily detectable under the fluorescence microscope. As the excitation and emission wavelengths of the fluorescent end product are between green and redAtty. Docket No.: 405002-549001WO wavelengths, SA-β-Gal-positive cells appear to be detectable in both channels. This kit would allow us to identify live senescent atrial cardiomyocytes so that we would be able to compare the electrical characteristics of these senescent cells and regular atrial cardiomyocytes as part of our electrophysiological investigations. We successfully used the SPiDER SA-β-Gal kit to identify senescent atrial cardiomyocytes (FIG.4A, FIG.4B). We used one experiment with aged atrial myocytes for quantification purposes and found that about 18.3% of cells stained positive with the SPiDER SA-β-Gal kit (FIG.4A). We did not detect any positive cells when we stained cells with the kit from a young rabbit atrial myocyte isolation (FIG.4B). Unfortunately, once the cells were labeled with the kit, we could not successfully patch them or obtain reliable cellular electrophysiological data such as resting membrane potential or APD as the kit treatment stiffened the plasma membrane and depolarized the resting membrane potential. We posit that changes in the intracellular ion balance rendered these cells unpatchable. Indeed, most of the cells died immediately after breaking the seal.

[0295] Aged Rabbit Atria Have Significantly Higher Numbers of Senescent Cells Compared to Young Rabbit Atria: We next determined the fraction of senescent cells in young and aged rabbit atria using Senescence-Associated β-Galactosidase (SA-β-Gal) staining (FIG.5A, FIG.5B). The results showed that left and right atria of aged rabbits contain significantly more SA-β-Gal+ cells (aged RA 14.7±8.9%, aged LA 11.9±9.5%) as compared to young rabbit atria (young RA 0.6±1.0%, young LA 1.1±2.3%, p<0.01) (FIG.5B). No significant difference was detected between the left and right atria within the same group. However, some variations within the aged group were detected. These variations reflect variability among aging rabbits similar to aging humans (138, 139). Interestingly, there was a trend for a larger number of senescent cells in the right atria that did not reach statistical significance.

[0296] Majority of the Senescent Cells in the Aged Rabbit Atria Are Atrial Cardiomyocytes: Based on their morphology, it appeared that the majority of the senescent cells identified in the aged atria were likely cardiomyocytes. To confirm this and assess the potential senescence of other cell types in the atria, we carried out immunohistochemistry (IHC) experiments with different cell type markers. The IHC experiments were done either solo or with SA-β-Gal staining in order to identify the senescent cell types in the rabbit atria. Different cell type-specific markers were used to identify the cells in the atria. Troponin T and α-actinin were used to identify atrial myocytes, vimentin and α-SMA for (myo)fibroblast detection, and CD68 for detection of macrophages. The IHC experiments proved useful in demontrating that the majority of the senescent cells in atria were indeed myocytes (data not shown). However, due to the difficulty in the analysis and quantification of the images, we next performed immunofluorescence (IF) experiments to identify the types of senescentAtty. Docket No.: 405002-549001WO cells. Frozen sections were co-stained with senescence marker γH2AX and different cell type-specific markers using double IF labeling. FIG.6A depicts representative images of an aged (top panel) and a young atrium (bottom panel) stained for the myocyte marker Desmin and senescence marker γH2AX. Nuclei exhibiting three or more γH2AX foci were scored as γH2AX positive (γH2AX+). The results show that most of the senescent cells stained positive for desmin. FIG.6B depicts aged rabbit atria stained for the myofibroblast marker αSMA and senescence marker γH2AX. The results show that relative smaller numbers of senescent cells are positive for αSMA as compared to Desmin. Of note, we excluded vessels from the study. Quantitation of the IF results revealed that the young atria had 79.3%±15.7 Desmin+ and 11.8%±5.5 αSMA+ cells, while aged atria had 75.2%±15.7 Desmin+ and 14.3%±2.2 αSMA+ cells (FIG.6C). Importantly, we observed a significant higher number of γH2AX+ nuclei in aged atria (21.5%±12.9) as compared to young rabbit atria (2.6%±2.2) (p<0.05). Furthermore, the results demonstrate that 78.8%±24.3 of the (nuclear) γH2AX-positive cells in aged atria were myocytes as defined by Desmin co-staining and their rod-shape typical of atrial myocytes (FIG.6D). In addition, 12.5%±8.6 of the γH2AX-positive cells stained positive for αSMA. Senescent macrophages (FIG.6E) and endothelial cells (data not shown) were also detected in the aged atria. However, their number is relatively small.

[0297] RT-qPCR and Western Blot Results Show Aged Rabbit Atria Have More p16 and p21 Expression Compared to Young Rabbit Atria: We next conducted RT-qPCR and Western blot assays to investigate the expression of senescence genes and SASP factors in young and aged atria. The results of RT-qPCR showed that the normalized transcript levels of p16 were 19.1 fold higher (p<0.01) in the aged LA, and 6.3 fold higher (p<0.01) in the aged RA as compared to the young atria. Normalized transcript levels of p21 in the aged RA were 2.6 fold higher (p<0.05) and a trended toward higher levels in the LA of aged group (FIG.12H, FIG.12I). Though a trend toward higher levels of transcripts of other SASP factors was observed in the aged group compared to the young, these differences did not reach statistical significance (data not shown). We next investigated the steady-state protein levels of p21, p53, and phosphorylated p53 using Western Blots. Normalized expression levels of p21 polypeptides were 2.7 fold higher in the aged LA (p<0.05) and 3.1 fold higher (p<0.01) in the aged RA as compared to the young atria. There were no significant differences in the protein levels of p53 or phospho-p53 (FIG.12I). Unfortunately, we could not identify a commercially available p16 antibody to investigate the protein levels of p16 in rabbit atria.

[0298] RNAseq Reveals Significantly Elevated Levels of Senescence and Inflammatory Pathways in the Aged Rabbits: High quality RNA was isolated from aged and young rabbit right atrial samples and bulk RNA sequencing (RNAseq) analysis was performed on right atria derived from young (n=5) and aged (n=5) rabbits. Right atria wereAtty. Docket No.: 405002-549001WO chosen as we saw more SA-β-Gal+ cells in right aged rabbit atria. Statistical analyses were done using DESeq2, which can compute normalized gene expression and differential expression statistics. Using a false discovery rate (FDR) cutoff of 0.05, we observed 247 upregulated and 116 downregulated genes in aged vs young animals. We next tested whether the SenMayo gene set, which comprises 125 senescence genes, is enriched in aged atria. Indeed, the SenMayo gene set (101) was significantly upregulated (p<0.05) in aged compared to young rabbit right atrium tissue (FIG.7A). Concordantly, Gene Set Enrichment Analysis (GSEA) of Kyoto Encyclopedia of Genes and Genomes (KEGG) (140) and Gene Ontology (GO) (141) pathways revealed that many of the most significantly enriched pathways were related to the inflammatory response. This included both innate immune response and adaptive immune response. This is suggestive of an inflammatory tissue milieu in the aged rabbit right atrium. One leading possibility is that the inflammatory response is in part comprised of, and in part derived from intereactions with the SASP, which is highly inflammatory. Some of the genes that were significantly overexpressed in the aged group are shown in the volcano plot’s right half, whereas some of the genes that were significantly less expressed in the aged group are shown in the left half (FIG.7B). RNAseq analysis also showed significantly elevated expression levels of senescence gene p16 (CDKN2A) in the aged atria (144.04%±99.7) compared to young (36.7%±6.8). We also checked for another senescence gene p21 (CDKN1A), however did not observe significant difference (FIG.7C).

[0299] Fibrosis Assesments in Aged and Young Rabbit Atria: We next investigated the degree of fibrosis in young and gaed atria using Masson’s Trichrome staining of 5 aged rabbits (betweeen 4-7 years old) and 7 young (between 5-11 months old) (FIG.19A). High indiviual variations within the aged group were observed during the analysis. The results (FIG.19B) show that 16.6%±12.1 of the aged left atria and 7.02.2%±6.2 of the aged right atria are fibrotic area, whereas 8.4%±3.1 of the young left atria and 9.9%±2.7 of the young right atria are fibrotic area. No significant difference was found between the aged and the young due to the large variation in fibrotic areas.

[0300] Islands of Senescent Cells Found in Human Atrial Samples: We next characterized the senescence profile of human atrial samples. In human left atrial samples, with SR we identified islands of senescent cells (FIG.20). Anonymized human right atrial appendages (RAAs) and left atrial appendages (LAAs) that were provided by Dr. David Van Wagoner (Cleveland Clinic) were analyzed using SA-β-Gal staining. The results show that there is a wide range of relative number of senescent cells in those samples as follows: 2.9%±3.7 in LAAs derived from patients with AF at the time of surgery, 1.97%± 2.01 in LAAs of patients with sinus rhythm (SR), 2.7%±3.5 in RAAs of patients with AF, and 1.6%±1.9 in the RAAs of patients with SR. Collectively, there are no significant differences in the numberAtty. Docket No.: 405002-549001WO of senescent cells in these biopsies from aged patients with either AF or SR (FIG.8A, FIG.8B).

[0301] Identification of the Types of Senescent Cells in Human RAA AF vs SR: We next used IF experiments to identify the senescent cells in human right atrial appendage samples taken during open heart surgery at Rhode Island Hospital by Dr. Sellke’s group. Anonymized human right atrial samples with AF (n=5) and with SR (n=5) were analyzed using IF experiments. Representative confocal images of human right atrial samples stained for myocyte marker Desmin and senescence marker γH2AX is shown in FIG.9A. The results show that the relative abundance of desmin-positive cells in AF group (60.4%±3.4) does not differ from that of SR group (67.6%±14.3). A representative confocal image of human right atrial samples stained for myofibroblast marker αSMA and senescence marker γH2AX is shown in FIG.9B. The results show that the relative abundance of αSMA positive cells in AF group (26.6%±10.8) does not differ from that of the the SR (18.7%±15.1).

[0302] Interestingly, there is a significant difference in the abundance of γH2AX+ nuclei between samples derived from patients with AF (39.5%±9.01) as compared to samples derived from patients with SR (17.9%±7.9) (p<0.01). This significant difference is also observed in the case of γH2AX+ nuclei that are either Desmin+ (AF 26.9%±5.06 in AF vs 11.7%±7.5 in SR, p<0.01) or αSMA+ nuclei (AF 8.6%±3.4 in AF, vs 3.14%±2.3 in SR, p<0.05) (FIG.9C). The abundance of CD68+ cells in AF and SR samples was similar (AF 3.1%±3.9, SR 3.2%±4.03 p=ns) (FIG.9D). Thus, AF is associated with a significant increase in oxidative damage to DNA as revealed by increase abundance of γH2AX+ nuclei. Of note, IHC experiments in aged and young human samples using p16 antibody revealed abundant p16-positive cells in the aged human atrium as compared to young human atrium (FIG.9E). Due to small number of young patient sample (n=2) quantitaive analysis was not performed.

[0303] LA Diamater Correlates with the Expression of SASP Factors: Dr. David Van Wagoner kindly shared with our group the results of bulk RNAseq data of LAA obtained during open heart surgery. We provided Dr. Van Wagoner with a list of senescence genes and SASP factors and the data were correlated with the rhythm phenotype during open heart surgery when the samples were collected, including a history of AF or SR. In addition, we correlated the abundance of senescence markers with LA diameter (a known risk factor AF) obtained by Echo. Bulk RNAseq was done using RNA derived from the left atrial appendage of 265 patients who underwent cardiac surgery at the Cleveland Clinics (142). Of the 265 samples (181 males and 84 females) 235 patient were of European origin and 30 patients were of African descent. The mean age was 60±12 years. Some of the factors showed positive correlation between them and male gender (e.g., MMP1, TGFβ2, and Edn3), some of the factors showed positive correlation between them and female gender (e.g., TGFβ1Atty. Docket No.: 405002-549001WO and TIMP1), while some factors showed positive correlation between them and both genders (e.g., VCAM1, SERPINE2, and THBS2). These results suggest that the major risk factor for development of AF correlates with an increase in the abundance of SASP factors. Of note, the history of AF or the presence of AF at the time of surgery was not associated with an abundance of SASP factors.

[0304] Fibrosis Assessments in Human RAAs and LAAs, AF vs SR: We next investigated the degree of fibrosis in the LAA and RAA derived from five patients in SR and 5 patients in AF using Masson’s Trichrome staining. Samples were flash frozen and embedded in OCT prior to cryosectioning (10µm). The results show that both patients with AF or SR groups had a relative large area of fibrosis in RAA as compared to LAA samples (FIG.21A, FIG.21B). Specifically, the AF group’s relative area of fibrosis was 19.0%±11.4 while the relative area of fibrosis of RAA samples derived from patients with and SR group was 16.5%±9.5 in their RAAs, while in the LAA samples of the AF group the relative fibrotic area was 8.9%±4.7 and SR group had 9.8%±8.1. The differences between the groups did not reach statistical significance.

[0305] Fisetin Reduces Senescent Cell Numbers in Left and Right Rabbit Atria: We next carried out a pilot study to investigate the effect of treatment with the senolytic drug Fisetin on atrial arrhythmias of the aged rabbits. In order to determine the dose of Fisetin we designed two treatment protocols. The first protocol included two days, each day with a daily doses of 20 mg / kg Fisetin or vehicle control followed by observation for three weeks. The second protocol included two cycles of two days, each day with a daily dose of 20 mg / kg Fisetin or vehicle control followed by observation for a week and then sacrificing the rabbits three weeks after the first dose (FIG.10A). Our protocol included pre- and post-treatment assestments of the cardiac function including surface ECG and Echocardiogram recordings. We also collected blood samples from our rabbits within 8–12 hours after the first dose of Fisetin / vehicle of each cycle and before the start and completion of the treatment. Upon conclusion of the study, we harvested the hearts by beating heart harvest (BHH) and compared cellular senescence in the atria of Fisetin-treated rabbits to that of control rabbits using SA-β-Gal assay. The results show that Fisetin treatment for two cycles eliminated most of the senescent cells from the left and right atria (from 6.09%±1.24 to 1.15%±09 in the LA and from 7.28%±3.34 to 1.23%±1.08 in the right atrium, p<0.05). By contrast, one cycle of two-day treatment with Fisetin failed to significantly eliminate the senescent cells in the LA (FIG.11A, FIG.11B). All samples were analyzed blindly. Based on these results we chose protocol B for our double blinded pilot study.

[0306] We next conducted a pilot study of 22 rabbits on protocol B, in which 11 rabbits were treated with Fisetin and 11 were treated with vehicle, The results show that 2 rabbits in the vehicle group developed spontaneous AF while none of the Fisetin groupAtty. Docket No.: 405002-549001WO developed AF. Of note, in the Fisetin group 4 out 11 developed PACs (Premature Atrial Contractions) that did not progress to AF (FIG.22A). Analysis of pre- and post-treatment ECGs revealed no significant diffrence between Fisetin and control group in PR, QRS, QT and RR intervals (FIG.22B). Echo results showed individual variations in the measurements of atrial function such as longitudinal atrial strain. No differences were detected between the left atrial strain, left atrial volume, or left atrial ejection fraction between Fisetin-treated vs vehicle control. Of note, we will need to increase the sample to 25 in each group to confirm these underpowered observations. Our preliminary data suggest that it is likely that Fisetin treatment will have no harmful effects on the aged rabbit atria despite eliminating almost all senescent cells including myocytes.

[0307] Optical mapping of 5 Fisetin and 5 vehicle treated rabbits did not show significant difference between the groups for the measured parameters which includes APD and APD dispersion, CV and CV dispersion at 80-90ms and 350ms cycle lengths (FIG.23A, FIG.23B). Programmed electrical stimulation (PES) induced only 1 out of 5 Fisetin treated rabbits, while 4 out of 5 vehicle-treated rabbits were inducible. The fifth vehicle treated rabbit had persisted AF during the entire OM study. Of note, optical mapping showed that Fisetin decreased reentry while it increased triggered activity (data not shown).

[0308] In a general summary, FIG.2A shows a characterization of atrial action potential and mapping of reentry during AF in aged rabbit heart in a photo of optical mapping location (box) in left atrium (Ant.: anterior; Post.: posterior), with FIG.2A (bottom) showing atrial APD and CV maps. The maps show a large heterogeneity in APDs and CVs. Focal activation (a, b) and reentry (c) maintain AF. Multiple AFs occurred lasting from a few seconds to >10 min and terminated spontaneously in this rabbit. FIG.18A and FIG.18B show representative ECG recordings from aged rabbits with sinus rhythm (FIG.18A) and AF (FIG.18B). The APD90 at 1.25 Hz stimulation rate of atrial myocytes from young and aged rabbits is provided with representative AP waveforms of atrial myocytes from young and aged rabbits; (FIG.3A; the AP stimulation rate was 1.25 Hz. (FIG.3B) Cumulative APD data from a total of 49 cells of 4 young and 4 aged rabbit hearts. Scatter plot shows Mean ± SEM.

[0310] Representative images of aged and young live atrial myocytes stained with SPiDER SA-β-Gal and imaged in the green channel; representative epifluorescence images of freshly isolated aged rabbit atrial myocytes (FIG.4A); and representative brightfield and epifluorescence images of freshly isolated young rabbit atrial myocytes (FIG.4B). SA-β-Gal- positive cells show intense green fluorescence and are marked with red rectangles. In contrast, regular cardiomyocytes exhibit only faint autofluorescence. Aged cardiomyocytes appear to have more lipofuscin, a pigmented molecule that is autofluorescent and accumulates over time in lysosomes of postmitotic cells, compared to young cardiomyocytes. Scale bars are 100 µm in FIG.4A and FIG.4B.Atty. Docket No.: 405002-549001WO

[0311] FIG.5A and FIG.5B show senescence in atria of aged and young rabbits with representative SA-β-Gal-stained images of aged (4y6m) and young animals (11m) (the 40x images are from the dotted areas of the 2x images, FIG.5A). The scale bar of aged animal’s 2x image is 6 mm. The scale bar of young animal’s 2x image is 4 mm. Scale bars of 40x images are 60 µm. Blue color represents SA-β-Gal positivity (FIG.5B) Scatter plot (Mean ± SEM) shows percentage of SA-β-Gal+ cells in aged and young atria. At least 5 randomly selected images at 40x per animal were used for quantification. **p<0.01, Mann- Whitney U test (n=5 aged and n=7 young rabbits).

[0312] FIG.6A, FIG.6B, FIG.6C, FIG.6D, and FIG.6E illustrate identification of the types of senescent cells in rabbit atria with identification of the types of senescent cells in rabbit atria. Representative IF confocal images showing staining for γH2AX and Desmin; Top row (FIG.6A): images from an aged atrium. Bottom row (FIG.6B): images from a young atrium (note: no γH2AX+ nuclei were detected in the region depicted from the young atrium). Representative IF confocal images show staining for γH2AX and αSMA in an aged atrium. Panels A and B: Left images (FIG.6B): DAPI in blue and γH2AX in red; center images (FIG.6B): DAPI in blue and Desmin in white or αSMA in green; right images (FIG.6B) include all channels together. All scale bars of confocal images are 10 μm. The large yellow rectangles are magnified regions indicated by the smaller yellow rectangles. Yellow arrows indicate cells that are positive for both γH2AX and the cell type-specific marker. Quantification of total percentage of Desmin+, αSMA+, and γH2AX+ nuclei in aged and young atria is provided in FIG.6C, and quantification of percentage of γH2AX+ nuclei that are either Desmin+ or αSMA+ in aged animals is in FIG.6D. Left and right atria were analyzed separately but no significant differences in the staining of γH2AX and Desmin / αSMA were observed (data not shown). n=5 for aged, n=5 for young animals. Scatter plots= Mean±SEM, *p<0.05, **p<0.01, Mann-Whitney U test. FIG.6E shows representative IF confocal images of atria showing staining for γH2AX F4 / 80 (note: no γH2AX+ nuclei that were also F4 / 80+ were detected in the region depicted). Scale bars are 5.50 μm. FIG.12H and FIG.12I show results of RT-qPCR and Western Blot experiments Figure 4-7: Results of RT-qPCR and Western Blot experiments. Scatter plots (FIG.12H, Mean ± SEM) show relative RNA expression levels of senescence genes p16 and p21 in the right and left atria of aged and young rabbits. On the left (FIG.12I): Representative right atrium blot images of 3 young and 3 aged rabbits. Y: young, A: aged, RA: right atrium, LA: left atrium. On the right (FIG.12I): Scatter plots (Mean ± SEM) show relative protein expression levels of senescence genes p21 and p53 in the right and left atria of aged and young rabbits * p<0.05, **p<0.01, Mann-Whitney U test (n=5 aged and n=5 young rabbits).

[0313] FIG.7A, FIG.7B, and FIG.7C show bulk RNAseq analysis of aged and young rabbit right atrial samples. GSEA shows (FIG.7A) that the SenMayo gene set isAtty. Docket No.: 405002-549001WO significantly enriched in aged vs young rabbit right atria (NES: normalized enrichment score). Volcano plot (FIG.7B) showing the genes that are significantly up and downregulated in aged compared to young atria. Scatter plots (FIG.7C) depict RNA level expression differences of senescence genes p16 (CDKN2A) and p21(CDKN1A) in aged versus young atria (***p<0.001). FIG.19A and FIG.19B illustrate fibrosis in atria of aged and young rabbits. Representative M. Trichrome images (FIG.19A) of aged (4y6m) and young rabbit atria (11m). The scale bar of aged animal’s 2x image is 6mm. The scale bar of young animal’s 2x image is 4mm. Scale bars of 40x images are 60µm. Quantification of percent %fibrosis per chamber (FIG.19B). Scatter plots: Mean ± SEM, n=5 aged and n=7 young rabbits, Mann-Whitney U test. FIG.20 shows senescent cell islands in human atrial samples. FIG.8A and FIG.8B show a senescence assessment of human RAA and LAA samples with and without AF. FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E show the IF and IHC data from human right atrial specimens with and without AF.

[0314] FIG.21A and FIG.21B illustrate fibrosis in human RAA and LAA samples. FIG.10A (and FIG.10B) illustrate fisetin application protocols. FIG.11A and FIG.11B shows the senescence assessment of the Fisetin treatments on aged rabbits. FIG.20 shows senescent cell islands in human atrial samples. Human left atrial sample from a 54-year-old female patient with SR stained for SA-β-Gal (counterstained with NFR). Senescent cell islands are marked with black rectangles on the left image. Magnified images are shown on the right. FIG.8A and FIG.8B show the senescence assessment of human RAA and LAA samples with and without AF. Representative SA-β-Gal-stained images of human RAA and LAA samples either with AF or sinus rhythm (SR) (counterstained with NFR, FIG.8A). Yellow arrows (FIG.8A) indicate areas with SA-β-Gal positivity. Scale bars are 10 μm. Scatter plot (FIG.8B, Mean + SEM) shows percentage of SA-β-Gal+ cells in the right and left appendages of patients with either SR or AF at the time of surgery. Age average of AF LAA and RAA is 63.8 years. Age average of SR LAA is 65.5 years and SR RAA is 63.6 years. One-way ANOVA with post-hoc Tukey test.

[0315] In FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E, IF and IHC data from human right atrial specimens with and without AF is provided. Representative IF confocal images (FIG.9A) at 60x of atria with AF showing staining for γH2AX and Desmin. Graph on the right (FIG.9A): quantification of percent Desmin+ cells. Representative IF confocal images (FIG.9B) of atria with AF showing staining for γH2AX and αSMA. Graph on the right (FIG.9B): quantification of percent αSMA+ cells. Quantification (FIG.9C) of total % γH2AX+ nuclei and % γH2AX+ nuclei that are either Desmin+ or αSMA+. Representative IF confocal images (FIG.9D) of atria with AF showing staining for γH2AX and CD68. Left images (FIG.9D): DAPI in blue and γH2AX in red; center images (FIG.9D): DAPI in blue and Desmin in white, αSMA in green, or CD68 in yellow; right images (FIG.9D) include allAtty. Docket No.: 405002-549001WO channels together. Bigger yellow rectangles show focused areas outlined by the smaller rectangles. Yellow arrows indicate cells that are positive for both γH2AX and the cell type- specific marker. Scale bars of confocal images are 20 μm. Scale bars of focused nuclear images are 10 μm. n=5 for AF, n=5 for SR. Age average (FIG.9D) of AF RAA is 65.8 years. Age average of SR RAA is 68.3 years. Error bars denote SEM, *p<0.05, **p<0.01, 2-tailed Exact test. FIG.9E shows representative images of female (F) and male (M) human atrial samples stained for p16 antibody. Counterstaining was done with NFR. Black rectangles indicate the positive areas for the staining. Scale bars are 10 μm.

[0316] In FIG.21A and FIG.21B fibrosis in human RAA and LAA samples. (A) Representative M. Trichrome images of RAA samples from patients with AF and SR. The scatter plot (Mean + SEM) on the right shows percentage area of fibrosis of the RAA samples. (B) Representative M. Trichrome images of LAA samples (AF and SR). The Scatter plot (Mean ± SEM) on the right indicates the percent fibrosis of LAA samples. All scale bars are 10 μm. n=5 for AF, n=5 for SR, 2-tailed Exact test.

[0317] Fisetin application protocols (FIG.10A, FIG.10B). Protocol A: Animals received Fisetin (20mg / kg) for 2 days throughout the protocol. Protocol B: Animals received either Fisetin (20mg / kg) or vehicle 2 days per week and for 2 weekly cycles throughout the protocol (green and blue arrows). BHH: beating heart harvest.

[0318] In FIG.11A and FIG.11B, senescence assessment of the Fisetin treatments on aged rabbits is shown. (A) Representative SA-β-Gal-stained images (counterstained with NFR staining). Blue color and blue arrows indicate areas with SA-β-Gal positive cells. Small scale bars are 10 μm. Big scale bar is 100 μm. (B) Quantification results of percent SA-β- Gal+ cells. LA: Left atrium, RA: Right atrium, Veh: Vehicle, 1-Cyc: 2 days-1 week of treatment, 2-Cyc: 2 days-2 weeks of treatment. n=3 for all groups. The age average per group is 4.4 years. Scatter plot: Mean ± SEM, *p<0.05. One-way ANOVA with post-hoc Tukey test.

[0319] In FIG.22A and FIG.22B, pre- and post-treatment ECG results of the pilot Fisetin study. (A) Tables of control and Fisetin groups showing their arrhythmia status pre- and post-treatment. (B) Showing PR, QRS, QT and RR analysis results of the treatment groups pre- and post-treatment. y-axis: millisecond. Scatter plots: Mean ± SEM, 2-tailed Exact test. FIG.23A and FIG.23B show Optical mapping results of the pilot Fisetin study. (FIG.23A) Scatter plots of APD and APD dispersion at 80-90ms and 350ms cycle lengths of Fisetin (white)-and vehicle-treated rabbits (red). (FIG.23B) Scatter plots (Mean ± SEM) show CV and CV dispersion at 80-90 and 350ms cycle lengths. Fisher exact test was used for the statistics.

[0320] Discussion: Although the role of cellular senescence has previously been investigated and the related pathogenesis of cardiovascular diseases have been described,Atty. Docket No.: 405002-549001WO the relationship between senescence, aging, and atrial arrhythmias is not well understood due to lack of proper larger animal models with spontaneous AF. Our experiments show accumulation of senescent cells with SA-β-Gal activity and DNA damage in the aged rabbit atria as compared to young rabbits. Human atrial tissue with AF presented significantly more DNA damage compared to human atrial tissue with SR. Most of these cells with DNA damage are cardiomyocytes, and their accumulation is associated with a marked increase in the expression senescence genes and SASP factors including markers of inflammation. We therefore propose that the accumulation of senescent cells and the SASP-mediated inflammatory response predisposes the atria to AF (FIG.24).

[0321] As mentioned earlier during the introduction of the aged rabbit AF model, one of the major problems in the field to study AF is the lack of more clinicaly relevant animal model which developes spontaneous AF with similar electrophysiological features to aged human heart. Koren lab has extensively studied electrophysiological features of the rabbit heart and recently validated aged rabbit heart as a reliable larger animal model for AF studies by mapping and documenting its atrial properties via OM experiments and ECG recordings. During OM experiments conducted on both young and aged rabbit atria we tried to stimulate arrhythmias by either with S1S2, S1S5 pacing protocol or stretching the atria and showed that all aged hearts were inducible.

[0322] According to our optical mapping experiments (FIG.2A), there is a substantial amount of variation in CV and APD in the aged rabbit group, which causes a greater APD dispersion. We also successfully mapped a spontaneous AF episode. The optical maps reveal that both reentery and multiple focal activity play an important role in maintaining the AF in aged rabbit atria, similar to AF in humans (64). Optical mapping studies of aged and young rabbits show that programmed electrical stimulation (PES) triggers significantly more AF in aged rabbit atria as compared to young atria (data not shown). To that end, we screened the entire aging colony for spontaneous arrhythmias using surface ECG. Our results documented a prevalence of 12.5% of spontaneous AF in the aging rabbit colony (FIG.18A, FIG.18B). Given that the prevalence of AF in the aged human population (>65) ranges between 10 to 15% (143), the prevalence of spontaneous AF obsverved in the aged rabbit colony supports our contention that the aged rabbit model is a unique animal model to investigate the pathogenesis of age-related AF, as well as the role of cell senescence in its pathogenesis. Importantly, this model will enable us to explore novel therapies for AF.

[0323] Our patch clamp experiments to study electrophysiological features of aged and young rabbit atrial cardiomyocytes included recordings of APD90and APD50of freshly isolated atrial cardiomyocytes. The results show no significant difference between the two groups (FIG.3A, FIG.3B) and correlate with our optical mapping data, which did not reveal differences in APD between young and aged rabbits either (data not shown). We expectedAtty. Docket No.: 405002-549001WO prolongation of APD of myocytes derived from aged atria as well as larger variance if indeed senescence is associated with marked changes in the electrical properties of the atrial myocytes. One possible explanation could be that cell isolation may select for healthier cells and that senescence cells would perish in the process of cell isolation. To test this hypothesis, we next isolated cells and stained them for SA-β-Gal activity. To that end, we treated the cardiomyocytes with the SPiDER SA-β-Gal kit to label the live senescent ones for subsequent patch clamp experiments (FIG.4A, FIG.4B). The results show that we can isolate senescent atrial myocytes and that these cells do not perish during isolation. However, we could not pacth clamp the treated cells as the treatment changed their cellular and electrical characteristics, likely due to blocking of lysosomal proton pumps resulting in acidification of the cytoplasm and changing the physical and electrical characteristics of their membranes. Nevertheless, the studies supported the overall quantitative assessment of the relative number of senescent myocytes in the atrium.

[0324] To the best of our knowledge, there is no study in the literature to this day showing senescence in the atria of a larger-animal model that recapitulates human electrophysiological features and shows similar prevalence of spontaneous AF. In the current study, we documented the presence of senescent cells in the aged rabbit atria using SA-β-Gal staining, which is widely accepted and commonly used for identifying senescent cells. The aged group included retired female breeders, aged 4 to 6 years, while the young group included rabbits aged 5 to 11 months (for reference, a rabbit year is taken to be roughly 14-15 human years). We documented accumulation of SA-β-Gal+ cells and their location in aged atria (FIG.5A, FIG.5B). Interestingly, the results showed large variance in senescent cell accumulations in both atrial chambers of aged rabbits reflecting the biological variation in the aging rabbit model. We noted heterogenous distribution of the senescent cells throughout the aged atria, which is consistent with the ‘zombie cell’ definition that is used for senescent cells (144). The term is used to denote the capability of senescent cells to induce senescence in neighbouring cells in a paracrine manner (92). As there is a paucity of commercially available retired male breeders, all experiments were done with female rabbits throughout this study. We were nevertheless able to carry out SA-β-Gal stainings on atrial samples derived from 4 male retired breederes over the course of this study. We identified senescent cells but their relative numbers were somewhat lower than in atria of aged female rabbits (data not shown) underscoring previously reported sex differences in senescence in humans and animals [reviewed in (145)].

[0325] Our immunofluorescence (IF) stainings using cell type-specific antibodies combined with the DNA damage marker γH2AX successfully documented the types of the senescent cells in the atria. The experiments were designed to identify most senescent cells found in the atria, namely cardiomyocytes (CMs), cardiac myofibroblasts (CMFs), endothelialAtty. Docket No.: 405002-549001WO cells, and macrophages. The results of these studies reveal that most abundant senescent cells were atrial cardiomyocytes followed by atrial myofibroblasts (FIG.6A, FIG.6B). The IF experiments did not reveal any significant differences in the overall cellular composition of aged vs. young atria (FIG.6C). However, the relative number of cells labeled with the DNA damage marker γH2AX was significantly higher in the aged group (FIG.6C). About 80% of all cells with persistent DNA damage were atrial cardiomyocytes (FIG.6D). Of note, vessel areas were excluded from the study as their inclusion would have skewed the results in favor of senescent CMFs. Echocardiograms revealed no significant differences in the atrial longitudinal strain between young and aged rabbit. We thus reason that senescent atrial cells do contract and provide mechanical support to the atria. The confocal images of the IF experiments showed the presence of both senescent and non-senescent macrophages (FIG.6E) as well as atrial endothelial cells (data not shown) in the aged atria. As the percentages of these cell types were significantly lower compared to the ones of CMs and CMFs, we did not carry out quantitative analyses. Our results are the first to document the presence of senescence cells in a larger-animal model with spontaneous AF. The results therefore extend the observations in several human studies that documented the presence of senescent atrial myocytes, myofibroblasts, and endothelial cells in atrial samples (129, 130, 146).

[0326] The RT-qPCR results revealed statistically significantly higher levels of the CDKIs p16 and p21 in the aged compared to young rabbits (FIG.12H, FIG.12I). Of note, p16 levels were elevated in both atrial chambers. In different samples derived from aged atria, we detected p16 levels that were 3 to more than 30 times higher than p16 levels in the young group, which is consistent with marked activation of this senescent pathway (FIG.12H). Western Blot experiments showed significantly higher levels of p21, suggesting that p21 protein levels in senescent cells are possibly regulated by a post-transcriptional mechanism, likely protein stability, as RNA level differences between the groups in both atria were not as distinct as p16 levels. We did not observe significant differences in p53 or phospho p53 levels (FIG.12I). Additional RT-qPCR experiments showed that transcript levels of CCL-2, TIMP1, TIMP2, TGF-β, MMP1, MMP3, and IL-6 were not statistically significant due to high variability in their expression in the atrial biopsies used for generating the RNA samples (data not shown). We therefore proceded with bulk RNA sequencing (RNAseq). The SenMayo gene set was found to be significantly enriched in the aged rabbit right atria (FIG.7A, FIG.7B, FIG.7C). Additionally, the RNAseq data show upregulation of inflammatory pathways and the innate immunity response, as well as genes related to complement and coagulation cascades. This inflammatory state correlates with the documented pro-inflammatory SASP factors in aged rabbit atria in comparison to young ones. Taken together with SA-β-Gal stains (FIG.5A, FIG.5B), these observations supportAtty. Docket No.: 405002-549001WO the hypothesis that senescent cell burden is increased with age in the rabbit right atrium and that this burden entails immune-mediated tissue dysfunction. In conclusion, analysis of the right atrial transcriptome suggests that the SASP-mediated inflammatory response is likely associated with the high pevalence of spontaneous AF in the aged rabbit colony.

[0327] Our rabbit data shows that all different cell types that were detected in this study can become senescent. This correlates well with the results of our human histological studies (FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E) and the literature showing different types of senescent cells found in human atrial samples (129, 130, 146). Previous publications have shown that fibroblasts and endothelial cells can undergo replicative senescence that is dependent on telomere shorthing or dysfunction. However, recent studies show that post-mitotic cells can undergo senescence as a response to stressors such as DNA damage and oxidative stress (147). This stress-induced premature senescence is telomere length independent. In this study, we show that most of the senescent cells in aged rabbit and human atria are myocytes (AMs). It has been shown that oxidative stress and mitochondrial damage contribute to AF progression (148-150). Of note, persistent DNA damage can occur even at long telomeres causing induction of senescence (151-153). Indeed, a recent study showed that telomere-associated DNA damage creating telomere- associated foci (TAF) can be associated with cellular senescence independent of telomere length both in vivo in aged mouse and human hearts and in vitro in cultured mouse embryonic cardiomyocytes (154). The same study showed that purified senescent CMs have a non-canonical SASP with elevation of SASP factors such as endothelin 3 (Edn3) and TGF- β2. This observation suggests that SASP factors expressed by senescent CMs may include molecules playing role in cardiac remodelling in addition to their pro-inflammatory role. However, interpretation of mechanistic studies of senescence in mouse hearts could mislead us as the aging process of mouse hearts differs from that of rabbit or human hearts. For example, aging mouse hearts undergo pathological hypertrophy, while aging rabbit hearts lack known markers of hypertrophy (Koren lab, unpublished data).

[0328] Atrial fibrosis is one of the key features of aging atria (FIG.19A, FIG.19B) and one of the most important substrates for the maintenance of AF. A study showed that senescence may play a role in atrial fibrosis-induced AF (155). Here, higher levels of atrial fibrosis and shorter telomeres were found in left atrial appendages derived from patients with AF. Moreover, another study showed that the accumulation of senescent cells is positively correlated with fibrotic lesions in patients with AF (130). In contrast to our study, the majority of senescent cells in this study were cardiac fibroblasts, with a small contribution from cardiomyocytes and endothelial cells. Well-known SASP factors contribute to fibrosis including TNFα, TGF-ß, NF-κB, ILs, and MMPs, which are also implicated in AF-associatedAtty. Docket No.: 405002-549001WO inflammation, fibroblast proliferation, and ultimately giving way to atrial electrical and structural remodelling.

[0329] Experiments on human and rabbit atrial samples were conducted simultaneously to identify respective senescence profiles and to investigate whether the senescence profile of rabbit atria recapitulated human atrial senescence. Similar to rabbit, the accumulation of senescent cells in human atria was heterogeneous, and some areas did not have any SA-β-Gal+ signals (FIG.20). Following quantitative analysis of SA-β-Gal assays was done on human right (RAA) and left atrial appendages (LAA) with AF or SR (FIG.8A). The results revealed different degrees of senescent cell presence in human samples ranging from 1% to 8%. AF samples showed a trend for more senescent cells, but statistical analysis did not reveal differences between the groups (FIG.8B). Of note, we were able to identify some small areas in some of the samples with almost 100% SA-β-Gal positivity closer to the epicardial region. The shape of these cells differs from atrial cells or fibroblasts (data not shown). Based on the cellular morphology of the senescent cells in these regions, we anticipate that they could be ganglionic plexus cells. This observation shoud be further explored in future studies.

[0330] IF experiments revealed no difference in the percentages of CMs and CMFs comparing AF and SR groups (FIG.9A, FIG.9B). However, we noted a significantly higher percentage of γH2AX+ cells in samples originating from AF compared to SR patients. Concordantly, significantly higher levels of both γH2AX+ myocytes and γH2AX+ myofibroblasts were seen in the AF group (FIG.9C). Surprisingly, we detected approximately 10-fold higher percentages of γH2AX+ nuclei than SA-β-Gal+ cells in both the AF and SR groups (cf. FIG.8B, FIG.9C). This discrepancy could be explained by the loss of β-galactosidase enzymatic activity in the interval between the initial tissue harvest, the flash freezing of tissue, embedding in OCT, and subsequent storage, or that not all cells with below a certain degree of DNA damage become senescent immediately. We also detected a small portion of CD68+ cells in the human RAA samples representing immun cells, some of which were also positive for senescence marker γH2AX (FIG.9D). There was no difference between the AF and SR groups in CD68+ cell percentage. These results indicate an active inflammatory process in the atria, which was confirmed by our RNAseq data in rabbit. In addition to these findings, our IHC experiments showed a signal accumulation of p16, a marker for senescence, in aging atria (AF and SR groups), suggesting that there is an increase in the senescence burden during aging in human atria (FIG.9E).

[0331] Hyperoxia generates reactive oxygen species (ROS) that cause oxidative damage, triggering DNA damage response (DDR) pathways and the activation of tumor suppressor proteins such as p53 (156). Gamma irradiation treatment causes DNA damage through double-strand breaks and ROS generation, activating DDR pathways, p53, and theAtty. Docket No.: 405002-549001WO p16-Rb pathway (157). Our results demonstrate that 40% oxygen treatment is a viable method to induce a high degree of senescence in a-EHTs. However, 5 Gy gamma irradiation had little effect on cell senescence (data not shown). In one example, combining gamma radiation with 40% oxygen resulted in a very significant reduction in the number of senescent cells (not shown). Our laboratory (35) recently demonstrated that myofibroblast senescence in the infarct border zone is proarrhythmogenic, while a group at Washington University in St. Louis reported gamma radiation (noninvasive cardiac radioablation) decreased the incidence of ventricular tachycardia in patients with heart failure and large MI (158). We speculate that gamma irradiation may have eliminated senescence cells in the scar and infarct border zone and thereby reducing the risk of arrhythmia. Surface and subsurface regions had different percentages of senescent cells and the core of the a-EHTs had the lowest number of senescent cells as tissue integrity in this region appeared to be poor compared to other regions. Confocal images acquired from IF slides revealed that, as expected, the majority of cells forming a-EHTs are cardiomyocytes (not shown). Altogether, these results show that we have validated a method to create a-EHTs with similar accumulation of senescent cells as observed in aged human and rabbit atria.

[0332] OM experiments (FIG.23A, FIG.23B) confirmed the ECG data as there were no significant difference in APD, CV and APD dispersion between the two groups. Programmed electrical stimulation of the two groups revealed that only 1 out 5 Fisetin- treated rabbits was inducible (atrial tachycardia) while 4 out of five vehicle-treated rabbits were inducible and developed AT. The fifth rabbit had spontaneous AF. Thus, both spontaneous arrhythmias and PES-induced arrhythmias showed a trend for higher prevalence in the vehicle-treated group. These results did not reach statistical significance. Of note, OM revealed that Fisetin-treated group were less prone to develop reentry circuits. However, the Fisetin group was more prone to develop enhanced triggered activity (Dr. Bum-Rak Choi – personal communication). Taken together these observations may explain the presence of multiple PACs that did not evolve into AF in the Fisetin-treated rabbits. The decreased inducibility and lower propensity for re-entry circuits in the Fisetin group suggest that the drugs may have an indirect antiarrhythmic effects or modulated factors involved in arrhythmia initiation and maintenance. The molecular underpinning of these phenomena will be the subject of further investigation.

[0333] Our pilot study yielded encouraging results even though the aged rabbits already have an “end stage” atrial disease including extensive fibrosis and evidence for inflammation. Of note, despite the elimination of senescent cells we did not observe a striking difference in longitudinal strain as a marker for atrial contractility. We reason that the next phase should entail using middle aged rabbits and treating them with Fisetin as a preventive therapy to abolish senescence, inflammation and thereby spontaneous andAtty. Docket No.: 405002-549001WO inducible arrhythmias. These studies will create a road map for preventive clinical studies to decrease the incidence of AF. It is reassuring that are pilot study in 11 rabbits was not associated with marked deleterious effects on the treatment group’s ECG, Echo and organ excitation as studied by OM. These observations are consistent with previous studies showing the effects of senolytic treatment on cardiac functions which also did not report any harmful effects (154, 159). As it has been shown that inflammation plays a key role in the pathophysiology of AF, targeting inflammation and its complications such as fibrosis represents a novel and promising strategy for preventing AF and likely additional age-related cardiovascular diseases.

[0334] Overall, AF in aged rabbit hearts replicates important clinical features of AF in the aged human population and is a unique animal model for investigating the pathogenesis of AF. Our findings show that the number of senescent cells (myocytes, myofibroblasts, macrophages) increase with age in both the left and right atria of aged rabbits and human atria. This accumulation is associated with detection of increased levels of senescence genes and SASP factors. The increased levels of SASP factors in dilated atria is a major risk factor for the development of AF, and the trend for the anti-arrhythmic effect of Fisetin suggest that SASP-mediated inflammatory response plays an important role in the pathogenesis of AF, and the use of senolytic therapy to mitigate the senescence burden in the atria may offer a novel strategy for modulating inflammation, fibrosis and reducing the incidence of AF.

[0335] In some aspects, the techniques described herein relate to a method for preventing or treating atrial fibrillation (AF) in a subject, the method including the steps of: (1) obtaining a subject in need of a prevention and / or a treating of AF, a subject suspected to be in need of a prevention and / or a treating of AF, or a normal healthy subject without a suspected need for the prevention and / or treating of AF; (2) administering a therapeutically effective amount of Formula 1 or a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2- phenylchromen-4-one) derivate such as 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4- one); Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one); Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one); Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8- tetrahydroxychromen-4-one); Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4- one); Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one); Isorhamnetin (3,5,7- trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one); Morin (2-(2,4-dihydroxyphenyl)- 3,5,7-trihydroxychromen-4-one); Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4- one); Natsudaidain (2-(3,4-dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4- one); Pachypodol (5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one); Quercetin (3,3′,4′,5,7-pentahydroxy-2-phenylchromen-4-one); Rhamnazin (3,5-dihydroxy-2- (4-hydroxy-3-methoxyphenyl)-7-methoxychromen-4-one); Rhamnetin (2-(3,4-Atty. Docket No.: 405002-549001WO dihydroxyphenyl)-3,5-dihydroxy-7-methoxychromen-4-one); Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin; or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of Formula 1 or 3- Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, Rhamnetin, Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin to the subject; whereby a risk of the subject acquiring AF or a prevalence of AF in the subject is lowered.

[0336] In some aspects, the techniques described herein relate to a method, wherein Formula 1 or the Fisetin derivative is administered in a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis; as measured by the mass of the non-salt and / or non-hydrate / solvate form and the mass of the subject in kg; or in a daily dose of about 100 mg; in a dose administered by a device in contact with the subject; or in a monthly dose.

[0337] In some aspects, the techniques described herein relate to a method, wherein the administration decreases a number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in an atrium of the subject.

[0338] In some aspects, the techniques described herein relate to a method, further including a spatial transcriptomic measurement of a cell composition in the subject, whereby the measurement elucidates the role of different cell types in modulating atrial senescence in the context of human atrial-like engineered heart tissues (a-EHTs).

[0339] In some aspects, the techniques described herein relate to a method, further including an optical mapping (OM) of a tissue from the subject, whereby the OM is utilized to record and visualize one or more electrophysiological properties in a myocardial preparations including Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, or cell culture monolayers, and wherein the OM is capable to reveal a marked reduction in a re-entrant wave, wherein the equivalent to OM is mapping the human atria using, for example, catheters.

[0340] In some aspects, the techniques described herein relate to a method, wherein the method is utilized to prevent AF in an aged population ≥ 50 years old, wherein the method is to prevent atrial fibrillation in subjects with paroxysmal atrial fibrillation; wherein the method is to prevent atrial fibrillation in subjects after radiofrequency ablation; wherein the method is to treat paroxysmal atrial fibrillation, persistent atrial fibrillation, or chronic atrial fibrillation; wherein the method is to prevent atrial fibrillation after an open heart surgery; wherein the method is to prevent AF in obese and / or overweight human subjects; whereinAtty. Docket No.: 405002-549001WO the method is executed to prevent atrial fibrillation in diabetes mellitus subjects; wherein the method is executed to treat and / or prevent AF related to obesity or AF risk due to obesity; wherein the method is executed to prevent a complication of persistent AF like stroke; wherein the method is executed to prevent AF in subjects with hypertension; or wherein the method is executed to prevent AF in subjects with ischemic heart disease, myocardial infarction, or heart failure.

[0341] In some aspects, the techniques described herein relate to a method, wherein the method is executed as a combination therapy with one or more therapeutic agents administered simultaneously with Formula 1 and / or a Fisetin derivative or before / after an administration of Formula 1 and / or a Fisetin derivative.

[0342] In some aspects, the techniques described herein relate to a method, wherein the subject is a subject in need of prevention of AF, and wherein the subject has one or more risk factors for developing AF, including age over 50 years, obesity, diabetes, hypertension, ischemic heart disease, heart failure, or a history of myocardial infarction.

[0343] In some aspects, the techniques described herein relate to a method, wherein the subject is suspected to be in need of prevention of AF based on the presence of one or more symptoms or diagnostic indicators of AF, including palpitations, shortness of breath, chest pain, fatigue, lightheadedness, or an irregular pulse.

[0344] In some aspects, the techniques described herein relate to a method, wherein the subject is a normal healthy subject without a suspected need for the prevention and / or treatment of AF, and wherein the method is used prophylactically to reduce the future risk of developing AF in the subject.

[0345] In some aspects, the techniques described herein relate to a method, wherein obtaining the subject includes identifying the subject as requiring or potentially requiring prevention and / or treatment of AF.

[0346] In some aspects, the techniques described herein relate to a method, wherein administering the therapeutically effective amount of the Fisetin derivate or the pharmaceutically acceptable form thereof includes administering the Fisetin derivate or the pharmaceutically acceptable form thereof orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation in an amount sufficient to lower the risk of the subject acquiring AF or the prevalence of AF in the subject.

[0347] In some aspects, the techniques described herein relate to a method, wherein the Fisetin derivate is Fisetin, and wherein Fisetin is administered in a dose of about 20 mg / kg to about 100 mg / kg daily, or in a dose of about 40 mg / kg to about 80 mg / kg daily, or in a dose of about 60 mg / kg daily, and wherein Fisetin is administered orally, parenterally,Atty. Docket No.: 405002-549001WO intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0348] In some aspects, the techniques described herein relate to a method, wherein the Fisetin derivate is 3-Hydroxyflavone, and wherein 3-Hydroxyflavone is administered in a dose of about 10 mg / kg to about 50 mg / kg daily, or in a dose of about 20 mg / kg to about 40 mg / kg daily, or in a dose of about 30 mg / kg daily, and wherein 3-Hydroxyflavone is administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0349] In some aspects, the techniques described herein relate to a method, wherein the Fisetin derivate is Azaleatin, and wherein Azaleatin is administered in a dose of about 5 mg / kg to about 25 mg / kg daily, or in a dose of about 10 mg / kg to about 20 mg / kg daily, or in a dose of about 15 mg / kg daily, and wherein Azaleatin is administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0350] In some aspects, the techniques described herein relate to a method, wherein the Fisetin derivate is Galangin, and wherein Galangin is administered in a dose of about 15 mg / kg to about 75 mg / kg daily, or in a dose of about 30 mg / kg to about 60 mg / kg daily.

[0351] In some aspects, the techniques described herein relate to a method, wherein the Fisetin derivate is Gossypetin, and wherein Gossypetin is administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, or in a dose of about 5 mg / kg to about 10 mg / kg daily.

[0352] In some aspects, the techniques described herein relate to a method, wherein the Fisetin derivate is Kaempferide, and wherein Kaempferide is administered in a dose of about 25 mg / kg to about 125 mg / kg daily, or in a dose of about 50 mg / kg to about 100 mg / kg daily.

[0353] In some aspects, the techniques described herein relate to a method, wherein the Fisetin derivate is Kaempferol, and wherein Kaempferol is administered in a dose of about 30 mg / kg to about 150 mg / kg daily, or in a dose of about 60 mg / kg to about 120 mg / kg daily.

[0354] In some aspects, the techniques described herein relate to a method, wherein the risk of the subject acquiring AF or a prevalence of AF in the subject is lowered is measured by a symptom reported by the subject; by a sign perceived by a healthcare provider; by a measurement of a device in contact with the subject and / or implanted with the subject; by an iWatch®; by a FitBit®; or by a remote measurement device. EXAMPLESAtty. Docket No.: 405002-549001WO

[0355] In addition to the scientific experiments discussed above, the following EXAMPLES are provided to illustrate the invention and should not be considered to limit its scope. EXAMPLE 1. DEFINING AND CHARACTERIZING THE TYPE AND QUANTITY OF SENESCENT CELLS

[0356] This EXAMPLE defines and characterizes the type and quantity of senescent cells in the atria of young and aged rabbits using multiple senescence biomarkers and correlates it with spontaneous and induced atrial fibrillation using ECG recordings in vivo and optical mapping ex vivo. The purpose of this EXAMPLE is also to verify that atrial fibrillation (AF) in aged rabbit hearts replicates important clinical features of atrial fibrillation in the elderly population such as automaticity, abnormal action potential duration (APD), discontinuous conduction, and reentry formation.

[0357] The inventors elucidated the molecular and cellular characteristics of senescent atrial myocytes (SAMs), senescent atrial myofibroblasts (SAMFs), and SASP- induced inflammation in aging rabbit atria using qPCR, single cell RNAseq / spatial transcriptomics, and cytokine arrays.

[0358] The inventors prospectively treat middle-aged rabbits with fisetin to further test if fisetin will abolish spontaneous and induced atrial fibrillation when the rabbit reached the age of four to five years.

[0359] A challenge to assaying atrial fibrillation mechanisms is the lack of a clinically relevant animal model with spontaneous atrial fibrillation. Atrial fibrillation induction in rodents is difficult because of the small size of the atrial tissue and species-dependent action potential characteristics. Larger animal models of atrial fibrillation were developed in sheep or dogs using tachy-pacing of atria to artificially trigger atrial fibrillation.

[0360] The inventors assayed a few aged rabbits (older than four years) and found spontaneous atrial fibrillation developing in four out of thirty two rabbits. This finding was unexpected because spontaneous atrial fibrillation in a large mammal is rare.

[0361] The inventors assayed the activation, action potential duration (e.g., FIG.3A, FIG.3B), and conduction velocity (CV) maps from aged rabbit atria. There is a large action potential duration dispersion between the posterior and anterior regions. The conduction velocity map also shows heterogeneity near the pulmonary vein. The bottom panels show a spontaneous transient atrial fibrillation episode captured with optical mapping. Activation maps show that multiple focal activities (maps 'a' and 'b') and rotors (map 'c') maintain atrial fibrillation. Four out of five aged rabbits were induced by either pacing or stretch. None of the four young rabbits was induced by pacing or stretch.

[0362] The inventors conducted patch clamp recordings on freshly isolated aged and young rabbit cardiomyocytes. See FIG.3A, FIG.3B. The inventors compared APD90of atrialAtty. Docket No.: 405002-549001WO myocytes derived from young rabbits to that of atrial myocytes derived from aged animals. The results showed no significant difference between these two groups. See FIG.3A, FIG.3B. In the young group, ADP90was equal to 97±62 milliseconds (n=26). In the aged group, ADP90was equal to 113±58 milliseconds (n = 23, p=0.35). Optical mapping assays did not reveal significant differences between the action potential duration of aged versus young atria.

[0363] To determine whether aged rabbits had senescent cells, the inventors next conducted senescence-associated β-galactosidase (SA-β-gal) staining, a widely accepted method for detecting cellular senescence. The inventors measured the positive senescent cells per atrial chamber. The result showed that the left and right atria of aged rabbits have many more SA-β-gal+cells than young rabbit atria. See FIG.5A, FIG.5B. There was a trend for a larger number of senescent cells in the right atria that did not reach statistical significance.

[0364] The inventors compared the electrical characteristics of senescent-aged atrial myocytes and regular-aged myocytes. The inventors labeled live senescent atrial cells with the SPiDER SA-β-gal kit (Dojindo Molecular Technologies Inc.) that stains live cells for SA- β-gal. About 18% of the atrial myocytes were stained positive with the SPiDER kit. See FIG.4A, FIG.4B. After the cells were labeled with the kit, the inventors could not patch them or obtain reliable cellular electrophysiological data such as resting membrane potential or action potential duration. The inventors posit that changes in intracellular pH rendered these cells unpatchable. Most cells died right after breaking the seal.

[0365] The inventors next conducted immunofluorescence (IF) assays to determine the type of the senescent cells that the inventors identified with SA-β-gal staining. The inventors used desmin as a myocyte marker, αSMA as a myofibroblast marker, vWF as an endothelial cell marker, and CD68 as a macrophage marker. The cell lineage markers are used with the senescence marker γH2AX. The results showed that most of the senescent cells were atrial myocytes, but there were also some senescent myofibroblasts, endothelial cells, and macrophages in the aged atria. Aged atria had many more γH2AX+nuclei compared to young rabbit atria. See FIG.6A, FIG.6B, FIG.6C, FIG.6D, and FIG.6E.

[0366] The inventors isolated high-quality RNA from aged and young rabbit right atrial samples. They performed bulk RNA- sequencing (RNAseq) analysis on the rabbit right atria derived from young (n = 5) and aged (n = 5) animals. The inventors started with right atrial samples’ analyses as the inventors saw more SA-β-gal+cells in the right-aged rabbit atrium. Using an FDR cutoff of 0.05, The inventors observed 247 upregulated and 116 downregulated genes in aged versus young animals. To determine whether aged tissue exhibits transcriptomic signatures indicative of a high burden of senescent cells, The inventors performed gene set enrichment analysis (GSEA) on the RNA-sequencing data.Atty. Docket No.: 405002-549001WO The inventors examined whether the SenMayo gene set would be enriched. This gene set was characterized by a consortium of researchers led by scientists from the Mayo Clinic. The SenMayo gene set comprises a panel of 125 genes manually curated through a literature review according to their informative value as senescence markers. See Saul et al., Nature Communications, 13, 4827 (2022)(41). The SenMayo gene set was significantly upregulated (p<0.05) in aged compared to young rabbit right atrium tissue. See FIG.7A, FIG.7B, and FIG.7C. Gene Set Enrichment Analysis (GSEA) of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways showed that many of the most significantly enriched pathways were related to the inflammatory response. The inventors posit that the inflammatory response is related to senescence-associated secretory phenotype (SASP), which is highly inflammatory. RNAseq analysis also showed significantly elevated senescence gene p16 (CDKN2A) expression levels in the aged atria compared to the young.

[0367] The inventors also checked for another senescence gene, p21, also known as CDKN1A. They did not observe significant differences due to high variations. See FIG.7A, FIG.7B, and FIG.7C.

[0368] RT-qPCR and Western blot results show that aged rabbit atria have more p16 and p21 expression than young rabbit atria. The inventors conducted RT-qPCR and Western blot assays to investigate the expression of senescence genes and SASP factors in young and aged atria. The results of RT-qPCR showed that the normalized transcripts levels of p16 were 19.1 fold higher (p<0.01) in the aged LA, and 6.3 fold higher (p<0.01) in the aged RA as compared to the young atria. Normalized transcript levels of p21 in the aged RA were 2.6 fold higher (p<0.05) and trended toward higher levels in the LA of the aged group. See FIG.12A, FIG.12B, FIG.12C, FIG.12D, FIG.12E, FIG.12F, FIG.12G, FIG.12H, and FIG.12I.

[0369] Though a trend toward higher levels of transcripts of other SASP factors was seen in the aged group compared to the young, these differences did not reach statistical significance.

[0370] The inventors next investigated the steady-state protein levels of p21, p53, and phosphorylated p53 using Western blots. Normalized expression levels of p21 polypeptides were 2.7-fold higher in the aged LA (p<0.05) and 3.1-fold higher (p<0.01) in the aged RA as compared to the young atria. The inventors created scatter plots to show relative RNA expression levels of senescence genes p16 and p21 in the right and left atria of aged and young rabbits. There were no significant differences in the protein levels of p53 or phospho-p53. The inventors created scatter plots to show relative protein expression levels of senescence genes p21 and p53 in the right and left atria of aged and young rabbits.Atty. Docket No.: 405002-549001WO

[0371] Unfortunately, no commercial p16 antibody was available to investigate the protein levels of p16 in rabbit atria.

[0372] The inventors posit that the SASP drives an inflammatory response. Treating senescence early on with a senolytic drug such as fisetin should reduce inflammation, fibrosis and could reduce the incidence of atrial fibrillation in the aged population. EXAMPLE 2. HUMAN STUDIES

[0373] This Example investigates the type and quantity of senescent atrial cells derived from adult patients (age < fifty years) and aged (> sixty-five years) patients with a history of either normal sinus rhythm (NSR) or atrial fibrillation. The inventors will compare single nuclei RNA sequencing (sn-RNAseq) in humans to that of rabbits and map the molecular signature of senescent atrial myocytes and senescent atrial myofibroblasts in human samples from patients in both age groups with and without atrial fibrillation.

[0374] The inventors next investigated the similarities between the senescence process in human aged atria as compared to rabbit aged atria.

[0375] The inventors first investigated the type and quantity of senescent cells in atria from adult males and females with an age greater than sixty-five years in either normal sinus rhythm (NSR) or atrial fibrillation. The human atrial samples were subjected to the same assays as the rabbit atria. EXAMPLE 1 shows that atrial fibrillation in aged rabbits is started by repetitive focal firing of action potentials in the atrium near the pulmonary vein, which often shows discontinuous conduction across severe fibrosis, precipitating long-lasting atrial fibrillation, replicating the features of atrial fibrillation seen in the clinical setting. The inventors posit that cellular senescence in the human atria underlies inflammation, fibrosis, and vulnerability to developing atrial fibrillation. The inventors conducted SA-β-gal assays on anonymized human right atrial appendages (RAA) and left atrial appendages (LAA) that were obtained from Dr. David Van Wagoner (Cleveland Clinic). These samples were obtained during open heart surgery of patients with either sinus rhythm or atrial fibrillation at the time of surgery. The inventors measured the number of senescent cells in these biopsies using SA-β-gal assays. The results showed a wide range of senescent cells (1-8%) in the RAA and LAA. See FIG.8A and FIG.8B. Like in rabbits, a trend for more senescent cells in the RA did not reach statistical significance. There were no differences in the number of senescent cells in these biopsies from aged patients with either atrial fibrillation or sinus rhythm. RNAseq of these samples showed a correlation between left atrium radius and upregulation of senescent markers, suggesting that processes that underlie a higher incidence of atrial fibrillation are associated with increases in senescence.

[0376] The inventors next performed immunofluorescence assays to trace the cell type of senescent cells in human right atrial samples. The results showed a significantAtty. Docket No.: 405002-549001WO difference in the percentage of γH2AX-positive (+) nuclei between atrial fibrillation and SR groups. This trend was also seen in γH2AX-positive nuclei that are desmin+or αSMA+. See FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E. The inventors identified CD68+cells in atrial fibrillation and SR samples with no significant difference. See FIG.9A, FIG.9B, FIG.9C, FIG.9D, and FIG.9E. Atrial fibrillation is associated with a significant increase in oxidative damage to DNA, as showed by an increased abundance of γH2AX-positive nuclei. Histochemistry assays with p16 antibodies showed abundant positive cells in the aged human atria compared to young.. EXAMPLE 3. SENOLYTIC ASSAYS OF AGED RABBITS WITH FISETIN

[0377] The inventors began pharmacologic assays to show that eliminating the senescent cells in aged rabbit atria of aged rabbits and examine the effects of this treatment on cardiac function and specifically on atrial arrhythmias. The inventors chose fisetin as the senolytic drug because there are several ongoing human clinical studies using this drug. The inventors first tested the effectiveness of several short-term protocols for eliminating senescence cells from the rabbit atria. The first protocol included two days of 20 mg / kg of fisetin poi each day and a three-week observation. The second protocol included two weekly cycles of two days a week of 20 mg / kg of fisetin po each day followed by observation for a week and then sacrificing the rabbits after three weeks. See FIG.10A and FIG.10B. The inventors gave the rabbits vehicle as a control. The rabbits underwent ECGs and echocardiograms before starting the protocol and at the end of the protocol. The inventors harvested hearts by beating heart harvest (BHH) and compared cell senescence in the atria of fisetin-treated rabbits to that of control rabbits using an SA-β-gal assay. The result showed that two cycles of fisetin eliminated most of the senescent cells from the left and right atria. One cycle was not enough to eliminate the senescent cells in the LA. See FIG.11A and FIG.11B. The inventors chose protocol B to assay sixteen aged rabbits, out of which eight were fed with fisetin and eight rabbits with vehicle control (protocol B). Five control and five fisetin rabbits were studied by optical mapping. Three vehicle control and three fisetin rabbits were sacrificed for biochemistry. Assessments of the ECG and echo recordings, optical mapping data, and arrhythmogenesis are analyzed. EXAMPLE 4. RELATIONSHIP BETWEEN SENESCENCE, FIBROSIS, INFLAMMATION, AND ATRIAL FIBRILLATION USING CARDIAC ATRIAL TISSUE FROM YOUNG AND AGED RABBITS, HUMANS, AND ENGINEERED HEART TISSUE.

[0378] The results of this EXAMPLE show that the aged rabbit replicates important clinical features of atrial fibrillation in the aged human population. Here we show that aged rabbits have a significant increase in the number of senescent myocytes and myofibroblastsAtty. Docket No.: 405002-549001WO in the atria. RNA-seq showed a group of SASP genes and factors enriched in the aged atria. We also examined human aged atrial tissue. The results show that patients with atrial fibrillation have a significantly higher level of the widely used senescence biomarker of DNA damage γH2AX as compared to the sinus rhythm (SR) group. Besides, SASP factor levels correlate with left atrial diameter, a well-known risk factor for AF. The inventors also examined the senolytic (selective elimination of senescent cells) effects of fisetin as a potential clinical treatment for atrial fibrillation. Fisetin treatment of aged rabbits eliminated nearly all senescent cells from the atria. A pilot assay showed a trend toward reduction in atrial fibrillation incidence and reentrant arrhythmias causing no harmful effect on atrial function, providing a possible clinical tool for future atrial fibrillation prevention.

[0379] The findings of this EXAMPLE show that the aged atria of both humans and rabbits show an accumulation of senescent cells and that rabbit atria express various senescence-associated genes. The work with fisetin suggests that targeting these senescent cells may represent a novel strategy for modulating inflammation and fibrosis and reducing the incidence of atrial fibrillation.EXAMPLE 5. METHOD TESTING

[0380] Various methods are developed and are generally summarized by the following list of example Descriptions.

[0381] Description 1. A method for preventing or treating atrial fibrillation (AF) in a subject, the method comprising the steps of: (1) obtaining a subject in need of a prevention and / or a treating of AF, a subject suspected to be in need of a prevention and / or a treating of AF, or a normal healthy subject without a suspected need for the prevention and / or treating of AF; (2) administering a therapeutically effective amount of Formula 1 or a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one) derivate such as 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one); Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5- methoxychromen-4-one); Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one); Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one); Kaempferide (3,5,7- trihydroxy-2-(4-methoxyphenyl)chromen-4-one); Kaempferol (3,4′,5,7-tetrahydroxy-2- phenylchromen-4-one); Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3- methoxyphenyl)chromen-4-one); Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4- one); Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one); Natsudaidain (2-(3,4- dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4-one); Pachypodol (5-hydroxy- 2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one); Quercetin (3,3′,4′,5,7- pentahydroxy-2-phenylchromen-4-one); Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3- methoxyphenyl)-7-methoxychromen-4-one); Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5- dihydroxy-7-methoxychromen-4-one); Astragalin, Azalein, Hyperoside, Isoquercitin,Atty. Docket No.: 405002-549001WO Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin; or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of Formula 1 or 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, Rhamnetin, Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin to the subject; whereby a risk of the subject acquiring AF or a prevalence of AF in the subject is lowered.

[0382] Description 2. The method of description 1, wherein Formula 1 or the Fisetin derivative is administered in a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis; as measured by the mass of the non-salt and / or non-hydrate / solvate form and the mass of the subject in kg; or in a daily dose of about 100 mg; in a dose administered by a device in contact with the subject; or in a monthly dose.

[0383] Description 3. The method of description 1, wherein the administration decreases a number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in an atrium of the subject.

[0384] Description 4. The method of description 1, further comprising a spatial transcriptomic measurement of a cell composition in the subject, whereby the measurement elucidates the role of different cell types in modulating atrial senescence in the context of human atrial-like engineered heart tissues (a-EHTs).

[0385] Description 5. The method of description 1, further comprising an optical mapping (OM) of a tissue from the subject, whereby the OM is utilized to record and visualize one or more electrophysiological properties in myocardial preparations including Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, or cell culture monolayers, and wherein the OM is capable to reveal a marked reduction in a re-entrant wave. In an example, mapping of the entire left atrium will be performed in sinus rhythm after transseptal access is achieved. Voltage map of the entire left atrium will be performed during sinus rhythm. Bipolar voltages will be recorded initially with a multipolar catheter with 2mm electrode spacing. All bipolar electrograms will be filtered between 30 and 500 Hz. Operators are required to use single point mapping in addition multipolar catheter to validate voltage at any region using contact force sensing catheter for validation, documented contact force of >5g and <25g will be targeted. Mean voltage for the entire left atrium as well as regions of the left atrium (posterior all, anterior wall, septum, floor and lateral wall / appendage) analyzed off-line. Low voltage will be defined as a location of >0.5x0.5cm in area that contains voltage <0.5mV. Low voltage within the wide antral isolation of the pulmonary veins and <0.5cm from a lesion used for vein isolation will be considered a result of the actual lesionAtty. Docket No.: 405002-549001WO and will not be considered a low voltage region. Maps will be saved and sent to a central laboratory and low voltage regions will be analyzed offline in a blinded fashion.

[0386] Description 6. The method of description 1, wherein the method is utilized to prevent AF in an aged population ≥ 50 years old, wherein the method is to prevent atrial fibrillation in subjects with paroxysmal atrial fibrillation; wherein the method is to prevent atrial fibrillation in subjects after radiofrequency ablation; wherein the method is to treat paroxysmal atrial fibrillation, persistent atrial fibrillation, or chronic atrial fibrillation; wherein the method is to prevent atrial fibrillation after an open heart surgery; wherein the method is to prevent AF in obese and / or overweight human subjects; wherein the method is executed to prevent atrial fibrillation in diabetes mellitus subjects; wherein the method is executed to treat and / or prevent AF related to obesity or AF risk due to obesity; wherein the method is executed to prevent a complication of persistent AF like stroke; wherein the method is executed to prevent AF in subjects with hypertension; or wherein the method is executed to prevent AF in subjects with ischemic heart disease, myocardial infarction, or heart failure.

[0387] Description 7. The method of description 1, wherein the method is executed as a combination therapy with one or more therapeutic agents administered simultaneously with Formula 1 and / or a Fisetin derivative or before / after an administration of Formula 1 and / or a Fisetin derivative.

[0388] Description 8. The method of description 1, wherein the subject is a subject in need of prevention of AF, and wherein the subject has one or more risk factors for developing AF, including age over 50 years, obesity, diabetes, hypertension, ischemic heart disease, heart failure, or a history of myocardial infarction.

[0389] Description 9. The method of description 1, wherein the subject is suspected to be in need of prevention of AF based on the presence of one or more symptoms or diagnostic indicators of AF, including palpitations, shortness of breath, chest pain, fatigue, lightheadedness, or an irregular pulse.

[0390] Description 10. The method of description 1, wherein the subject is a normal healthy subject without a suspected need for the prevention and / or treatment of AF, and wherein the method is used prophylactically to reduce the future risk of developing AF in the subject.

[0391] Description 11. The method of description 1, wherein obtaining the subject comprises identifying the subject as requiring or potentially requiring prevention and / or treatment of AF.

[0392] Description 12. The method of description 1, wherein administering the therapeutically effective amount of the Fisetin derivate or the pharmaceutically acceptable form thereof comprises administering the Fisetin derivate or the pharmaceutically acceptable form thereof orally, parenterally, intravenously, intraperitoneally, transdermally,Atty. Docket No.: 405002-549001WO intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation in an amount sufficient to lower the risk of the subject acquiring AF or the prevalence of AF in the subject.

[0393] Description 13. The method of description 1, wherein the Fisetin derivate is Fisetin, and wherein Fisetin is administered in a dose of about 20 mg / kg to about 100 mg / kg daily, or in a dose of about 40 mg / kg to about 80 mg / kg daily, or in a dose of about 60 mg / kg daily, and wherein Fisetin is administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0394] Description 14. The method of description 1, wherein the Fisetin derivate is 3- Hydroxyflavone, and wherein 3-Hydroxyflavone is administered in a dose of about 10 mg / kg to about 50 mg / kg daily, or in a dose of about 20 mg / kg to about 40 mg / kg daily, or in a dose of about 30 mg / kg daily, and wherein 3-Hydroxyflavone is administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0395] Description 15. The method of description 1, wherein the Fisetin derivate is Azaleatin, and wherein Azaleatin is administered in a dose of about 5 mg / kg to about 25 mg / kg daily, or in a dose of about 10 mg / kg to about 20 mg / kg daily, or in a dose of about 15 mg / kg daily, and wherein Azaleatin is administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0396] Description 16. The method of description 1, wherein the Fisetin derivate is Galangin, and wherein Galangin is administered in a dose of about 15 mg / kg to about 75 mg / kg daily, or in a dose of about 30 mg / kg to about 60 mg / kg daily.

[0397] Description 17. The method of description 1, wherein the Fisetin derivate is Gossypetin, and wherein Gossypetin is administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, or in a dose of about 5 mg / kg to about 10 mg / kg daily.

[0398] Description 18. The method of description 1, wherein the Fisetin derivate is Kaempferide, and wherein Kaempferide is administered in a dose of about 25 mg / kg to about 125 mg / kg daily, or in a dose of about 50 mg / kg to about 100 mg / kg daily.

[0399] Description 19. The method of description 1, wherein the Fisetin derivate is Kaempferol, and wherein Kaempferol is administered in a dose of about 30 mg / kg to about 150 mg / kg daily, or in a dose of about 60 mg / kg to about 120 mg / kg daily.

[0400] Description 20. The method of description 1, wherein the risk of the subject acquiring AF or a prevalence of AF in the subject is lowered is measured by a symptom reported by the subject; by a sign perceived by a healthcare provider; by a measurement of a device in contact with the subject and / or implanted with the subject; by an iWatch®; by a FitBit®; or by, for example, a remote measurement device.Atty. Docket No.: 405002-549001WO

[0401] Referring to FIG.25A, in step 100, the prophetic (planned) process begins with obtaining a subject in need of prevention and / or treatment of atrial fibrillation (AF), a subject suspected to be in need of prevention and / or treatment of AF, or a normal healthy subject without a suspected need for the prevention and / or treatment of AF. This step identifies the target population for the method of preventing or treating AF. The subject can be identified as requiring or potentially requiring prevention and / or treatment of AF, which is further elaborated in sub-step 100-a. It is important to note that in FIG.25A, FIG.25B, FIG. 26A, and FIG.26B, we cannot yet do the assays described under 104, 106, and 108 in humans, as we do not have access to the human atrial tissue. Instead, we will measure senescence in circulating white blood cells; paroxysmal Atrial Fibrillation with at least 2 documented episodes on ECG or monitor within the last 6 months. Commercially available home ECG systems (kardia, applewatch, etc.) are acceptable. All tracings used to enroll patients in the study will be reviewed by the ECG core lab prior to enrollment.

[0402] Sub-step 100-a (FIG.25A) involves identifying the subject as requiring or potentially requiring prevention and / or treatment of AF. This identification process ensures that the appropriate individuals are selected for the subsequent treatment steps. The subject may have one or more risk factors for developing AF, including age over 50 years, obesity, diabetes, hypertension, ischemic heart disease, heart failure, or a history of myocardial infarction. These risk factors help in pinpointing individuals who are more likely to benefit from the preventive or therapeutic measures.

[0403] The method aims to lower the risk of the subject acquiring AF or the prevalence of AF in the subject. This is achieved by administering a therapeutically effective amount of a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one) derivative such as 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one); Azaleatin (2-(3,4- dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one); Galangin (3,5,7-trihydroxy-2- phenylchromen-4-one); Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen- 4-one); Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one); Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one); Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy- 3-methoxyphenyl)chromen-4-one); Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen- 4-one); Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one); Natsudaidain (2-(3,4- dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4-one); Pachypodol (5-hydroxy- 2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one); Quercetin (3,3′,4′,5,7- pentahydroxy-2-phenylchromen-4-one); Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3- methoxyphenyl)-7-methoxychromen-4-one); Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5- dihydroxy-7-methoxychromen-4-one); Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin,Atty. Docket No.: 405002-549001WO Icariin, or Troxerutin; or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of these compounds to the subject.

[0404] The administration of these compounds is intended to prevent or treat AF by reducing the number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in the atrium of the subject. This reduction in senescent cells is a mechanism by which the risk or prevalence of AF is lowered.

[0405] In summary, step 100 and its sub-step 100-a focus on identifying and obtaining the appropriate subjects for the method of preventing or treating AF. This involves recognizing individuals with specific risk factors and administering a therapeutically effective amount of Fisetin derivatives to lower the risk or prevalence of AF by targeting senescent cells in the atrium.

[0406] In step 102 (FIG.25A), the method involves administering a therapeutically effective amount of Formula 1 or a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2- phenylchromen-4-one) derivative. The compounds involved in this step include Fisetin, 3- Hydroxyflavone, Azaleatin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, Rhamnetin, Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, and Troxerutin.

[0407] The administration associated with these compounds is administering a therapeutically effective amount to prevent or treat atrial fibrillation (AF). Specifically, the method involves administering a therapeutically effective amount of a small molecule Fisetin or its derivatives to the subject. The purpose of this administration is to prevent or treat AF.

[0408] The specific details of the compounds include the following: Fisetin: Administered in a dose of about 20 mg / kg to about 100 mg / kg daily, or in a dose of about 40 mg / kg to about 80 mg / kg daily, or in a dose of about 60 mg / kg daily. Fisetin can be administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0409] - 3-Hydroxyflavone: Administered in a dose of about 10 mg / kg to about 50 mg / kg daily, or in a dose of about 20 mg / kg to about 40 mg / kg daily, or in a dose of about 30 mg / kg daily.3-Hydroxyflavone can be administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0410] - Azaleatin: Administered in a dose of about 5 mg / kg to about 25 mg / kg daily, or in a dose of about 10 mg / kg to about 20 mg / kg daily, or in a dose of about 15 mg / kg daily. Azaleatin can be administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.Atty. Docket No.: 405002-549001WO

[0411] - Galangin: Administered in a dose of about 15 mg / kg to about 75 mg / kg daily, or in a dose of about 30 mg / kg to about 60 mg / kg daily.

[0412] - Gossypetin: Administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, or in a dose of about 5 mg / kg to about 10 mg / kg daily.

[0413] - Kaempferide: Administered in a dose of about 25 mg / kg to about 125 mg / kg daily, or in a dose of about 50 mg / kg to about 100 mg / kg daily.

[0414] - Kaempferol: Administered in a dose of about 30 mg / kg to about 150 mg / kg daily, or in a dose of about 60 mg / kg to about 120 mg / kg daily.

[0415] The method also includes administering these compounds in a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis. The purpose of administering these specific doses is to determine the effective dosage for preventing or treating AF.

[0416] The overall goal of this step is to administer the appropriate compounds to the subject to lower the risk or prevalence of atrial fibrillation (AF). The method ensures that the subject receives a therapeutically effective amount of the small molecule Fisetin or its derivatives for the prevention or treatment of AF.

[0417] In sub-step 102-a (FIG.26A, FIG.26B), the method involves administering a therapeutically effective amount of specific compounds to a subject. The compounds include Fisetin, 3-Hydroxyflavone, Azaleatin, Galangin, Gossypetin, Kaempferide, and Kaempferol. These compounds are administered in a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis. The purpose of this administration is to determine an effective dosage for preventing or treating atrial fibrillation (AF).

[0418] The administration of a therapeutically effective amount of these compounds is for preventing or treating AF. The specific details of these compounds, such as their chemical structures and therapeutic properties, are for their effectiveness. For instance, Fisetin is administered in a dose of about 20 mg / kg to about 100 mg / kg daily, or in a dose of about 40 mg / kg to about 80 mg / kg daily, or in a dose of about 60 mg / kg daily. Similarly, 3- Hydroxyflavone is administered in a dose of about 10 mg / kg to about 50 mg / kg daily, or in a dose of about 20 mg / kg to about 40 mg / kg daily, or in a dose of about 30 mg / kg daily. Azaleatin is administered in a dose of about 5 mg / kg to about 25 mg / kg daily, or in a dose of about 10 mg / kg to about 20 mg / kg daily, or in a dose of about 15 mg / kg daily. Galangin is administered in a dose of about 15 mg / kg to about 75 mg / kg daily, or in a dose of about 30 mg / kg to about 60 mg / kg daily. Gossypetin is administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, or in a dose of about 5 mg / kg to about 10 mg / kg daily. Kaempferide is administered in a dose of about 25 mg / kg to about 125 mg / kg daily, or in a dose of aboutAtty. Docket No.: 405002-549001WO 50 mg / kg to about 100 mg / kg daily. Kaempferol is administered in a dose of about 30 mg / kg to about 150 mg / kg daily, or in a dose of about 60 mg / kg to about 120 mg / kg daily.

[0419] These compounds are administered in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation. The administration of these compounds in specific doses and forms ensures that the subject receives an effective amount to lower the risk or prevalence of AF. The method aims to administer the appropriate compounds to the subject to lower the risk or prevalence of AF, thereby preventing or treating the condition effectively.

[0420] In sub-step 102-b (FIG.26A), the focus is on administering Fisetin in a dose of about 20 mg / kg to about 100 mg / kg daily. The administration associated with Fisetin includes administering a therapeutically effective amount of Fisetin to the subject to prevent or treat atrial fibrillation (AF). The specific details for Fisetin in this context are highly detailed. Fisetin is administered in a dose of about 20 mg / kg to about 100 mg / kg daily, or in a dose of about 40 mg / kg to about 80 mg / kg daily, or in a dose of about 60 mg / kg daily. The administration routes for Fisetin include oral, parenteral, intravenous, intraperitoneal, transdermal, intramuscular, subcutaneous, intranasal, sublingual, or inhalation. The purpose of administering Fisetin in these specific doses and forms is to lower the risk or prevalence of AF in the subject.

[0421] The method involves administering a therapeutically effective amount of a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one) derivative. This includes administering Fisetin in a dose of about 20 mg / kg to about 100 mg / kg daily, which is for determining the effective dosage to prevent or treat AF. The administration of Fisetin in these specified doses and forms ensures that the subject receives an appropriate amount to achieve the desired therapeutic effect, which is to lower the risk or prevalence of AF.

[0422] The detailed attributes and administration routes highlight the flexibility and precision required in the treatment regimen. By specifying the dose ranges and administration methods, the method ensures that the therapeutic efficacy of Fisetin is maximized while minimizing potential side effects. This comprehensive approach to administering Fisetin underscores the importance of tailored treatment plans in managing and preventing AF in subjects.

[0423] In sub-step 102-c, the focus is on the administration of 3-Hydroxyflavone. Specifically, 3-Hydroxyflavone is administered in a dose of about 10 mg / kg to about 50 mg / kg daily. This administration can be done in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation. The purpose of administering 3-Hydroxyflavone in these specific doses and forms is to ensure a therapeutically effective amount is delivered to the subject to lower the risk or prevalence of atrial fibrillation (AF).Atty. Docket No.: 405002-549001WO

[0424] The administration of a therapeutically effective amount of 3-Hydroxyflavone is for preventing or treating AF. This involves ensuring that the compound is delivered in a manner that maximizes its efficacy while minimizing potential side effects. The specified dose range of 10 mg / kg to 50 mg / kg daily is designed to provide an optimal balance between therapeutic benefits and safety.

[0425] The administration of 3-Hydroxyflavone, along with other small molecule Fisetin derivatives, aims to lower the risk of the subject acquiring AF or reduce the prevalence of AF in the subject. This is achieved by targeting the underlying mechanisms that contribute to AF, such as reducing the number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in the atrium of the subject.

[0426] The method also includes the potential for combination therapy, where 3- Hydroxyflavone can be administered simultaneously with other therapeutic agents or before / after the administration of other compounds. This approach enhances the overall treatment efficacy and provides a comprehensive strategy for managing AF.

[0427] Overall, sub-step 102-c highlights the importance of precise dosing and administration methods for 3-Hydroxyflavone to achieve the desired therapeutic outcomes in preventing or treating atrial fibrillation.

[0428] In sub-step 102-d (FIG.26A), Azaleatin is administered in a dose of about 5 mg / kg to about 25 mg / kg daily, or in a dose of about 10 mg / kg to about 20 mg / kg daily, or in a dose of about 15 mg / kg daily. The administration routes include oral, parenteral, intravenous, intraperitoneal, transdermal, intramuscular, subcutaneous, intranasal, sublingual, or inhalation. This sub-step is part of the broader step 102, which involves administering a therapeutically effective amount of Formula 1 or a small molecule Fisetin derivative to prevent or treat atrial fibrillation (AF) in a subject.

[0429] The administration for Azaleatin is administering a therapeutically effective amount, which is aimed at preventing or treating atrial fibrillation. The administration is described as administering a therapeutically effective amount of a small molecule Fisetin or its derivatives to the subject to prevent or treat AF.

[0430] The specific details for Azaleatin include the dose ranges mentioned above, which are for determining the effective dosage for preventing or treating AF. The administration reiterates the administration of a therapeutically effective amount of Azaleatin, among other derivatives, to the subject.

[0431] Implicitly, the process involves identifying (e.g., FIG.25A) the subject in need of AF prevention or treatment, preparing the appropriate dosage form of Azaleatin, and selecting the suitable administration route based on the subject's condition and preferences. The logical flow ensures that the subject receives the correct dosage to achieve the desired therapeutic effect, thereby lowering the risk or prevalence of AF.Atty. Docket No.: 405002-549001WO

[0432] In step 102-e (FIG.26B), Galangin is administered in a dose of about 15 mg / kg to about 75 mg / kg daily. The administration of a therapeutically effective amount of Galangin is aimed at preventing or treating atrial fibrillation (AF). The specific details of Galangin include its administration in a dose range of about 15 mg / kg to about 75 mg / kg daily, or in a dose of about 30 mg / kg to about 60 mg / kg daily. This administration is part of a broader method that involves administering a therapeutically effective amount of a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one) derivative, such as Galangin, to the subject. The purpose of this administration is to lower the risk or prevalence of AF in the subject.

[0433] The method for preventing or treating AF involves obtaining a subject in need of prevention and / or treatment of AF, a subject suspected to be in need of prevention and / or treatment of AF, or a normal healthy subject without a suspected need for the prevention and / or treatment of AF. The administration of Galangin, as part of this method, is intended to be therapeutically effective in reducing the risk or prevalence of AF in the subject. The administration can be carried out in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0434] The overall goal of this method is to provide an approach to managing AF by utilizing specific doses of Galangin and other small molecule Fisetin derivatives to achieve a therapeutic effect. This method is designed to be flexible in terms of dosage and administration routes, ensuring that it can be tailored to the needs of individual subjects to effectively prevent or treat AF.

[0435] In sub-step 102-f (FIG.26B), Gossypetin is administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, or in a dose of about 5 mg / kg to about 10 mg / kg daily. The administration of Gossypetin, a small molecule Fisetin derivative, is aimed at preventing or treating atrial fibrillation (AF) in a subject. The method involves administering a therapeutically effective amount of Gossypetin to the subject to lower the risk or prevalence of AF. The specific dosage range for Gossypetin is to ensure its efficacy in reducing the incidence of AF. This administration can be done in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation, ensuring flexibility in the method of delivery to suit different patient needs and conditions. The goal is to achieve a therapeutically effective amount that is sufficient to lower the risk of the subject acquiring AF or the prevalence of AF in the subject.

[0436] In step 102-g, Kaempferide is administered in a dose of about 25 mg / kg to about 125 mg / kg daily, or in a dose of about 50 mg / kg to about 100 mg / kg daily. The administration of Kaempferide is part of a broader method aimed at preventing or treatingAtty. Docket No.: 405002-549001WO atrial fibrillation (AF) in a subject. This method involves administering a therapeutically effective amount of a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one) derivative, which includes Kaempferide among other compounds.

[0437] The administration of Kaempferide is intended to prevent or treat AF by lowering the risk or prevalence of AF in the subject. The specific details of Kaempferide administration include the dosage range, which is for determining the effective amount needed to achieve the desired therapeutic effect. The method ensures that the subject receives an appropriate dose to lower the risk or prevalence of AF effectively.

[0438] The administration of Kaempferide, as well as other small molecule Fisetin derivatives, is carried out in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation. This flexibility in administration routes allows for tailored treatment plans based on the subject's specific needs and conditions.

[0439] Overall, the method described in step 102-g is part of an approach to managing AF, utilizing specific dosages and administration routes to ensure the therapeutic effectiveness of Kaempferide and other related compounds.

[0440] In step 102-h (FIG.26B), Kaempferol is administered in a dose of about 30 mg / kg to about 150 mg / kg daily, or in a dose of about 60 mg / kg to about 120 mg / kg daily. The administration of Kaempferol is aimed at preventing or treating atrial fibrillation (AF) in a subject. The method involves administering a therapeutically effective amount of Kaempferol, which is a small molecule Fisetin derivative, to the subject. The purpose of this administration is to lower the risk or prevalence of AF in the subject.

[0441] The specific details of Kaempferol include its administration in a dose range of about 30 mg / kg to about 150 mg / kg daily, or about 60 mg / kg to about 120 mg / kg daily. This administration can be carried out in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation. The goal is to ensure that the subject receives a therapeutically effective amount of Kaempferol to achieve the desired outcome of preventing or treating AF.

[0442] The method emphasizes the importance of administering a therapeutically effective amount of a small molecule Fisetin derivative, such as Kaempferol, to the subject. This administration is for lowering the risk or prevalence of AF in the subject. The detailed dosage ranges and forms of administration provided ensure that the method can be effectively implemented to achieve the desired therapeutic outcomes.

[0443] In step 104 (FIG.25A), the administration decreases a number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in an atrium of the subject. The primary components involved in this step are senescent atrial myocytes andAtty. Docket No.: 405002-549001WO senescent atrial myofibroblasts. The administration associated with these components is to decrease their number, which is for reducing the senescence in the atrium and thereby potentially mitigating the risk or severity of atrial fibrillation (AF).

[0444] The administration of decreasing the number of senescent atrial myocytes and senescent atrial myofibroblasts is aimed at reducing senescent cells. This reduction is significant because senescent cells contribute to the aging and dysfunction of cardiac tissues, which can exacerbate conditions like AF. By targeting these cells, the method aims to improve the overall health and function of the atrial tissue.

[0445] The specific details related to this step include the purpose of reducing the number of senescent cells in the atrium. This is achieved through the administration of a therapeutically effective amount of small molecule Fisetin or its derivatives, such as 3- Hydroxyflavone, Azaleatin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, Rhamnetin, Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin. These compounds are administered in various forms, including pharmaceutically acceptable salts, hydrates, and solvates, to ensure their efficacy in reducing senescent cells.

[0446] The method involves administering these compounds in specific doses to achieve the desired therapeutic effect. For instance, Fisetin is administered in a dose of about 20 mg / kg to about 100 mg / kg daily, or in a dose of about 40 mg / kg to about 80 mg / kg daily, or in a dose of about 60 mg / kg daily. Similarly, 3-Hydroxyflavone is administered in a dose of about 10 mg / kg to about 50 mg / kg daily, or in a dose of about 20 mg / kg to about 40 mg / kg daily, or in a dose of about 30 mg / kg daily. Azaleatin is administered in a dose of about 5 mg / kg to about 25 mg / kg daily, or in a dose of about 10 mg / kg to about 20 mg / kg daily, or in a dose of about 15 mg / kg daily. Galangin is administered in a dose of about 15 mg / kg to about 75 mg / kg daily, or in a dose of about 30 mg / kg to about 60 mg / kg daily. Gossypetin is administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, or in a dose of about 5 mg / kg to about 10 mg / kg daily. Kaempferide is administered in a dose of about 25 mg / kg to about 125 mg / kg daily, or in a dose of about 50 mg / kg to about 100 mg / kg daily. Kaempferol is administered in a dose of about 30 mg / kg to about 150 mg / kg daily, or in a dose of about 60 mg / kg to about 120 mg / kg daily.

[0447] The administration of these compounds is intended to lower the risk or prevalence of AF by reducing the number of senescent cells in the atrium, thereby improving the overall health and function of the cardiac tissue. This step is in the method for preventing or treating AF, as it directly targets the cellular mechanisms that contribute to the development and progression of the condition.Atty. Docket No.: 405002-549001WO

[0448] In step 106 (FIG.25A), the method further comprises a spatial transcriptomic measurement of a cell composition in the subject. This measurement elucidates the role of different cell types in modulating atrial senescence in the context of human atrial-like engineered heart tissues (a-EHTs). The spatial transcriptomic measurement is for understanding the cellular landscape and how various cell types contribute to the aging process within the atrium. This step involves advanced techniques to map the spatial distribution of gene expression within the tissue, providing insights into the cellular mechanisms underlying atrial fibrillation (AF).

[0449] The spatial transcriptomic measurement is performed to elucidate the role of different cell types in modulating atrial senescence. This involves analyzing the spatial arrangement and gene expression profiles of cells within the atrial tissue, which can reveal how specific cell types contribute to the development and progression of AF. By understanding these cellular interactions, researchers can identify potential targets for therapeutic intervention and develop more effective treatments for AF.

[0450] In the context of human atrial-like engineered heart tissues (a-EHTs), this measurement is particularly valuable. a-EHTs are laboratory-grown tissues that mimic the structure and function of the human atrium, providing a controlled environment for studying AF. The spatial transcriptomic measurement in a-EHTs allows researchers to observe how different cell types interact and influence each other in a setting that closely resembles the human heart. This can lead to the identification of key cellular players in atrial senescence and the development of targeted therapies to prevent or treat AF.

[0451] Overall, step 106 highlights the importance of spatial transcriptomic measurement in advancing our understanding of atrial senescence and improving the treatment of AF. By elucidating the role of different cell types in modulating atrial senescence, this step provides insights that can inform the development of more effective therapeutic strategies.

[0452] In step 108 (FIG.25A), the method involves an optical mapping (OM) of a tissue from the subject. The OM is utilized to record and visualize one or more electrophysiological properties in myocardial preparations, including Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, or cell culture monolayers. The OM is capable of revealing a marked reduction in a re-entrant wave.

[0453] Optical mapping (OM) is a technique used to record and visualize electrophysiological properties in various myocardial preparations. This method is particularly useful in understanding the electrical activity of the heart and identifying abnormalities such as re-entrant waves, which are often associated with arrhythmias like atrial fibrillation (AF). The Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, and cell culture monolayers are specific types of myocardial preparations thatAtty. Docket No.: 405002-549001WO provide different levels of detail and context for the OM. Langendorff-perfused isolated hearts allow for the study of the whole heart in a controlled environment, while coronary- perfused wedge preparations focus on specific regions of the heart, and cell culture monolayers provide a simplified model for cellular-level studies.

[0454] The primary purpose of using OM in this context is to record and visualize electrophysiological properties, which can help in understanding the electrical behavior of the heart tissue under different conditions. This technique is particularly valuable in research and clinical settings for diagnosing and studying the mechanisms of AF. By revealing a marked reduction in a re-entrant wave, OM can provide insights into the effectiveness of treatments aimed at preventing or treating AF. This reduction indicates a stabilization of the heart's electrical activity, which is a positive outcome in the management of AF.

[0455] In summary, step 108 involves the use of optical mapping to record and visualize electrophysiological properties in myocardial preparations, with the goal of revealing a marked reduction in re-entrant waves, thereby contributing to the understanding and treatment of atrial fibrillation.

[0456] In step 110 (FIG.25A), the method is utilized to prevent atrial fibrillation (AF) in various populations and conditions. This step encompasses several sub-steps, each targeting a different group or condition associated with AF. The method is designed to prevent AF in an aged population ≥ 50 years old, as the risk of AF increases with age. The method also aims to prevent atrial fibrillation in subjects with paroxysmal atrial fibrillation, a condition characterized by sudden, irregular heartbeats that can lead to more severe forms of AF if not managed properly.

[0457] Additionally, the method is intended to prevent atrial fibrillation in subjects after radiofrequency ablation, a common procedure used to treat AF by destroying the heart tissue causing the irregular heartbeat. This preventive measure is to avoid recurrence of AF post-procedure. The method also addresses the treatment of paroxysmal atrial fibrillation, persistent atrial fibrillation, or chronic atrial fibrillation, providing an approach to managing different stages and severities of AF.

[0458] For patients undergoing open heart surgery, the method aims to prevent atrial fibrillation, a common complication post-surgery that can lead to increased morbidity and prolonged hospital stays. The method is also tailored to prevent AF in obese and / or overweight human subjects, recognizing the link between obesity and increased AF risk. Similarly, it targets diabetes mellitus subjects, as diabetes is a significant risk factor for developing AF.

[0459] The method is executed to treat and / or prevent AF related to obesity or AF risk due to obesity, addressing the metabolic and inflammatory changes associated with obesity that contribute to AF. It also aims to prevent complications of persistent AF, such asAtty. Docket No.: 405002-549001WO stroke, which is a severe and life-threatening consequence of untreated or poorly managed AF.

[0460] Furthermore, the method is designed to prevent AF in subjects with hypertension, another major risk factor for AF, and in subjects with ischemic heart disease, myocardial infarction, or heart failure, conditions that significantly increase the risk of developing AF. By targeting these populations and conditions, the method provides an approach to preventing and managing AF, thereby reducing the overall burden of this arrhythmia on patients and healthcare systems.

[0461] In step 112 (FIG.25B), the method is executed as a combination therapy with one or more therapeutic agents administered simultaneously with Formula 1 and / or a Fisetin derivative or before / after an administration of Formula 1 and / or a Fisetin derivative. The primary components involved in this step are therapeutic agents, Formula 1, and Fisetin derivative.

[0462] The administration associated with these components involves administering a therapeutically effective amount of a small molecule Fisetin or its derivatives to the subject. This administration is aimed at preventing or treating atrial fibrillation (AF). The specific details of the Fisetin derivative include a therapeutically effective amount, which is for lowering the risk or prevalence of AF in the subject. The Fisetin derivative can be administered in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0463] The combination therapy approach enhances the treatment efficacy by utilizing multiple therapeutic agents. This method ensures that the subject receives a treatment regimen that targets AF from multiple angles, potentially increasing the overall effectiveness of the therapy. The combination therapy can be tailored to the specific needs of the subject, considering factors such as the severity of AF, the presence of comorbidities, and the subject's response to previous treatments.

[0464] In summary, step 112 involves a combination therapy approach where therapeutic agents are administered alongside Formula 1 and / or a Fisetin derivative. This method aims to enhance the treatment efficacy for preventing or treating AF by utilizing a therapeutically effective amount of the Fisetin derivative in various forms. The combination therapy is designed to lower the risk or prevalence of AF in the subject, providing a treatment regimen.

[0465] In step 114 (FIG.25B), the subject is identified as a subject in need of prevention of atrial fibrillation (AF), and the subject has one or more risk factors for developing AF, including age over 50 years, obesity, diabetes, hypertension, ischemic heart disease, heart failure, or a history of myocardial infarction. The method involves obtaining a subject in need of a prevention and / or a treating of AF, a subject suspected to be in need ofAtty. Docket No.: 405002-549001WO a prevention and / or a treating of AF, or a normal healthy subject without a suspected need for the prevention and / or treating of AF. The purpose of this step is to identify the target population for the method of preventing or treating atrial fibrillation (AF).

[0466] The subject is administered a therapeutically effective amount of a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one) derivate such as 3- Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one); Azaleatin (2-(3,4-dihydroxyphenyl)-3,7- dihydroxy-5-methoxychromen-4-one); Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one); Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one); Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one); Kaempferol (3,4′,5,7-tetrahydroxy-2- phenylchromen-4-one); Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3- methoxyphenyl)chromen-4-one); Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4- one); Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one); Natsudaidain (2-(3,4- dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4-one); Pachypodol (5-hydroxy- 2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one); Quercetin (3,3′,4′,5,7- pentahydroxy-2-phenylchromen-4-one); Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3- methoxyphenyl)-7-methoxychromen-4-one); Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5- dihydroxy-7-methoxychromen-4-one); Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin; or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, Rhamnetin, Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin to the subject. The purpose of administering these compounds is to lower the risk or prevalence of atrial fibrillation (AF) in the subject.

[0467] The administration of these compounds is done in a therapeutically effective amount, which is defined as a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis. For specific compounds, the doses are as follows: Fisetin is administered in a dose of about 20 mg / kg to about 100 mg / kg daily, 3-Hydroxyflavone is administered in a dose of about 10 mg / kg to about 50 mg / kg daily, Azaleatin is administered in a dose of about 5 mg / kg to about 25 mg / kg daily, Galangin is administered in a dose of about 15 mg / kg to about 75 mg / kg daily, Gossypetin is administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, Kaempferide is administered in a dose of about 25 mg / kg to about 125 mg / kg daily, and Kaempferol is administered in a dose of about 30 mg / kg to about 150 mg / kg daily. These compounds canAtty. Docket No.: 405002-549001WO be administered orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0468] The method aims to prevent or treat atrial fibrillation (AF) by targeting subjects with specific risk factors and administering appropriate doses of small molecule Fisetin derivatives to lower the risk or prevalence of AF.

[0469] In step 116, the subject is suspected to be in need of prevention of atrial fibrillation (AF) based on the presence of one or more symptoms or diagnostic indicators of AF, including palpitations, shortness of breath, chest pain, fatigue, lightheadedness, or an irregular pulse. The method involves identifying the subject as requiring or potentially requiring prevention and / or treatment of AF. This identification is for targeting the appropriate population for the method of preventing or treating AF.

[0470] The subject is identified based on the presence of specific symptoms or diagnostic indicators. These symptoms include palpitations, shortness of breath, chest pain, fatigue, lightheadedness, or an irregular pulse. These indicators are for determining the need for AF prevention and ensuring that the method is applied to those who are most likely to benefit from it.

[0471] The method involves administering a therapeutically effective amount of a small molecule Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one) derivative such as 3- Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one); Azaleatin (2-(3,4-dihydroxyphenyl)-3,7- dihydroxy-5-methoxychromen-4-one); Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one); Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one); Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one); Kaempferol (3,4′,5,7-tetrahydroxy-2- phenylchromen-4-one); Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3- methoxyphenyl)chromen-4-one); Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4- one); Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one); Natsudaidain (2-(3,4- dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4-one); Pachypodol (5-hydroxy- 2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one); Quercetin (3,3′,4′,5,7- pentahydroxy-2-phenylchromen-4-one); Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3- methoxyphenyl)-7-methoxychromen-4-one); Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5- dihydroxy-7-methoxychromen-4-one); Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin; or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of these compounds to the subject. The purpose of administering these compounds is to lower the risk of the subject acquiring AF or to reduce the prevalence of AF in the subject.

[0472] The method ensures that the subject receives a therapeutically effective amount of these compounds, which is for achieving the desired outcome of preventing orAtty. Docket No.: 405002-549001WO treating AF. The administration of these compounds can be done in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation. The specific doses and forms of administration are tailored to ensure the effectiveness of the treatment.

[0473] Overall, step 116 involves an approach to identifying and treating subjects at risk of AF by administering specific compounds in therapeutically effective amounts to lower the risk or prevalence of AF.

[0474] In step 118 (FIG.25B), the focus is on the subject, specifically a normal healthy subject without a suspected need for the prevention and / or treatment of atrial fibrillation (AF). The method is used prophylactically to reduce the future risk of developing AF in the subject. The administration involves obtaining a subject who is a normal healthy individual and administering a therapeutically effective amount of a small molecule Fisetin or its derivatives to the subject. The purpose of this administration is to prevent or treat AF.

[0475] The components involved in this step include the subject and the normal healthy subject. The administration of a therapeutically effective amount of a small molecule Fisetin or its derivatives to the subject is for this step. This includes compounds such as 3- Hydroxyflavone, Azaleatin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, Rhamnetin, Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin. These compounds can be administered in various forms, including orally, parenterally, intravenously, intraperitoneally, transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by inhalation.

[0476] The specific details of the administration include a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis. For example, Fisetin can be administered in a dose of about 20 mg / kg to about 100 mg / kg daily, or in a dose of about 40 mg / kg to about 80 mg / kg daily, or in a dose of about 60 mg / kg daily. Similarly, 3-Hydroxyflavone can be administered in a dose of about 10 mg / kg to about 50 mg / kg daily, or in a dose of about 20 mg / kg to about 40 mg / kg daily, or in a dose of about 30 mg / kg daily. Azaleatin can be administered in a dose of about 5 mg / kg to about 25 mg / kg daily, or in a dose of about 10 mg / kg to about 20 mg / kg daily, or in a dose of about 15 mg / kg daily. Galangin can be administered in a dose of about 15 mg / kg to about 75 mg / kg daily, or in a dose of about 30 mg / kg to about 60 mg / kg daily. Gossypetin can be administered in a dose of about 2.5 mg / kg to about 12.5 mg / kg daily, or in a dose of about 5 mg / kg to about 10 mg / kg daily. Kaempferide can be administered in a dose of about 25 mg / kg to about 125 mg / kg daily, or in a dose of about 50 mg / kg to about 100 mg / kg daily. Kaempferol can be administered in a dose of about 30 mg / kg to about 150 mg / kg daily, or in a dose of about 60 mg / kg to about 120 mg / kg daily.Atty. Docket No.: 405002-549001WO

[0477] The method aims to lower the risk of the subject acquiring AF or the prevalence of AF in the subject. This is measured by a symptom reported by the subject, a sign perceived by a healthcare provider, or a measurem...

Claims

Atty. Docket No.: 405002-549001WO CLAIMS We claim:

1. A method for preventing or treating atrial fibrillation (AF) in a subject, the method comprising the steps of: (1) obtaining a subject in need of a prevention and / or a treating of AF, a subject suspected to be in need of a prevention and / or a treating of AF, or a normal healthy subject without a suspected need for the prevention and / or treating of AF; (2) administering a therapeutically effective amount of a compound of formula 1, a pharmaceutically acceptable salt of formula 1, and / or a pharmaceutically acceptable hydrate and / or solvate and / or a prodrug of formula 1 to the subject:, Formula 1; wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms as indicated in Formula 1; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl; whereby a risk of the subject acquiring AF or a prevalence of AF in the subject is lowered.

2. The method of claim 1, wherein Formula 1 further comprises a tautomer and / or an isomer thereof wherein atom 9 is a tautomeric -OH or atom 10 is a tautomeric =O as is represented by Formula 1 Tautomer below:Atty. Docket No.: 405002-549001WO, Formula 1 Tautomer; wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl.

3. The method of claim 1, wherein Formula 1 comprises one of the following compounds:, 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one);, Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one);, Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one);, Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one);, Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one);, Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one);, Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Natsudaidain (2-(3,4-dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4-one);, Pachypodol (5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one);, Quercetin (3,3′,4′,5,7-pentahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxychromen-4-one);, Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxychromen-4-one); or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, or Rhamnetin.

4. The method of claim 1, further comprising atom 10 is replaced by a glycoside.

5. The method of claim 1, further comprising Formula 1 comprises Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin.

6. The method of claim 1, wherein the risk of the subject acquiring AF or a prevalence of AF in the subject is lowered is measured by a symptom reported by the subject; by a sign perceived by a healthcare provider; by a measurement of a device in contact with the subjectAtty. Docket No.: 405002-549001WO and / or implanted with the subject; by an iWatch®; by a FitBit®; or by a remote measurement device.

7. The method of claim 1, whereby a vulnerability of the subject acquiring AF or a prevalence of AF in the subject is lowered.

8. The method of claim 1, wherein Formula 1 is administered in a dose in the range from about 1 mg / kg to about 100 mg / kg on a daily, weekly, or 20 mg / kg for 1 or 2 days on a monthly basis; as measured by the mass of the non-salt and / or non-hydrate / solvate form and the mass of the subject in kg; or in a daily dose of about 100 mg; in a dose administered by a device in contact with the subject; or in a monthly dose.

9. The method of claim 1, wherein the administering is executed for a time period of longer than one year.

10. The method of claim 1, wherein the administration decreases a number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in an atrium of the subject.

11. The method of claim 10, wherein one or more persistent SAMs and / or SAMFs increase an arrhythmic propensity with an aging of the subject, and wherein the decreases of claim 10 is measured as a comparison to the number of the SAMs and / or SAMFs that would be measured in the same subject without the administering during the aging of the subject.

12. The method of claim 1, further comprising a spatial transcriptomic measurement of a cell composition in the subject, whereby the measurement elucidates the role of different cell types in modulating atrial senescence in the context of human atrial-like engineered heart tissues (a-EHTs).

13. The method of claim 1, whereby the method prevents an accumulation of senescent cells in the atria.

14. The method of claim 1, wherein the method is executed including a monthly dose of Formula 1 to the subject.

15. The method of claim 1, whereby the method reduces an incidence and / or a duration of spontaneous AF episodes.Atty. Docket No.: 405002-549001WO 16. The method of claim 1, further comprising an optical mapping (OM) of a tissue from the subject, whereby the OM is utilized to record and visualize one or more electrophysiological properties in myocardial preparations including Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, or cell culture monolayers, and wherein the OM is capable to reveal a marked reduction in a re-entrant wave.

17. The method of claim 1, further comprising the method is executed for a time period of longer than five years, and a fibrosis of the atrium is reduced compared to the same atrium of the same subject without the method of claim 1.

18. The method of claim 1, wherein the method prevents a disease of aging, an atrial senescence of a myofibroblast, and atrial senescence of a myocyte, an inflammation, or a fibrosis.

19. The method of claim 1, wherein the administration is a senolytic therapy that is an administration of Formula 1 to a person in need thereof.

20. The method of claim 19, wherein the method is utilized to prevent AF in an aged population ≥ 50 years old, wherein the method is to prevent atrial fibrillation in subjects with paroxysmal atrial fibrillation; wherein the method is to prevent atrial fibrillation in subjects after radiofrequency ablation; wherein the method is to treat paroxysmal atrial fibrillation, persistent atrial fibrillation, or chronic atrial fibrillation; wherein the method is to prevent atrial fibrillation after an open heart surgery; wherein the method is to prevent AF in obese and / or overweight human subjects; wherein the method is executed to prevent atrial fibrillation in diabetes mellitus subjects; wherein the method is executed to treat and / or prevent AF related to obesity or AF risk due to obesity; wherein the method is executed to prevent a complication of persistent AF like stroke; wherein the method is executed to prevent AF in subjects with hypertension; or wherein the method is executed to prevent AF in subjects with ischemic heart disease, myocardial infarction, or heart failure.

21. The method of claim 1, wherein the method is executed as a combination therapy with one or more therapeutic agents administered simultaneously with Formula 1 or before / after an administration of Formula 1.

22. The method of claim 1, wherein the method is utilized for treating AF in an aging population.Atty. Docket No.: 405002-549001WO 23. A drug screening platform for measuring one or more effects of a proposed therapeutic agent (candidate) on AF, the platform comprising a heart cell, tissue, or a human atrial-like engineered heart tissue (a-EHTs) and a compound of formula 1, a pharmaceutically acceptable salt of formula 1, and / or a pharmaceutically acceptable hydrate and / or solvate of formula 1:, Formula 1; wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms as indicated in Formula 1; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl; whereby a screening of the candidate for a risk of AF or a prevalence of AF in the cell or tissue in the platform is enabled.

24. The drug screening platform of claim 23, further comprising a control unit including the heart cell or the a-EHTs without the presence of Formula 1; whereby the control unit enables a testing of the candidate on the same cell or a-EHTs without the presence of Formula 1.

25. The drug screening platform of claim 23, wherein Formula 1 further comprises a tautomer and / or an isomer thereof wherein atom 9 is a tautomeric -OH or atom 10 is a tautomeric =O as is represented by Formula 1 Tautomer below:Atty. Docket No.: 405002-549001WO, Formula 1 Tautomer; wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl.

26. The drug screening platform of claim 23, wherein Formula 1 comprises one of the following compounds:, 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one);, Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one);, Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one);, Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one);, Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one);, Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one);, Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Natsudaidain (2-(3,4-dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4-one);, Pachypodol (5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one);, Quercetin (3,3′,4′,5,7-pentahydroxy-2-phenylchromen-4-one);Atty. Docket No.: 405002-549001WO, Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxychromen-4-one);, Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxychromen-4-one); or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin, Rhamnazin, or Rhamnetin.

27. The drug screening platform of claim 23, further comprising atom 10 is replaced by a glycoside.

28. The drug screening platform of claim 23, further comprising Formula 1 comprises Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin.

29. The drug screening platform of claim 23, wherein the risk an AF or a prevalence of AF in the cell, tissue, or a-EHTs is measured by one or more analytical methods performed on theAtty. Docket No.: 405002-549001WO platform.

30. The drug screening platform of claim 23, wherein the candidate decreases a number of senescent atrial myocytes (SAMs) and / or senescent atrial myofibroblasts (SAMFs) in a cell, tissue, or a-EHTs of the platform.

31. The drug screening platform of claim 23, further comprising a spatial transcriptomic measurement of a cell composition in the subject, whereby the measurement elucidates the role of different cell types in modulating atrial senescence in the context of the cell, tissue, or human atrial-like engineered heart tissues (a-EHTs).

32. The drug screening platform of claim 23, further comprising an optical mapping (OM) of a tissue from the subject, whereby the OM is utilized to record and visualize the electrophysiological properties in a variety of myocardial preparations such as Langendorff- perfused isolated hearts, coronary-perfused wedge preparations, and cell culture monolayers will reveal marked reduction in reentrant waves.

33. The drug screening platform of claim 23, wherein the screening is executed as a combination therapy with one or more therapeutic agents administered simultaneously with Formula 1 or before / after an administration of Formula 1.

34. A supplement for preventing or treating atrial fibrillation (AF) in a subject, the supplement comprising: a therapeutically effective amount of a compound of formula 1, a pharmaceutically acceptable salt of formula 1, and / or a pharmaceutically acceptable hydrate and / or solvate of formula 1 to the subject:Atty. Docket No.: 405002-549001WO, Formula 1; wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms as indicated in Formula 1; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl; wherein the supplement is provided in a pharmaceutical formulation.

35. The supplement of claim 34, wherein Formula 1 further comprises a tautomer and / or an isomer thereof wherein atom 9 is a tautomeric -OH or atom 10 is a tautomeric =O as is represented by Formula 1 Tautomer below:, Formula 1 Tautomer;Atty. Docket No.: 405002-549001WO wherein 1, 2, 3, 4, 4a, 9, 10, and 1* are each atoms; wherein 5, 6, 7, 8, 2*, 3*, 4*, 5*, and 6* are each independently a substituent attached by a single chemical, covalent bond to Formula 1; wherein each substituent independently comprises: -H, -OCH3, -OH; -I, -Br, -F, or Cl.

36. The supplement of claim 34, wherein Formula 1 comprises one of the following compounds:, 3-Hydroxyflavone (3-hydroxy-2-phenylchromen-4-one);, Azaleatin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-5-methoxychromen-4-one);,Atty. Docket No.: 405002-549001WO Fisetin (3,3′,4′,7-tetrahydroxy-2-phenylchromen-4-one);, Galangin (3,5,7-trihydroxy-2-phenylchromen-4-one);, Gossypetin (2-(3,4-dihydroxyphenyl)-3,5,7,8-tetrahydroxychromen-4-one);,Atty. Docket No.: 405002-549001WO Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one);, Kaempferol (3,4′,5,7-tetrahydroxy-2-phenylchromen-4-one);, Isorhamnetin (3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one);, Morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one);Atty. Docket No.: 405002-549001WO, Myricetin (3,3′,4′,5′,5,7-hexahydroxy-2-phenylchromen-4-one);, Natsudaidain (2-(3,4-dimethoxyphenyl)-3-hydroxy-5,6,7,8-tetramethoxychromen-4-one);, Pachypodol (5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxychromen-4-one);Atty. Docket No.: 405002-549001WO, Quercetin (3,3′,4′,5,7-pentahydroxy-2-phenylchromen-4-one);, Rhamnazin (3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxychromen-4-one);, Rhamnetin (2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxychromen-4-one); or a pharmaceutically acceptable salt of and / or a pharmaceutically acceptable hydrate and / or solvate of 3-Hydroxyflavone, Azaleatin, Fisetin, Galangin, Gossypetin, Kaempferide, Kaempferol, Isorhamnetin, Morin, Myricetin, Natsudaidain, Pachypodol, Quercetin,Atty. Docket No.: 405002-549001WO Rhamnazin, or Rhamnetin.

37. The supplement of claim 34, further comprising atom 10 is replaced by a glycoside.

38. The supplement of claim 34, further comprising Formula 1 comprises Astragalin, Azalein, Hyperoside, Isoquercitin, Kaempferitrin, Myricitrin, Quercitrin, Robinin, Rutin, Spiraeoside, Xanthorhamnin, Amurensin, Icariin, or Troxerutin.