Use of retinoids for the treatment of atrial fibrillation.

Abstract

The present invention relates to methods of treating atrial fibrillation or preventing or reversing atrial remodeling, comprising administering a retinoid, such as all-trans retinoic acid (ATRA).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 306,955, filed February 4, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Technical field]

[0002] The present invention relates to methods for treating atrial fibrillation or preventing or reversing atrial remodeling, comprising administering a retinoid, such as, for example, all-trans retinoic acid (ATRA). [Background technology]

[0003] Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice, affecting an estimated 2.7 to 6.1 million people in the United States alone. 2、3 (Non-Patent Documents 1, 2). Atrial fibrillation adversely affects all cardiovascular outcomes and increases the risk of stroke, morbidity, mortality, and hospitalization. 4、5 (Non-Patent Documents 3, 4) The annual cost of hospitalization for atrial fibrillation in the United States is estimated at $6.65 billion per year. 7 (Non-Patent Document 5). The two main treatment options for rhythm control (sinus rhythm maintenance) of atrial fibrillation (AF) are antiarrhythmic drugs (AADs) and catheter ablation therapy. AADs are less effective in maintaining sinus rhythm and have fatal proarrhythmic side effects. Catheter ablation is more effective in maintaining sinus rhythm, but the procedure is invasive and has the potential for major adverse events. 9-11 (Non-Patent Documents 6 to 8). Importantly, none of the treatments address the underlying left atrial myopathy that causes atrial fibrillation (AF) and promotes its progression to persistent AF. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Morillo, CA, Banerjee, A., Perel, P., Wood, D. & Jouven, X. Atrial fibrillation: the current epidemic. J Geriatr Cardiol 14, 195-203 (2017). [Non-Patent Document 2] January, CT, et al. 2014 AHA / ACC / HRS guideline for the management of patients with atrial fibrillation: a report of the American College of Cardiology / American Heart Association Task Force on Practice Guidelines and the Heart Rhythm Society. J Am Coll Cardiol 64, e1-76 (2014). [Non-Patent Document 3] Marrouche, NF, et al. Catheter Ablation for Atrial Fibrillation with Heart Failure. N Engl J Med 378, 417-427 (2018). [Non-Patent Document 4] Bhat, A., et al. Drivers of hospitalization in atrial fibrillation: A contemporary review. Heart Rhythm 17, 1991-1999 (2020). [Non-Patent Document 5] Wolowacz, SE, Samuel, M., Brennan, VK, Jasso-Mosqueda, JG & Van Gelder, IC The cost of illness of atrial fibrillation: a systematic review of the recent literature. Europace 13, 1375-1385 (2011). [Non-Patent Document 6] Singh, BN, et al. Amiodarone versus sotalol for atrial fibrillation. N Engl J Med 352, 1861-1872 (2005). [Non-Patent Document 7] Kochiadakis, GE, et al. Amiodarone, sotalol, or propafenone in atrial fibrillation: which is preferred to maintain normal sinus rhythm? Pacing Clin Electrophysiol 23, 1883-1887 (2000). [Non-Patent Document 8] Packer, DL, et al. Effect of Catheter Ablation vs Antiarrhythmic Drug Therapy on Mortality, Stroke, Bleeding, and Cardiac Arrest Among Patients With Atrial Fibrillation: The CABANA Randomized Clinical Trial. JAMA 321, 1261-1274 (2019). Summary of the Invention [Means for solving the problem]

[0005] In one aspect, the present invention relates to a method for treating atrial fibrillation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a retinoid or a pharma- ceutically acceptable salt thereof, hi certain embodiments, the subject has been determined to be deficient in said retinoid.

[0006] In another aspect, the present invention relates to a method for treating atrial fibrillation in a subject in need thereof, comprising the steps of: a) determining a level of a retinoid in a serum sample obtained from the subject; b) comparing the level of the retinoid determined in step (a) with a control level of said retinoid; and c) administering a therapeutically effective amount of said retinoid or a pharma- ceutically acceptable salt thereof to the subject determined to be deficient in said retinoid based on the comparison in step (b). In certain embodiments, treatment with the retinoid or a pharma-ceutically acceptable salt thereof is continued until the subject is no longer determined to be deficient in said retinoid by repeating steps (a)-(b).

[0007] In certain embodiments of any of the above methods, the subject has been diagnosed with heart failure. In certain embodiments of any of the above methods, the subject has been diagnosed with hypertension.

[0008] In another aspect, the present invention relates to a method for preventing or reversing atrial remodeling in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a retinoid or a pharma- ceutically acceptable salt thereof, hi certain embodiments, the subject has been determined to be deficient in said retinoid.

[0009] In another aspect, the present invention relates to a method for preventing or reversing atrial remodeling in a subject in need thereof, comprising: a) determining a level of a retinoid in a serum sample obtained from the subject; b) comparing the level of the retinoid determined in step (a) with a control level of said retinoid; and c) administering a therapeutically effective amount of said retinoid or a pharma- ceutically acceptable salt thereof to the subject determined to be deficient in said retinoid based on the comparison in step (b). In certain embodiments, treatment with said retinoid or a pharma- ceutically acceptable salt thereof is continued until the subject is no longer determined to be deficient in said retinoid by repeating steps (a)-(b).

[0010] In certain embodiments of the above two methods, the subject has been diagnosed with acute onset heart failure.

[0011] In certain embodiments of any of the above methods, the retinoid is retinoic acid. In certain embodiments of any of the above methods, the retinoid is all-trans retinoic acid (ATRA).

[0012] In certain embodiments of any of the above methods, the subject is a human. In certain embodiments of any of the above methods, the subject is over 65 years of age.

[0013] In certain embodiments of any of the above methods, the retinoid or a pharma- ceutically acceptable salt thereof is administered or delivered to the atrium.

[0014] In certain embodiments of any of the above methods, the retinoid or a pharma- ceutically acceptable salt thereof is administered in combination with one or more additional therapeutic agents.

[0015] These and other aspects of the invention will become apparent to those skilled in the art in the following description, claims and drawings. [Brief description of the drawings]

[0016] [Figure 1A] Figures 1A-1C show RNA-seq analysis shows differentially expressed genes in mouse left atrium (LA) following transverse aortic constriction banding (TAC) and angiotensin II (AngII) infusion. 1 Figure 1A is a Venn diagram showing the overlap of 3364 differentially expressed genes between the TAC and AngII experiments. [Figure 1B] FIG. 1B shows pairwise correlation of log fold changes of differential gene expression in AngII versus TAC banding experiments (R=0.88, P<2.2×10-16). [Figure 1C] Figure 1C: KEGG pathway analysis of up-regulated genes involved in signaling pathways from the group of 3364 overlapping genes in Figure 1A. [Diagram 2] The retinoic acid (RA) synthesis pathway is shown. Circulating retinol is converted to all-trans-RA (ATRA) through successive oxidation steps. ALDH1A2 (also called RALDH2) catalyzes the final step of RA biosynthesis. RA then activates the nuclear retinoic acid receptor (RAR) and retinoid X receptor (RXR), which bind as heterodimers to retinoic acid response elements (RAREs) to activate the transcription of target genes. ATRA levels are tightly regulated by CYP26A1, CYP26B1, and CYP26C1, which catabolize ATRA for elimination. [Figure 3A] Figures 3A and 3B show RNA expression profiling of human left atrial tissue. Figure 3A shows that lower ALDH1A2 expression in the left atrium (LA) correlates with reduced left ventricular ejection fraction (LVEF), indicating reduced ATRA biosynthesis in the LA with heart failure. [Figure 3B] FIG. 3B shows that higher CYP26B1 expression in the LA is associated with persistent atrial fibrillation (AF) and that increased ATRA degradation contributes to LA remodeling leading to AF. [Figure 4A]Figures 4A-4E show electrocardiogram assessment of transverse aortic coarctation (TAC)-strangulated mice treated with vehicle (V) or ATRA 10 mg / kg administered daily via intraperitoneal injection over the 2-week study period. n=15-18 / group. Data represent mean ± SEM. One-way ANOVA was used, followed by Tukey's test for multiple comparisons to compare differences between experimental groups. *p-value <0.05, **p-value <0.005, ***p-value <0.001, ****p-value <0.0001. (Figure 4A) Heart rate (HR) is increased in the TAC+V and TAC+ATRA cohorts. [Figure 4B] The PR interval is shortened in the TAC+V and TAC+ATRA cohorts. ATRA does not significantly change the PR interval in the TAC+ATRA vs. TAC+V cohorts. [Figure 4C] P-wave duration is prolonged in the TAC+V cohort. ATRA treatment prevents P-wave prolongation in the TAC+ATRA cohort. [Figure 4D] QRS duration is prolonged in the TAC+V and TAC+ATRA cohorts. [Figure 4E] The HR-corrected QT (QTc) interval is prolonged in the TAC+V and TAC+ATRA cohorts. [Figure 5A]Figures 5A-5E show cardiac structural and functional assessment using transthoracic echocardiograms in sham-operated (Sham) or transverse aortic coarctation (TAC)-banded cohorts treated with vehicle (V) control or ATRA 10 mg / kg administered daily via intraperitoneal injection over a 2-week period. M-mode comparison of left ventricular (LV) Sham+V, Sham+ATRA, TAC+V, and TAC+ATRA. All measurements are performed in the parasternal LV long axis view. All scale bars are 1 mm. n=6 for Sham+V and Sham+ATRA groups and n=13 for TAC+V and TAC+ATRA groups (Figures 5B-5D). n=13 for each group in Figure 5E. One-way ANOVA was used followed by Tukey's test for multiple comparisons to compare differences between experimental groups. *p-value<0.05, **p-value<0.005, ***p-value<0.001, ****p-value<0.0001. (FIG. 5A) Left ventricular (LV) wall thickness, represented by the arrows, is increased in both the TAC+V and TAC+ATRA cohorts. [Figure 5B] Normalized LV wall thickness [Figure 5C] Left ventricular (LV) fractional shortening (FS). FS is equally reduced in TAC+V and TAC+ATRA hearts. [Figure 5D] LV strain measured using a speckle tracking algorithm. LV strain is equally reduced in TAC+V and TAC+ATRA hearts. [Figure 5E] Reservoir strain values ​​were measured in the left atrium (LA).12 TAC+V LA showed a reduction in reservoir strain. TAC+ATRA hearts showed significant improvement in reservoir strain parameters. [Figure 6A]Figures 6A-6E show cardiac optical mapping of TAC banded mice treated with ATRA. Optical mapping of Langendorff-perfused hearts from Sham+V, TAC+V, and TAC+ATRA hearts. Isochrones are drawn at 1 ms intervals. Data represent mean ± SEM. n=4-5 / group. One-way ANOVA was used, followed by Tukey's test for multiple comparisons to compare differences between experimental groups. *p-value <0.05, **p-value <0.005, ***p-value <0.001, ****p-value <0.0001. (Figure 6A) Representative left atrial activation map at 37 °C. Hearts were paced from the right atrium with a basic stimulation period (BCL) of 100 ms. [Figure 6B] Calculated LA conduction velocity (CV). [Figure 6C] Calculated left ventricular CV in TAC+V hearts after an atrial burst pacing protocol [Figure 6D] 6. ECG tracing of sustained atrial arrhythmia in a TAC+V heart after an atrial burst pacing protocol. [Figure 6E] Left atrial activation map of a sustained atrial arrhythmia shown in panel (Figure 6D). [Figure 7] Histological examination of TAC-strangulated hearts treated with ATRA 10 mg / kg daily via intraperitoneal injection over a 2-week period. (A) Trichrome staining of left atrial sections. (B) Higher magnification of the atrial region selected from panel (A) above. Treatment with ATRA limits interstitial and perivascular fibrosis associated with TAC-strangulation. (C) Collagen volume fraction measured by ImageJ analysis. n=5 / group. Data represent mean ± SEM. One-way ANOVA was used, followed by Tukey's test for multiple comparisons to compare differences between experimental groups. *p-value<0.05, **p-value<0.005, ***p-value<0.001, ****p-value<0.0001 [Figure 8]Connexin-43 (Cx43) expression in TAC-strangulated hearts treated with ATRA. Sham+V, Sham+ATRA, TAC+V, and TAC+ATRA hearts were perfusion-fixed and paraffin-embedded. Sections were probed with antibodies against Cx43, N-cadherin (N-CAD), and DAPI nuclear stain. Sham+V and Sham+ATRA hearts show similar expression and localization of Cx43 in the intercalated disc as evidenced by colocalization of Cx43 and N-CAD. TAC+V LA shows reduced Cx43 expression and relocalization of Cx43 to the lateral membrane. TAC+ATRA shows robust Cx43 expression in the intercalated disc. Scale bar: 20 μm [Figure 9A] Figures 9A-9E show the effect of ATRA on gene expression profiles in TAC-lacerated hearts. (Figure 9A) Heatmap of differentially expressed genes (DEGs, p<0.05) between TAC+ATRA and TAC+V (n=5 / group). [Figure 9B] (FIG. 9B) Venn diagram of the total number of up- or down-regulated DEGs (p<0.05 in each group) in [TAC+ATRA_vs_TAC+V] vs. [TAC_vs_Sham] by Venny2.1. Reciprocally expressed genes (952 up-regulated genes and 1035 down-regulated genes) in [TAC+ATRA_vs_TAC+V] vs. [TAC_vs_Sham] were used for functional analysis. [Figure 9C] Gene Ontology (GO) classification of up-regulated DEGs in Rectome pathways and biological process categories analyzed by Enrichr (enrichment analysis). Dashed lines represent -log10 (P=0.05). (#) Number of annotated genes. [Figure 9D]Gene Ontology (GO) classification of down-regulated DEGs in Rectome pathways and biological process categories analyzed by Enrichr (enrichment analysis). Dashed lines represent -log10 (P=0.05). (#) Number of annotated genes. [Figure 9E] Heatmap of genes involved in a curated list of I-VI from functional analysis showing reciprocal change between TAC vs. Sham and TAC+ATRA vs. TAC. [Figure 10A] Figures 10A-10H show electrocardiogram and histological evaluation of transverse aortic coarctation (TAC)-strangled mice with delayed vehicle (dV) treatment versus delayed ATRA (dATRA) treatment. In this experiment, test mice were TAC-strangled and left untreated for 2 weeks. At week 2, TAC mice were treated with dV or dATRA for 2 weeks, making the total study period 4 weeks. n=5-8 / group, equal gender distribution. Data represent mean ± SEM. One-way ANOVA was used, followed by Tukey's test for multiple comparisons to compare differences between experimental groups. *p value < 0.05, **p value < 0.005, ***p value < 0.001, ****p value < 0.0001. (Figure 10A) Heart rate (HR) is not altered by dATRA treatment. [Figure 10B] The PR interval is not altered by dATRA treatment. [Figure 10C] dATRA treatment significantly shortens P wave duration. [Figure 10D] QRS complex duration does not change with dATRA treatment. [Figure 10E] QTc interval is not altered by dATRA treatment [Figure 10F] Trichrome staining of paraffin-embedded left atrial sections. [Figure 10G] High magnification view of a selected atrial region from the above panel of Figure 10F. Delayed treatment with ATRA reverses interstitial and perivascular fibrosis associated with TAC entrapment. [Figure 10H] Measured collagen volume fraction by ImageJ analysis. [Figure 11A] Figures 11A-11D show electrocardiogram evaluation of sham versus transverse aortic coarctation (TAC)-constricted mice treated with vehicle (V) or various doses of ATRA (2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg administered daily via intraperitoneal injection over a period of 2 weeks). Of note, ATRA 20 mg / kg showed increased ventricular ectopy, indicating potential cardiotoxicity at this high dose. n=8-10 / group. Data represent mean ± SEM. One-way ANOVA was used, followed by Tukey's test for multiple comparisons to compare differences between experimental groups. *p-value <0.05, **p-value <0.005, ***p-value <0.001, ****p-value <0.0001. (Figure 11A) Heart rate (HR) does not change significantly in the TAC+V vs. TAC+ATRA cohorts over the ATRA dose range of 2 mg / kg to 10 mg / kg. [Figure 11B] The PR interval does not change significantly in the TAC+V vs. TAC+ATRA cohorts across the ATRA dose range of 2 mg / kg to 10 mg / kg. [Figure 11C] The shortening of P-wave duration is ATRA dose-dependent: the maximal shortening of P-wave duration is seen at an ATRA dose of 10 mg / kg, with no further shortening at a dose of 20 mg / kg. [Figure 11D] QRS duration does not change significantly in the TAC+V vs. TAC+ATRA cohorts across the ATRA dose range of 2 mg / kg to 10 mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention relates to methods for treating atrial fibrillation or preventing or reversing atrial remodeling, comprising administering a retinoid, such as, for example, all-trans retinoic acid (ATRA).

[0018] definition In order to facilitate an understanding of the principles and features of various embodiments of the present invention, various exemplary embodiments are described below. Although exemplary embodiments of the present invention are described in detail, it should be understood that other embodiments are also contemplated. Therefore, the present invention is not intended to be limited in scope to the details of the configuration and arrangement of the components described in the following description or examples. The present invention is capable of other embodiments and can be implemented or carried out in various ways. Also, in describing the exemplary embodiments, specific terminology is used for the sake of clarity.

[0019] The term "about" or "approximately" means within a statistically meaningful range of values. Such ranges can be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, even more preferably within 5%. The allowable variation encompassed by the term "about" or "approximately" depends on the particular system being tested and can be easily understood by those skilled in the art.

[0020] The terms "a," "an," and "the" do not denote a limitation of quantity, but rather denote the presence of "at least one" of the referenced item.

[0021] The terms "patient," "individual," "subject," and "animal" are used interchangeably herein and refer to mammals, including, but not limited to, humans and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0022] The term "treat" a condition, disorder or condition or "treatment" thereof includes: (1) preventing or delaying the appearance of at least one clinical or subclinical symptom of the condition, disorder or condition developing in a subject who may be afflicted with or susceptible to the condition, disorder or condition, but who has not yet experienced or displayed a clinical or subclinical symptom of the condition, disorder or condition; or (2) inhibiting the condition, disorder or condition, i.e., arresting, reducing or delaying the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., regressing the condition, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0023] "Effective amount" as applied to a dose or amount refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. It should be noted that when a combination of active ingredients (active ingredients) is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the condition being treated, the particular drug(s) used, the mode of administration, etc.

[0024] A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition, disorder or pathology, or to delay or minimize one or more symptoms associated with a condition, disorder or pathology. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall treatment, reduces or avoids symptoms or causes of a condition, and / or enhances the therapeutic effectiveness of another therapeutic agent.

[0025] In certain embodiments, pharmaceutically acceptable salts include the salts of acidic or basic groups present in the compounds of the present disclosure.As used herein, the term "pharmaceutically acceptable salts" refers to the salts of the compounds of the present invention that are safe for application in subjects.Pharmaceutically acceptable acid salts include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,11-methylene-bis-(2-hydroxy-3-naphthoate)) salt. Certain compounds of the present disclosure can form pharma- ceutically acceptable salts with various amino acids.Suitable base salts include, but are not limited to, aluminum salt, calcium salt, lithium salt, magnesium salt, potassium salt, sodium salt, zinc salt, and diethanolamine salt.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Berge, SM et al, Journal of Pharmaceutical Science, 1977, 66, 1, 1-19.

[0026] In the context of the medical field, the term "prevention" includes any activity that reduces the burden of mortality or morbidity due to a disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the onset of a disease, secondary and tertiary levels of prevention include activities aimed at preventing the progression of the disease and the appearance of symptoms, as well as reducing the negative effects of an already established disease by restoring function and reducing disease-related complications.

[0027] The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications as commonly accomplished in the art or as described herein.The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry, and the experimental procedures and techniques thereof described herein are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0028] Additionally, in describing the exemplary embodiments, terminology is employed for the sake of clarity. Each term is intended to have its broadest meaning as understood by one of ordinary skill in the art and to encompass all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[0029] Methods of the Invention In one aspect, the present invention relates to a method for treating atrial fibrillation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a retinoid or a pharma- ceutically acceptable salt thereof, hi certain embodiments, the subject has been determined to be deficient in said retinoid.

[0030] In another aspect, the present invention relates to a method for treating atrial fibrillation in a subject in need thereof, comprising: a) determining a level of a retinoid in a serum sample obtained from the subject; b) comparing the level of the retinoid determined in step (a) with a control level of said retinoid; and c) administering a therapeutically effective amount of said retinoid or a pharma- ceutically acceptable salt thereof to the subject determined to be deficient in said retinoid based on the comparison in step (b). In certain embodiments, treatment with said retinoid or a pharma- ceutically acceptable salt thereof is continued until the subject is no longer determined to be deficient in said retinoid by repeating steps (a)-(b).

[0031] In another aspect, the present invention relates to a method for preventing or reversing atrial remodeling in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a retinoid or a pharma- ceutically acceptable salt thereof, hi certain embodiments, the subject has been determined to be deficient in said retinoid.

[0032] In another aspect, the present invention relates to a method for preventing or reversing atrial remodeling in a subject in need thereof, comprising: a) determining a level of a retinoid in a serum sample obtained from the subject; b) comparing the level of the retinoid determined in step (a) with a control level of said retinoid; and c) administering a therapeutically effective amount of said retinoid or a pharma- ceutically acceptable salt thereof to the subject determined to be deficient in said retinoid based on the comparison in step (b). In certain embodiments, treatment with said retinoid or a pharma- ceutically acceptable salt thereof is continued until the subject is no longer determined to be deficient in said retinoid by repeating steps (a)-(b).

[0033] Quantification of retinoids can be performed by any method known in the art and described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, for example, Yang, N., et al. Cardiac retinoic acid levels decline in heart failure. JCI Insight 6 (2021). In certain embodiments, retinoids can be measured by, for example, LC-MRM (liquid chromatography with multiple reaction monitoring), UHPLC-MS / MS (ultra-high performance liquid chromatography), HPLC / MS n , LC-MS / MS, GC / MS, or LC / diode array detector-atmospheric pressure chemical ionization / MS / MS.

[0034] In certain non-limiting embodiments, the retinoid is retinol (vitamin A), retinal (retinaldehyde), retiferol, tretinoin (retinoic acid), isotretinoin, alitretinoin (9-cis-retinoic acid), etretinate, acitretin, adapalene, bexarotene, tazarotene, trifarotene. In certain embodiments, the retinol is a retinol isomer. In certain embodiments, the retinol isomer is all-trans retinol, 13-cis-retinol, 11-cis-retinol, 9-cis-retinol, 3,4-didehydro-retinol, 3,4-didehydro-13-cis-retinol; 3,4-didehydro-11-cis-retinol; or 3,4-didehydro-9-cis-retinol.

[0035] In certain non-limiting embodiments, retinoids are compounds that contain retinoid structures, retinoid metabolites, or substances that can be metabolized to retinoids or retinoid metabolites.Retinoids further include compounds that are analogs or mimics of retinoids or retinoid metabolites, or substances that can be metabolized to analogs or mimics of retinoids or retinoid metabolites.In certain embodiments, retinoids are compounds that are analogs or mimics of retinoids or retinoid metabolites, or substances that can be metabolized to analogs or mimics of retinoids or retinoid metabolites, as described in U.S. Patents 5,648,563; 5,648,385; 5,618,839; 5,559,248; 5,616,712; 5,616,597; 5,602,135; 5,599,819; 5,556,996; 5,534,516; 5,5 The retinoid may be any of the retinoids disclosed in US Pat. Nos. 16,904; 5,498,755; 5,470,999; 5,468,879; 5,455,265; 5,451,605; 5,426,118; 5,407,937; 5,399,586; 5,399,561; or 5,391,753.

[0036] In certain embodiments, the retinoid is retinoic acid. In certain embodiments, the retinoid is all-trans retinoic acid (ATRA).

[0037] In certain embodiments, ATRA is [ka] or a pharma- ceutically acceptable salt thereof.

[0038] In certain embodiments, the subject has been diagnosed with heart failure. In certain embodiments, the subject has been diagnosed with hypertension. In certain embodiments, the subject has been diagnosed with acute onset heart failure.

[0039] In certain embodiments, the subject is a human. In certain embodiments, the subject is over 65 years of age.

[0040] In certain embodiments, the retinoid or a pharma- ceutically acceptable salt thereof is administered or delivered to the atrium.

[0041] In certain embodiments, the retinoid, or a pharma- ceutically acceptable salt thereof, is administered in combination with one or more additional therapeutic agents.

[0042] The route of administration may be any mode of administration known in the art, including, but not limited to, injection into the involved tissue, intra-arterially, intravenously, via an implanted device, parenterally, topically, subcutaneously, intradermally, transdermally (e.g., via a transdermal patch), via internal application during surgery, intramuscularly, intraperitoneally, buccally, intrathecally, intracranially, or orally, etc. In a preferred embodiment, the retinoid or a pharma- ceutically acceptable salt thereof is administered or delivered to the atrium of the heart.

[0043] The retinoids of the present invention, or pharma- ceutically acceptable salts thereof, can be encapsulated or otherwise protected from gastric or other secretions, if desired.

[0044] In certain embodiments, the retinoid of the present invention or a pharma- ceutically acceptable salt thereof may be administered in conjunction with other treatments. In certain embodiments, the retinoid of the present invention or a pharma- ceutically acceptable salt thereof may be administered in conjunction with treatments for, but not limited to, atrial fibrillation, heart failure, hypertension, and / or acute onset heart failure. In certain embodiments, the other treatments may be, but are not limited to, defibrillation, including electrical and / or pharmacological defibrillation, surgery, coronary artery bypass surgery, repair or replacement of heart valves, catheterization, use of an implantable cardioverter defibrillator (ICD), cardiac resynchronization therapy (CRT), use of a ventricular assist device (VAD), heart transplantation, palliative care, beta blockers (e.g., atenolol, metoprolol, or bisoprolol), calcium channel blockers, and the like. blockers (e.g., amlodipine or diltiazem), digoxin, antiarrhythmics, anticoagulants (e.g., warfarin, apixaban, dabigatran, edoxaban or rivaroxaban), angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril, benazepril, lisinopril or captopril), angiotensin II receptor blockers (e.g., losartan, valsartan or candesartan), diuretics (e.g., furosemide), aldosterone antagonists (e.g., spironolactone or eplerenone), positive inotropes, hydralazine and isosorbide dinitrate (BiDil), vericiguat, diuretics The therapeutic agent may be an antihypertensive drug (diuretic), an alpha blocker (e.g., doxazosin or prazosin), an alpha-beta blocker (e.g., carvedilol or labetalol), a renin inhibitor (e.g., aliskiren), a vasodilator (e.g., hydralazine or minoxidil), a centrally acting drug (e.g., clonidine, guanfacine or methyldopa), and combinations thereof.

[0045] The dosage administered will depend upon the route of administration, the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0046] The effective dose of the retinoid or its pharma- ceutically acceptable salt for the therapeutic uses discussed above can be determined using methods known to those skilled in the art. The effective dose can be determined, preferably in vitro, to identify optimal dose ranges using any of the various methods described herein. In one embodiment, an aqueous solution of the retinoid or its pharma- ceutically acceptable salt is administered by intraperitoneal injection. Each dose can range from about 0.001 μg / kg body weight to about 100 mg / kg body weight, more preferably about 0.1 μg / kg to 20 mg / kg body weight. The administration schedule can vary from only once, to once a week to daily or twice a day (or more), depending on many clinical factors.

[0047] Suitable, non-limiting examples of the dosage of the retinoid or a pharma- ceutically acceptable salt thereof according to the present invention, or a composition comprising such a retinoid or a pharma- ceutically acceptable salt thereof, are about 1 ng / kg to about 1000 mg / kg, for example, about 1 mg / kg to about 100 mg / kg, for example, about 5 mg / kg to about 50 mg / kg. Other representative dosages of a compound or composition of the invention include about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, or 1000 mg / kg.

[0048] In certain embodiments, the retinoid or a pharma- ceutically acceptable salt thereof is administered at a dose of about 2 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg, hi certain embodiments, the retinoid or a pharma- ceutically acceptable salt thereof is administered intraperitoneally.

[0049] In certain embodiments, the retinoid or a pharma- ceutically acceptable salt thereof may be administered hourly, daily, weekly, monthly, yearly, or as a one-time delivery. In certain embodiments, the retinoid or a pharma-ceutically acceptable salt thereof may be administered daily by intraperitoneal injection for a period of 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0050] In certain embodiments of the present invention, the method may further comprise administering pharma- ceutically acceptable carrier to the subject during administration of retinoid or its pharma- ceutically acceptable salt.Carrier may be diluent, aerosol, topical carrier, aqueous solution, nonaqueous solution or solid carrier.As used herein, the term "suitable pharma-ceutically acceptable carrier" includes any standard pharma-ceutically acceptable carrier, such as phosphate buffered saline, water, emulsion such as oil / water emulsion or triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules. EXAMPLES

[0051] The present invention is also described and demonstrated by the following examples. However, the use of these and other examples anywhere in this specification is merely illustrative and in no way limits the scope and meaning of the invention or the exemplified terms. Similarly, the present invention is not limited to the specific preferred embodiments described herein. Indeed, many modifications and variations of the present invention will be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the present invention. Therefore, the present invention should be limited only by the appended claims and the full scope of equivalents to which such claims are entitled.

[0052] Example 1. Identification of ATRA as a master regulatory switch capable of protecting against pathological atrial remodeling. Atrial fibrillation (AF) is a multifactorial disease in which genetic predisposition coupled with pathological stressors induce electrical and structural changes in the left atrium (LA) that promote AF. Maladaptive changes, including conduction delays, inflammation, and fibrosis, are known as atrial remodeling. 12,13 When exposed to pressure overload conditions, such as hypertensive heart disease, valvular heart disease, and diastolic and systolic heart failure (HF), the left atrium (LA) becomes particularly vulnerable to the remodeling process. Remodeled atria exhibit reduced conduction velocity (CV) as a result of reduced expression of the myocardial sodium channel Nav1.5 (encoded by Scn5a) and reduced sodium current (I Na ) decrease 14 , reduced expression and mislocalization of the high-conductance gap junction proteins connexin 43 and 40 (Cx43 and Cx40, encoded by Gja1 and Gja5, respectively) from the intercalated disc. 15,16 , as well as increased fibrosis 17 , which impairs conduction by disrupting the continuity of muscle fibers. Na are major determinants of membrane excitability in cardiomyocytes, and Cx43 and Cx40 gap junctions (or gap junctions) facilitate passive conductance between cardiomyocytes 18、19 The remodeling process tends to be progressive, resulting in atrial myopathy that initiates AF and drives its progression from paroxysmal (self-terminating) to persistent.

[0053] As mentioned in the Background section, neither antiarrhythmic drugs (AADs) nor catheter ablation therapy address the underlying left atrial myopathy that causes atrial fibrillation (AF) and promotes its progression to persistent form. To address this important unmet need, the inventors have identified novel therapies that can use atrial developmental signaling pathways to prevent and reverse the pathological atrial remodeling process that leads to AF.

[0054] To better define the atrial remodeling process, we performed a comparative transcriptomic analysis of two cardiac pressure overload models: transverse aortic coarctation (TAC) banding and angiotensin II (AngII) infusion (Figure 1A). We found that the LA undergoes remodeling in a highly reproducible manner (Figure 1B). The major biological processes upregulated in both models included the pro-inflammatory pathway (chemokine signaling, transforming growth factor-β (TGF-β) signaling, B cell receptor signaling, tumor necrosis factor (TNF) signaling, and Toll-like receptor (TLR) signaling), and the profibrotic pathway (extracellular matrix (ECM) receptor interactions and focal adhesions). These pro-inflammatory and pro-fibrotic pathways underlie the molecular basis of structural remodeling. The main downregulated biological processes were metabolic pathways (respiratory transport chain and fatty acid oxidation in mitochondria) and pathways affecting the expression of rapid conduction genes (adrenergic signaling, arrhythmogenic cardiomyopathy, and ERBB signaling pathways in cardiomyocytes). Downregulation of rapid conduction genes, such as Scn5a, Gja1 / Cx43, and Gja5 / Cx40, contributes to electrical remodeling. The highly uniform manner in which LA remodels indicates the existence of master regulatory switches to prevent inflammation / fibrosis, metabolic abnormalities, and conduction disease. Herein, it was hypothesized that cardiac pressure overload turns off these regulatory switches, causing pathological remodeling.

[0055] To identify these master regulatory switches that may serve as therapeutic targets for atrial remodeling, we investigated signaling pathways that are essential for atrial development. Atrial myocytes originate from progenitor cells in the dorsal secondary heart field (SHF), which contribute to the inflow tract of the linear heart tube. Retinoic acid signaling is important for the differentiation of SHF precursors into atrial myocytes. 21Endogenous retinoids are derived from vitamin A through successive oxidative steps to generate the active metabolite all-trans retinoic acid (ATRA), the predominant form of retinoic acid (Figure 2). Mice deficient in aldehyde dehydrogenase 1 family member A2 (ALDH1A2, also known as RALDH2), which oxidizes the final step in retinoic acid biosynthesis, die at embryonic day 10.5 (E10.5) and exhibit severe defects in atrial growth. 20 Oral maternal supplementation with ATRA rescued the atrial defect, indicating that the atria are responsive to circulating ATRA. On the other hand, chick embryos exposed to excess ATRA developed enlarged atria. 22 The importance of retinoic acid in the differentiation of human atrial myocytes has been demonstrated using human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). 23,24 Addition of retinoic acid effectively increased the population of atrial myocytes from the progenitor cell pool. 23-25 .

[0056] Based on the importance of ATRA signaling in atrial myocyte differentiation and chamber formation, it was hypothesized herein that ATRA has an essential role in maintaining atrium electrical and structural homeostasis as well as cardioprotection against pathological atrial remodeling. In support of the maintenance hypothesis, expression levels of Aldh1a1 and Aldh1a2 (ATRA biosynthesis) were 8-fold and 10-fold higher, respectively, in atrial myocytes compared to ventricular myocytes in postnatal day 21 (P21) hearts. Meanwhile, expression levels of Cyp26b1 (ATRA degradation) were 14-fold lower in atrial myocytes compared to ventricular myocytes in P21 hearts. These data suggest that continuous ATRA enrichment is required to maintain the postnatal atria. In support of a role for ATRA in cardioprotection, ATRA levels are reduced by 39% and 32%, respectively, in the ventricular myocardium of HF patients and in a guinea pig HF model. 26ATRA treatment could improve left ventricular systolic function and attenuate ventricular fibrosis in a guinea pig HF model. Consistent with this finding, ATRA reduces proliferation, collagen secretion, and TGF-β production in dissociated neonatal rat cardiac fibroblasts exposed to AngII. 27 In diabetic kidneys, ATRA inhibits the production of inflammatory cytokines, chemokines, adhesion molecules, and growth factors by downregulating the TLR signaling pathway involving the transcription factor NF-κB. 28 Although ATRA presents itself as a regulatory switch that can protect against pathological atrial remodeling, no studies have examined the role of ATRA signaling in the postnatal atria.

[0057] The present inventors have uncovered an unrecognized role for ATRA in maintaining the electrical and structural integrity of the LA under pressure overload conditions. Analysis of the Cleveland Clinic Biobank reveals that ALDH1A2, an essential ATRA biosynthetic enzyme, is downregulated in the LA of patients with reduced left ventricular ejection fraction (LVEF), and that CYP26B1, which catalyzes the degradation of ATRA, is upregulated in the LA of patients with persistent AF. These data suggest that downregulation of ATRA contributes to the process of atrial remodeling that leads to AF. In support of this hypothesis, it has been shown herein that ATRA treatment is highly effective in preventing electrical and structural remodeling in the LA due to cardiac pressure overload. Using TAC strangulation as a model of cardiac pressure overload, it has been shown herein that ATRA treatment normalizes atrial conduction properties, improves left atrial function parameters, and prevents atrial fibrosis. In addition, ATRA therapy prevented the remodeling of Cx43 gap junctions in the LA of TAC-strangulated hearts, as evidenced by normalization of Cx43 expression in the intercalated disc. RNA-seq analysis revealed that ATRA treatment upregulated genes important for atrial conduction and metabolism and downregulated proinflammatory signaling pathways (AngII signaling, TLR signaling, platelet-derived growth factor (PDGF) signaling, TNF signaling, and interleukin-1b (IL-1b) signaling), and pathways involved in ECM deposition. Finally, it is shown herein that ATRA treatment can reverse left atrial fibrosis and improve atrial conduction parameters, even when initiated 2 weeks after TAC strangulation, which has important clinical implications for patients presenting with HF.

[0058] Having implicated ATRA as a master regulatory switch that prevents and reverses atrial remodeling during cardiac pressure overload, we investigate the mechanisms underlying ATRA-mediated protection. Our hypotheses are that i) endogenous ATRA maintains the electrical and structural integrity of the adult left atrium, ii) cardiac pressure overload reduces endogenous ATRA levels in the LA and promotes pathological remodeling, iii) therapeutic ATRA blocks key proinflammatory cytokine / chemokine / growth factor production pathways that drive fibrosis, and iv) therapeutic ATRA normalizes left atrial conduction, in part, by restoring sodium conductance and gap junction conductance. To test these hypotheses, the following studies were performed:

[0059] 1:Investigating the role of endogenous ATRA in maintaining normal atrial physiology, the effect of pressure overload on endogenous ATRA levels, and the therapeutic potential of increasing endogenous ATRA levels to prevent TAC-induced LA remodeling.

[0060] 2: To clarify the anti-inflammatory mechanism by which ATRA prevents and reverses atrial fibrosis, and to study the ability of ATRA to reverse atrial fibrosis in more advanced stages of HF.

[0061] 3: Study to clarify the ionic mechanism by which ATRA improves atrial conduction parameters and study its effect on atrial arrhythmia susceptibility.

[0062] This disclosure identifies ATRA as a novel therapeutic agent that can prevent and reverse atrial remodeling in cardiac pressure overload conditions. This finding represents an important advance in the field of cardiac electrophysiology, since there are currently no effective therapeutic agents that target the molecular basis underlying atrial remodeling.

[0063] We will study how cardiac pressure overload affects ATRA expression and how altered production or degradation of ATRA contributes to pathological remodeling, as well as the underlying mechanisms by which ATRA confers protection against atrial conduction disease, inflammation, and fibrosis, which are the main causes of atrial myopathy.

[0064] Example 2. Left atrial expression of ALDH1A2 is lower in patients with reduced LVEF and CYP26B1 expression is increased in patients with persistent AF. PolyA+RNA sequencing was performed on left atrial appendage (LAA) tissue from a total of 265 subjects (235 subjects of European descent and 30 subjects of African descent, mean age 60 ± 12 years, 181 males). Expression of ALDH1A2 (ATRA biosynthesis) was lower in the LA of patients with reduced LVEF, indicating that atrial ATRA synthesis is reduced in HF patients (Figures 3A-3B). Expression of CYP26B1 (ATRA degradation) was higher in the LA of patients with persistent AF compared to patients in sinus rhythm or paroxysmal AF, suggesting that increased degradation of endogenous ATRA may be involved in the transition from paroxysmal to persistent AF.

[0065] SUMMARY:Decreased expression of left atrial ALDH1A2 in patients with HF and increased expression of left atrial CYP26B1 in patients with persistent AF suggest that reduced LA ATRA levels, due to decreased biosynthesis or increased degradation, may contribute to atrial remodeling leading to AF.

[0066] Example 3. ATRA protects against pathological atrial remodeling in TAC-choke mice. To clarify the role of ATRA in cardioprotection against pathological LA remodeling induced by pressure overload, we used the TAC strangulation model. TAC strangulation creates a fixed afterload obstruction in the systemic circulation and serves as an excellent model for drug testing, since the surgically induced coarctation cannot be reversed by drug therapy. Two weeks of TAC strangulation have previously been shown to cause electrical and structural remodeling of the left atrium in a highly reproducible manner prior to the development of overt HF. 1 For the protection study of ATRA, the study design consisted of C57B1 / 6 wild-type male and female mice equally divided into four groups: i) Sham+V (sham-operated (Sham)+vehicle control), ii) Sham+ATRA, iii) TAC+V (TAC+vehicle control), and iv) TAC+ATRA, with a two-week study period. ATRA (10 mg / kg / day, intraperitoneal (IP) injection) or vehicle control was first administered 30 min before TAC or Sham surgery, and then daily for two weeks.

[0067] Electrocardiography (ECG) was performed at 2 weeks in the Sham+V, Sham+ATRA, TAC+V, and TAC+ATRA cohorts to examine the effect of ATRA treatment on atrial and ventricular activation and repolarization parameters. In the ECG, P wave duration is an index of atrial activation time, PR interval is an index of atrial-ventricular activation time including atrioventricular nodal conduction time, QRS wave duration is an index of ventricular activation time, and HR corrected QT (QTc) interval is an index of ventricular repolarization time. There were no differences in ECG measurements between the Sham+V and Sham+ATRA groups, indicating that ATRA treatment had no discernible effect on cardiac activation or repolarization parameters under normotensive conditions (Figures 4A-4E). TAC-choke mice treated with vehicle control (TAC+V) had increased heart rate (HR), prolonged P wave duration, QRS wave duration, and QTc interval duration, and shortened PR interval compared to the Sham group. In TAC-choke mice treated with ATRA (TAC+ATRA), P wave duration was shortened to normal levels, whereas HR, PR interval, QRS complex, and QTc interval duration were unchanged compared to the TAC+V cohort.

[0068] Summary: ATRA normalizes atrial activation time in TAC-choke mice but has no effect on HR or ventricular activation and repolarization times. These findings indicate that ATRA has a profound protective effect against atrial electrical remodeling despite fixed afterload obstruction and ECG evidence of ventricular electrical remodeling.

[0069] Example 4. ATRA improves left atrial functional characteristics in TAC-choke mice. Transthoracic echocardiography (TTE) was performed after 2 weeks in the Sham+V, Sham+ATRA, TAC+V, and TAC+ATRA cohorts to examine the effect of ATRA treatment on atrial and ventricular structure and function. There were no differences in LA and left ventricular (LV) echocardiographic measurements between the Sham+V and Sham+ATRA groups, indicating that ATRA treatment has no discernible effect on global cardiac structure or function under normotensive conditions (Figure 5A-5E). The TAC+V and TAC+ATRA groups had similar left ventricular hypertrophy (LVH) and reduced left ventricular systolic function as measured by LV fractional shortening and longitudinal strain analysis compared to the Sham+V and Sham+ATRA groups (Figure 5B-D). These findings indicate that ATRA did not affect the left ventricular hypertrophy response to pressure overload or the reduction in left ventricular systolic function. Next, the effect of TAC constriction on left atrial function was measured using reservoir train analysis. 8,30 Left atrial reservoir strain analysis is a sensitive method to study left atrial mechanics in both relaxation (conduit strain) and contraction (systolic strain) in an angle-independent manner. Left atrial mechanics in the TAC+ATRA group were significantly improved compared to the TAC+V group, although values ​​did not return to normal levels (Figure 5E).

[0070] Summary: ATRA improves left atrial functional parameters but does not affect LVH or left ventricular function. These findings indicate that ATRA improves left atrial myocardial physiology despite fixed afterload obstruction and ventricular structural remodeling.

[0071] Example 5. ATRA normalizes left atrial conduction velocity (CV) in TAC-choke mice. Optical mapping was then performed on Langendorff-perfused Sham+V, TAC+V, and TAC+ATRA hearts to assess left atrial and left ventricular conduction properties (Figures 6A-6E). Representative isochronal maps of the Sham+V, TAC+V, and TAC+ATRA left atrium are shown in Figure 6A. Consistent with the prolongation of P-wave duration on ECG in the TAC+V group, left atrial CV was significantly decreased in TAC+V hearts compared to Sham+V hearts (Figures 6A, 6B). In the TAC+ATRA group, left atrial CV was significantly increased to normal levels (CV = 0.52 ± 0.02 m / s for Sham+V, 0.30 ± 0.03 m / s for TAC+V, and 0.50 ± 0.02 m / s for TAC+ATRA, p < 0.05, one-way ANOVA) (Figure 6B). Left ventricular CV in Sham+V, TAC+V, and TAC+ATRA hearts was not significantly different among the three groups.

[0072] Atrial arrhythmogenicity was evaluated by performing an atrial burst pacing protocol in TAC+V and TAC+ATRA hearts. 6 Atrial burst pacing induced sustained atrial arrhythmias (>30 s duration) in 1 of 3 TAC hearts, whereas 0 of 3 TAC+ATRA hearts showed sustained atrial arrhythmias. Induction of sustained atrial tachycardia in a TAC+V heart is shown in Figure 6C. Optical maps of the LA in TAC+V during atrial tachycardia showed abnormal activation wavefronts emanating from the LA body (Figure 6D).

[0073] Summary: ATRA normalizes left atrial CV in TAC-constricted hearts.

[0074] Example 6. ATRA protects against left atrial fibrosis in TAC-strangled mice. To investigate the mechanism by which ATRA restores normal LA CV, changes in LA fibrosis burden were assessed using ImageJ analysis on Masson's trichrome stained sections and quantified throughout the LA area per section. 1As shown in Figure 7, there was no difference in fibrosis levels as measured by collagen volume fraction between Sham+V and Sham+ATRA samples, indicating that ATRA does not affect fibrosis under normotensive conditions. Both the TAC+V and TAC+ATRA groups showed left atrial hypertrophy, consistent with increased intracardiac pressure. TAC+V LAs had significantly increased collagen volume fraction in the interstitial and perivascular regions compared with the Sham+V and Sham+ATRA groups. Of note, TAC+ATRA LAs showed decreased left atrial interstitial and perivascular fibrosis and significantly decreased collagen volume fraction compared with the TAC+V LAs. Collagen volume fraction in TAC+ATRA LAs was not significantly different from the Sham groups (5.4 ± 0.1% for Sham+V, 5.3 ± 0.1% for Sham+ATRA, 16.6 ± 2.5% for TAC+V, and 8.3 ± 0.6% for TAC+ATRA).

[0075] Summary: ATRA protects against atrial fibrosis in pressure-overloaded hearts.

[0076] Example 7. ATRA prevents Cx43 remodeling in the LA of TAC-confined hearts. Because Cx43 remodeling is a known contributor to conduction disease in pressure-overloaded hearts, we investigated whether ATRA treatment protected against Cx43 gap junction remodeling using immunofluorescence staining in Sham+V, Sham+ATRA, TAC+V, and TAC+ATRA LA sections. Sham+V and Sham+ATRA LA showed similar levels of Cx43 expression and proper localization at the intercalated disc, as evidenced by co-immunostaining with N-cadherin (N-CAD) (Figure 8). In TAC+V LA, Cx43 expression appeared to be reduced and relocalized to the lateral membrane (i.e., gap junction remodeling). In TAC+ATRA LA, Cx43 expression was robust and properly localized to the intercalated disc.

[0077] Summary: ATRA treatment prevents Cx43 remodeling in pressure-overloaded hearts.

[0078] Example 8. ATRA regulates a transcriptional program in pressure-overloaded LA that protects against electrical and structural remodeling. To clarify the ATRA-dependent transcriptional program in the LA that protects against pressure-induced remodeling, we performed RNA-seq on LA samples from TAC+V and TAC+ATRA hearts (Figures 9A-9E). Principal component analysis (PCA) and Euclidean plots of the global gene expression data showed a clear separation of TAC+V and TAC+ATRA samples. Using a threshold criterion of P<0.05, we obtained 4133 transcripts that were differentially expressed between TAC+ATRA and TAC+V LA samples (2075 transcripts were upregulated and 2058 transcripts were downregulated). Heatmap analysis of the 4133 genes showed excellent separation of gene expression by treatment group (Figure 9A). We then performed a comparative analysis of differentially expressed genes (DEGs) from the [TAC+ATRA vs. TAC+V] dataset compared to the [TAC vs. Sham] dataset, generating the Venn diagram in Figure 9B. Based on Venn diagram analysis, 1987 DEGs (952 upregulated and 1035 downregulated genes) were identified in the [TAC+ATRA vs. TAC+V] dataset that ran in the opposite direction to the [TAC vs. Sham] dataset, representing the “normalization” of adversely remodeled genes.

[0079] Next, we performed Gene Ontology (GO) biological process and REACTOME pathway analysis using Enrichr on the significantly up-regulated and down-regulated genes that were "normalized" with ATRA treatment (Figure 9C, 9D). The major biological processes significantly up-regulated with ATRA treatment included pathways involved in mitochondrial energy production [mitochondrial ATP synthesis-coupled electron transport, fatty acid oxidation] and signaling pathways involved in cardiac / atrial development [WNT ligand biosynthesis and transport, retinoic acid-mediated signaling, sarcomere organization, cardiac development]. Major biological processes and pathways significantly downregulated with ATRA treatment include regulation of ECM production [extracellular structure organization, ECM organization], proinflammatory signaling [response to interleukin-1 (IL-1), regulation of interleukin-6 (IL-6) production, positive regulation of TNF superfamily cytokine production, regulation of inflammatory response, regulation of I-kB kinase / NF-kB signaling, cytokine signaling in the immune system, regulation of mast cell degranulation, signaling by PDGF], and regulation of vasoconstriction [regulation of systemic arterial blood pressure]. In Figure 9E, representative DEGs (upper panel) in TAC-strangulated LA that were "normalized" by ATRA therapy (lower panel) were categorized by biological process.

[0080] Example 9. ATRA therapy downregulates key inflammatory signaling pathways in pressure-overloaded LA. The present pathway analysis showed that ATRA therapy downregulated three key mechanisms of inflammation in pressure-overloaded LA: i) the TLR / NF-kB / NLRP3 signaling pathway for the production and activation of proinflammatory cytokines (IL-1b), ii) mast cell-dependent PDGF-A activation, and iii) AngII signaling activation. ATRA downregulated the expression of Toll-like receptor 2 (TLR2), NF-kB signaling components, NLRP3 (NACHT, LRR, and PYD domain-containing protein 3), and IL-1b (Figure 9D, 9E). Importantly, TLR2, NF-kB, NLRP3, and IL-1b have all been shown to be upregulated in AF patients. 31-35 In diabetic nephropathy, ATRA has been shown to block the production of cytokines, chemokines, and growth factors through inhibition of TLR / NF-kB signaling. 28 In addition, TLR2 36 and TLR4 28,34 transcriptionally regulates the expression of the NLRP3 inflammasome, which converts proinflammatory cytokines, such as Pro-IL-1b (the precursor of IL-1b) to their activated forms, such as IL-1b, via activated caspase-I (Casp1-p20 and Casp1-p10)-dependent cleavage 37 Another signaling pathway that has been shown to play an important role in atrial fibrosis during cardiac pressure overload is via mast cell-dependent PDGF-A secretion. 6 PDGF-A has been shown to stimulate cell proliferation and collagen synthesis in cardiac fibroblasts. 6The present pathway analysis shows that ATRA treatment downregulates adipocyte degranulation and PDGF signaling pathways, resulting in decreased expression of PDGF-A and PDGF-B (Figures 9D, 9E). Finally, it is noteworthy that angiotensin-converting enzyme (ACE), which is upregulated by TAC strangulation, was significantly downregulated by ATRA therapy (Figures 9D, 9E). Activation of AngII signaling pathway in LA by pressure overload is a potent inducer of inflammation, fibrosis, and Cx43 gap junction remodeling. Blockade of AngII signaling can reduce atrial fibrosis and partially rescue Cx43 remodeling. 38,39 .

[0081] Summary: ATRA downregulates key inflammatory pathways involved in atrial fibrosis and electrical remodeling.

[0082] Example 10. Delayed ATRA treatment reverses pathological atrial remodeling in the late stages of cardiac pressure overload. To investigate the ability of ATRA to reverse atrial remodeling, we designed a new study in which ATRA therapy was initiated 2 weeks after TAC or Sham surgery. This model more accurately reflects the clinical symptoms of HF patients. For the ATRA reversal study, C57B1 / 6 wild-type male and female mice were equally divided into three groups: i) Sham+dV (delayed vehicle administration start), ii) TAC+dV, iii) TAC+dATRA (delayed ATRA administration start). Mice underwent Sham or TAC surgery and were left untreated for 2 weeks. Administration of ATRA (10 mg / kg / day, IP injection) or vehicle control was initiated 2 weeks after TAC or Sham surgery and continued for 2 weeks, making the total study period 4 weeks. At the end of the 4-week study, ECG and histological analysis were performed. In the TAC+dV cohort, there was an increase in HR, as well as prolongation of P wave duration, PR interval, QRS duration, and QTc interval on the ECG (Figure 10A-E). In the TAC+dATRA group, P wave duration was significantly shortened compared to the TAC+dV group, while PR interval, QRS duration, and QTc interval were not significantly different compared to the TAC+dV group.

[0083] We next quantified LA fibrosis burden using ImageJ analysis in Masson's trichrome stained sections (Figure 10F-H). TAC+dV LA at 4 weeks measured a collagen volume fraction of 21.9±1.8% compared to 16.6±2.5% in TAC+V LA at 2 weeks (Figure 10H and Figure 7C), indicating the development of progressive fibrosis. Of note, delayed ATRA treatment in TAC+dATRA LA reduced collagen volume fraction to 10.9±1.1% at 4 weeks, which is lower than the expected level measured in TAC+V LA at 2 weeks (Figure 10H and Figure 7C). These data indicate that delayed ATRA therapy can reverse the fibrotic process in the LA of pressure-overloaded hearts.

[0084] Summary: ATRA treatment reduces atrial electrical remodeling and reverses atrial fibrosis caused by cardiac pressure overload.

[0085] Example 11. ATRA treatment shows a dose-response curve in normalizing atrial electrocardiogram parameters in TAC-choke mice. To examine the dose-dependent effects of ATRA on atrial conduction parameters, electrocardiograms (ECGs) were performed on the Sham+V, TAC+V, and TAC+ATRA cohorts at week 2 using various doses of ATRA (2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg administered daily by intraperitoneal injection). There was no significant dose-dependent effect of ATRA on heart rate, PR interval, or QRS duration, but ATRA treatment dose-dependently reduced P-wave prolongation (Figures 11A-11D). Maximal shortening of P-wave duration was seen at an ATRA dose of 10 mg / kg, with no further shortening at the 20 mg / kg dose. Notably, ventricular ectopy was increased at 20 mg / kg ATRA, indicating possible cardiotoxicity at this high dose.

[0086] Summary: ATRA dose-dependently normalizes atrial activation time in TAC-choke mice.

[0087] Example 12. Further Experiments 1:To investigate the role of endogenous ATRA in maintaining normal atrial physiology, the effect of pressure overload on endogenous ATRA levels, and the therapeutic potential of increasing endogenous ATRA levels to prevent TAC-induced LA remodeling.

[0088] Hypothesis: Endogenous ATRA is required to maintain normal atrial physiology, TAC banding reduces ATRA levels in the LA and blood, and inhibition of the ATRA degradation pathway restores ATRA levels in the LA and prevents atrial remodeling.

[0089] 1.1 Investigation of the maintenance role of ATRA in atrial electrical and structural homeostasis. The present data show enrichment of Aldh1a1 and Aldh1a2 expression and relative deficiency of Cyp26b1 expression in postnatal atrial myocytes compared with ventricular myocytes, indicating that ATRA signaling remains enriched in postnatal atria. Herein, it is hypothesized that ATRA, which has an essential role in atrial specification, continues to have a maintenance role in atrial electrical and structural integrity in the adult heart. Differential expression of ALDHA1A1, ALDHA1A2, and CYP26B1 at the protein level in the atria and ventricles of the adult heart is confirmed using Western blot and immunofluorescence staining. ATRA levels in the atria, ventricles, and plasma are then quantified. Quantification of endogenous ATRA levels is performed. To investigate the role of ATRA in maintaining the electrical and structural integrity of adult atria, wild-type C57B1 / 6 male and female mice were cultured with 10-kDa siRNA, which has previously been shown to efficiently block ATRA biosynthesis in vivo. 40Treat with the ALDH1A1 and ALDH1A2 specific inhibitor WIN18446 (2 mg / g diet) for a total of 4 weeks. Quantify ATRA levels in atria, ventricles, and plasma after treatment with WIN18446 to confirm endogenous inhibition. Evaluate the effect of ATRA depletion on ECG and TTE parameters. Quantify the degree of fibrosis in the atria and ventricles using Masson's Trichrome staining. Based on electrophysiological or fibrotic changes in the LA, perform optical mapping to evaluate conduction and repolarization parameters. Differential gene expression of ion channels or profibrotic pathways will also be evaluated similarly to the TAC+ATRA experiments.

[0090] 1.2 Study of the effect of cardiac pressure overload on endogenous ATRA levels in LA and plasma. ATRA levels have been reported to be decreased in failing ventricular myocardium by mass spectrometry in patients with idiopathic dilated cardiomyopathy (IDCM) and in a guinea pig HF model. 26 In the NYU Metabolomics Core Resource Laboratory, ATRA levels will be quantified in the LA and plasma of Sham- and TAC-strangled mice as described in section 1.1 above. Paired analyses of plasma and LA tissue ATRA levels will be performed to evaluate whether plasma ATRA levels correlate with LA tissue ATRA levels. This data will provide information on whether plasma ATRA levels are a good surrogate indicator of LA ATRA levels. The relationship of atrial and plasma ATRA levels to the degree of atrial electrical and structural remodeling will also be studied.

[0091] 1.3 Study of the effect of the CYP26 inhibitor talarozole in preventing electrical and structural remodeling of the atrium due to cardiac pressure overload. Left atrium CYP26B1 transcript levels were elevated in patients with persistent AF. Similarly, Cyp26b1, as well as Cyp26a1 and Cyp26c1, were significantly upregulated in the LA of TAC-strangulated hearts at 2 weeks. These data suggest that increased ATRA degradation may be responsible for ATRA deficiency in the LA in humans and mice, contributing to atrial remodeling. To test this hypothesis, it has been shown that talarozole effectively increases ATRA levels in rodents. 41 Block all CYP26 isoforms using the pan-CYP26 inhibitor talarozole. Based on previously published data 41 The first dose (2.5 mg / kg) of talarozole or vehicle control is administered 12 hours before TAC constriction, twice daily for 2 weeks. Two weeks after TAC, physiological testing is performed using ECG and TTE. Histological analysis of the LA is also performed to quantify the fibrotic burden. If talarozole has a positive effect on atrial conduction parameters, optical mapping is performed to measure conduction and repolarization parameters. The effect of talarozole on ATRA levels is also evaluated in the LA as described in 1.1 and 1.2.

[0092] Discussion: Based on the present findings, inhibition of endogenous ATRA is expected to have adverse effects on atrial conduction parameters, leading to activation of proinflammatory and profibrotic pathways, albeit less severe than TAC strangulation. By simultaneously measuring plasma and left atrial ATRA levels after TAC strangulation, we will determine whether plasma ATRA levels are a good surrogate indicator of left atrial ATRA levels. We will also determine whether plasma and left atrial ATRA levels directly correlate with the severity of atrial conduction disease and fibrosis. This will provide information to guide potential therapies in patients with cardiac pressure overload conditions. Finally, we will determine whether inhibition of CYP26B1 can increase left atrial ATRA levels to a therapeutic threshold that can prevent pressure-induced remodeling. The measured left atrial ATRA levels may be unchanged or higher with TAC strangulation. However, the present data showing a beneficial response with ATRA replacement therapy indicate that, regardless of the endogenous ATRA levels reached, they are insufficient to prevent adverse remodeling due to pressure overload.

[0093] 2: To clarify the anti-inflammatory mechanism by which ATRA prevents and reverses atrial fibrosis, and further to investigate the ability of ATRA to reverse atrial fibrosis in advanced stages of HF.

[0094] Hypothesis: ATRA therapy prevents and reverses atrial fibrosis by negatively regulating proinflammatory cytokine / chemokine / growth factor pathways in both early and more advanced stages of HF.

[0095] 2.1 Study of the effects of ATRA treatment on proinflammatory cytokines, chemokines and mediators in pressure-overloaded LA. Our RNA-seq analysis shows that ATRA reduces fibrosis by reducing the expression of proinflammatory cytokines, chemokines, cell adhesion molecules, and growth factors. To reveal the major inflammatory pathways regulated by ATRA in pressure-overloaded LA, we test the expression of 60 proinflammatory cytokines, chemokines, and growth factors at the protein level using an antibody array kit panel (RayBio® C-Series Mouse Inflammation Antibody Array 1 Kit, and Mouse Growth Factor Array C3). Using this unbiased protein-level screen, we identify the proinflammatory cascades most significantly affected by ATRA treatment and correlate this data with the inflammatory pathways identified by our comparative RNA-seq analysis.

[0096] 2.2 Study of the effect of ATRA treatment on mast cell-dependent PDGF signaling. To evaluate the effect of ATRA treatment on mast cell recruitment and activation in the LA of TAC-banded hearts, staining with toluidine blue (0.1%; Sigma-Aldrich) and avidin conjugated to a fluorescent dye (rhodamine-labeled avidin, 1:100; Vector Laboratories) was performed to quantify the number and activity levels of mast cells in the LA of tissue sections. 6 Avidin conjugated to a fluorescent dye binds to negatively charged heparin proteoglycans and identifies degranulated mast cells. The effect of ATRA on the levels of PDGF-A and PDGF-B is assayed using growth factor array data from 2.1. Findings from this study provide information on how ATRA prevents mast cell recruitment or activation in pressure-overloaded LA.

[0097] 2.3 Study of the effect of ATRA treatment on the TLR2 / NF-kB / Il-1b / NLRP3 inflammasome pathway. The effect of ATRA treatment on the expression levels of TLRs (TLR2 and TLR4), NLRP3, and activated caspase-1 (Casp1-p20) in Sham+V, TAC+V, and TAC+ATRA is studied by Western blot. Changes in IL-1b expression are assayed using the inflammation array kit used in 2.1. The effect of ATRA on NF-kB activation status is also examined by 1) quantifying the protein levels of IkB-a, an NF-kB inhibitor; 2) quantifying the phosphorylation (activation) status of NF-kB / p65; and 3) quantifying the nuclear localization status of NF-kB / p65. This allows us to determine the effect of ATRA on TLR-dependent signaling pathways that serve as regulatory hubs for the NLRP3 inflammasome as well as the production hubs for proinflammatory cytokines, chemokines, and growth factors.

[0098] 2.4 Study of the effect of ATRA treatment on the renin-angiotensin system (RAS) signaling pathway. Activation of the RAS signaling pathway in the LA by pressure overload is a potent inducer of atrial inflammation, fibrosis, and electrical remodeling. AngII promotes inflammation by increasing the production of proinflammatory cytokines such as IL-6, TNF, and chemotactic signals that recruit immune cell migration. Angiotensin-converting enzyme (ACE) is upregulated by TAC strangulation and significantly downregulated with ATRA therapy, providing an additional mechanism for inflammation reduction. The effect of ATRA treatment on ACE expression is verified at the protein level. The effect of ATRA treatment on circulating AngII levels in serum of Sham, Sham+ATRA, TAC, and TAC+ATRA mice is also studied by using an AngII enzyme immunoassay kit (RAB0010, Sigma-Aldrich). The effect of ATRA therapy on IL-6 and TNF expression is also examined using the inflammation array kit in 2.1.

[0099] 2.5 Investigating the ability of ATRA to reverse atrial fibrosis in more advanced stages of HF. As the delayed ATRA initiation model more accurately reflects the clinical picture of HF, we investigate the effect of ATRA on atrial remodeling when initiated at progressively later time points using TAC constriction. The present data show that ATRA can reverse atrial fibrosis when administration is initiated 2 weeks after TAC constriction. To investigate whether ATRA can reverse fibrosis at a later time point after TAC constriction, administration of ATRA therapy or vehicle control is initiated 4 or 6 weeks after TAC constriction and treatment is continued for 2 weeks. The effect of ATRA on preventing progression or reversing atrial remodeling is first examined using ECG and TTE. Fibrosis in the LA is quantified using ImageJ analysis of Masson's Trichrome stained sections. Optical mapping is also performed to quantify changes in atrial and ventricular conduction velocity and repolarization parameters.

[0100] Discussion: The above experiments explore the mechanisms underlying the potent anti-inflammatory and anti-fibrotic effects of ATRA in pressure-overloaded LA.

[0101] 3: To clarify the ionic mechanism by which ATRA improves atrial conduction parameters and to investigate its effect on atrial arrhythmia susceptibility.

[0102] Hypothesis: ATRA therapy improves atrial conduction parameters, in part, by normalizing sodium conductance and gap junction conductance. Improvement of atrial conduction parameters with ATRA therapy reduces susceptibility to atrial arrhythmias.

[0103] 3A.1 Quantification of Nav1.5 expression in ATRA-treated LA of TAC-confined hearts. Scn5a expression is decreased at the RNA and protein levels in the LA after 2 weeks of TAC-confined hearts. 1ATRA treatment of TAC-banded hearts increases Scn5a expression in the left atrium and improves conduction parameters. Western blot analysis is performed to quantify Nav1.5 levels in the LA of TAC+ATRA and TAC+V groups. Immunofluorescence staining is also used to evaluate whether ATRA treatment properly localizes Nav1.5 to the membrane.

[0104] 3A.2 Patch clamp assay to evaluate the effect of ATRA treatment on sodium currents in TAC-banded left atrial myocytes. To perform a comprehensive biophysical evaluation of sodium currents in TAC+ATRA and TAC+V left atrial myocytes. 1,18,42 .

[0105] 3A.3 Investigation of the effect of ATRA on the ErbB4 signaling pathway, a regulator of Scn5a expression. The ErbB4 signaling pathway, which enhances Scn5a expression in atrial myocardium, is downregulated in TAC-strangulated LA. 1 Preliminary data obtained herein show that ATRA treatment prevents the downregulation of ErbB4 in the LA by TAC strangulation at the protein level. The activation status of ErbB4 (phospho-ErbB4-Y1284) and its heterodimerization partner ErbB2 (phospho-ErbB2-Tyr1248) is examined in the LA of TAC+ATRA and TAC+V hearts. The expression and activity levels of downstream effectors of ErbB4 signaling, such as the MAPK ERK1 / 2 and the transcription factor ETV1, are also studied. 43,44 .

[0106] 3B.1 Study of the ATRA-dependent correction mechanism of Cx43 remodeling in LA. The present data showed that ATRA treatment prevented Cx43 remodeling in LA. Ca 2+Phosphorylation of key serine residues (S325 / S328 / S330) of Cx43 (pS-Cx43) by calmodulin protein kinase II (CaMKII) and casein kinase 1d (CK1d) has been shown to be important for assembly into gap junctions and maintenance at the intercalated disc. 45,46 It is hypothesized herein that ATRA therapy prevents Cx43 remodeling by restoring phosphorylation of Cx43 at S325 / S328 / S330 sites. The percentage of total Cx43 phosphorylated at S325 / S328 / S330 is quantified in LA samples from Sham, Sham+ATRA, TAC, and TAC+ATRA hearts using antibodies.

[0107] 3B.2 Study of the role of ATRA in improving CaMKII oxidation status and ROS production to protect against Cx43 remodeling. Oxidized CaMKII plays a key role in Cx43 hypophosphorylation and remodeling 46 It is hypothesized herein that ATRA treatment partially protects LA from Cx43 remodeling by reducing oxidized CaMKII levels. Western blot analysis with an antibody against oxidized CaMKII (Met281 / 282, MilliporeSigma, 07-1387; rabbit) is used to quantify oxidized CaMKII levels in the left atria of Sham+V, Sham+ATRA, TAC+V, and TAC+ATRA. TAC strangulation is a known inducer of reactive oxygen species (ROS) production. To assess whether ATRA treatment reduces ROS activation, we used the fluorescent ROS sensor 2',7'-dichlorodihydrofluorescein diacetate (H 2 The study was conducted using left atrial myocytes isolated from hearts treated with Sham+V, Sham+ATRA, TAC+V, and TAC+ATRA loaded with 1000x DCFDA. 46 Mitochondrial ROS production is also measured using MitoSOX fluorescence with flow cytometry. 47,48 .

[0108] 3B.3 Study of the effect of ATRA treatment on CK1d protein stabilization. Our RNA-seq analysis showed no significant changes in CK1d transcript abundance with ATRA treatment, but this does not exclude changes at the protein level. CK1d protein levels are quantified in Sham, Sham+ATRA, TAC, and TAC+ATRA left atria using Western blot analysis.

[0109] 3B.4 Study of the effect of ATRA treatment on the renin-angiotensin system (RAS) signaling pathway on Cx43 remodeling. Activation of the RAS signaling pathway in the LA by pressure overload is a potent inducer of Cx43 gap junction remodeling. Blockade of the RAS pathway by inhibiting AngII synthesis (angiotensin-converting enzyme inhibitors, ACEIs) or directly blocking the angiotensin II receptor (angiotensin II receptor antagonists, ARBs) significantly reduced Cx43 remodeling. The effect of ATRA treatment on ACE expression and AngII production at the protein level is examined as described in 2.5.

[0110] 3C Evaluation of the effect of ATRA therapy on arrhythmia susceptibility in TAC-banded hearts. Ambient arrhythmia burden in the atria and ventricles of TAC-banded hearts treated with ATRA or vehicle control will be quantified by using implantable telemetry 49 A comprehensive electrophysiological study will be performed to assess baseline electrophysiological properties and further to assess arrhythmia susceptibility using in vivo atrial arrhythmia provocation testing. 44 Complete optical mapping experiments by studying the effects of ATRA therapy on conduction parameters, repolarization properties, and arrhythmia susceptibility using programmed stimulation protocols on Langendorff-perfused hearts. 18,42,50 .

[0111] Discussion: Based on the present findings, it is predicted herein that ATRA normalizes sodium channel behavior and restores a signaling environment that prevents gap junction remodeling in pressure-overloaded hearts. It will be determined whether ATRA reduces oxidized CaMKII levels by restoring pS-Cx43 levels and reducing ROS activation. It will also be determined whether ATRA stabilizes CK1d protein levels. It is anticipated that ATRA therapy reduces RAS activation by decreasing ACE levels in pressure-overloaded LA. Knowledge gained from this study provides mechanistic insight into how ATRA treatment corrects left atrium electrical remodeling.

[0112] SUMMARY: This study addresses how ATRA modulates key inflammatory and ionic pathways to maintain normal adult atrial physiology, as well as its therapeutic benefit in preventing and reversing pathological atrial remodeling that leads to AF. As there are currently no effective therapies that directly target atrial remodeling, this discovery represents an important advancement with high therapeutic potential. References

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[0114] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Furthermore, it should be understood that all values ​​are approximate and are provided for illustrative purposes. Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Claims

1. A composition for preventing and / or treating atrial arrhythmia in a subject in need thereof, the composition comprising a retinoid or a pharmaceutically acceptable salt thereof.

2. The atrial arrhythmia is (a) atrial fibrillation; or (b) Atrial tachycardia 2. The composition of claim 1, wherein:

3. The composition described in claim 1, wherein administration of the retinoid or a pharmaceutically acceptable salt thereof shortens the P-wave duration of the subject as determined by an electrocardiogram (ECG).

4. A composition for preventing or reversing atrial remodeling in a subject in need thereof, the composition comprising a retinoid or a pharmaceutically acceptable salt thereof.

5. The composition described in claim 4, wherein the atrial remodeling is characterized by a prolongation of P-wave duration as determined by an electrocardiogram (ECG).

6. the atrial remodeling (a) atrial fibrosis; or (b) Atrial myopathy The composition of claim 4 comprising:

7. A composition for preventing or treating a conduction disease in a subject in need thereof, the composition comprising a retinoid or a pharmaceutically acceptable salt thereof.

8. A composition for improving conduction parameters in a subject in need thereof, the composition comprising a retinoid or a pharmaceutically acceptable salt thereof.

9. The retinoid (a) retinoic acid or all-trans retinoic acid (ATRA); and / or (b) administered at a dose of about 0.1 μg / kg to about 20 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 100 mg / kg, or about 5 mg / kg to about 50 mg / kg; and / or (c) administered or delivered to the atrium; and / or (d) administered by oral, intravenous, subcutaneous, or intramuscular routes; and / or (e) encapsulated; and / or (f) The composition of any one of claims 1 to 8, administered in combination with one or more additional therapeutic agents.

10. The subject is (a) determined to be deficient in said retinoid; and / or (b) diagnosed with acute-onset heart failure; and / or (c) diagnosed with hypertension; and / or (d) is human; and / or (e) over 65 years of age.

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