Application of antiarrhythmic drugs to stem cell-derived cardiomyocytes and uses thereof

Abstract

The present invention relates to antiarrhythmic agents for use in methods for treating heart failure by transplantation of stem cell-derived cardiomyocytes, antiarrhythmic cardiomyocyte populations, methods for obtaining antiarrhythmic cardiomyocyte populations by in vitro exposure of stem cell-derived cardiomyocytes to antiarrhythmic agents, and / or medical uses of antiarrhythmic cardiomyocyte populations in preventing arrhythmias and treating heart failure. In particular, the present invention relates to transplantation of antiarrhythmic cardiomyocyte populations or co-administration of transplanted stem cell-derived cardiomyocytes with antiarrhythmic agents in vivo.

Description

[Technical Field]

[0001] The present invention relates generally to the field of stem cells, and more particularly to an antiarrhythmic cardiomyocyte cell population, a method for obtaining the antiarrhythmic cardiomyocyte cell population, and its medical use in the prevention or reduction of arrhythmias resulting from transplantation of stem cell-derived cardiomyocytes in the treatment of heart failure. The present invention also relates to an antiarrhythmic agent for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. [Background technology]

[0002] The heart is one of the body's least regenerative organs. As a result, when cardiac injury occurs, myocardial cells die, leaving behind scarred areas that cannot contract. This leads to reduced pumping power, heart failure, and increased morbidity and mortality. Heart disease is a leading cause of death worldwide.

[0003] Human pluripotent stem cells can differentiate into cardiomyocytes and are being investigated for repair of damaged hearts when cardiomyocytes are lost or fail.

[0004] In the few cases in which cardiomyocytes were injected endocardially into non-human primate hearts, several types of arrhythmias were detected during the first 4–6 weeks after engraftment: sustained ventricular tachycardia, nonsustained ventricular tachycardia, and accelerated idiopathic ventricular rhythm.

[0005] The reason for these arrhythmias is currently unknown, but it is not surprising that they occur transiently, as all cardiomyocytes have the ability to contract and beat. It is expected that transplanted cells will beat on their own before integrating with the host myocardium. Furthermore, scarred areas of the myocardium, especially the border zone, are known to induce arrhythmias in patients. Injection of materials into the scar may itself be arrhythmogenic.

[0006] In monkeys, arrhythmias occurring after cell infusion have been described as being of a different type and origin (derived from ectopic lesions) than conventional arrhythmias occurring after acute myocardial infarction and heart failure (reentry mechanism). This clearly defines the need to treat cell transplantation-induced arrhythmias and the fact that the arrhythmias would not have occurred if the cells had not been infused. Therefore, this type of arrhythmia may be considered a new type of condition caused by cell transplantation, and specific treatments for this type of arrhythmia do not yet exist.

[0007] To regenerate cardiac muscle tissue in vivo after cardiac injury, various strategies have been explored, including the generation of various types of pluripotent stem cell-derived cardiac lineage cells, such as early cardiovascular progenitor cells, immature beating cardiomyocytes, and more mature, e.g., heterotypic tissue-engineered cardiac constructs. Generally, all approaches to generating such cells in vitro result in cardiomyocytes with a relatively immature phenotype, resembling fetal cells from the first and second trimesters of pregnancy in terms of their gene expression profile, cell morphology, sarcoplasmic histology, electrophysiological properties, and resulting contractile force. Notably, several of the various strategies have been shown to primarily produce cells capable of engraftment and maturation after transplantation. However, overall efficiency has been limited. This is particularly due to a limited understanding of the underlying mechanisms mediating engraftment and maturation after transplantation, highlighting the need for further research, particularly to identify additional therapeutics for transplanted cardiomyocytes and promote their development and integration.

[0008] The present invention aims to address the problem of arrhythmias caused by engraftment of stem cell-derived cardiomyocytes in methods for treating heart failure. In particular, the present invention aims to promote the integration of transplanted stem cells into the host myocardium, for example, to avoid, prevent, and / or reduce arrhythmias. Summary of the Invention [Means for solving the problem]

[0009] The above-mentioned objects are achieved by the aspects of the present invention. Furthermore, the present invention can also solve further problems that will become apparent from the disclosure of the exemplary embodiments.

[0010] In its broadest aspects, the present invention relates to in vitro and in vivo approaches for preventing or reducing or treating arrhythmias resulting from transplantation of stem cell-derived cardiomyocytes and treating heart failure in patients.

[0011] In one aspect, the present invention relates to a method for obtaining a population of antiarrhythmic cardiomyocytes, comprising culturing stem cell-derived cardiomyocytes in a medium comprising one or more antiarrhythmic agents.

[0012] In one aspect, the present invention relates to a population of antiarrhythmic cardiomyocytes for use as a medicament.

[0013] In one aspect, the present invention relates to a population of anti-arrhythmic cardiomyocytes for use in the treatment of heart failure.

[0014] In one aspect, the present invention relates to an anti-arrhythmic cardiomyocyte cell population for use in preventing or reducing arrhythmias resulting from transplantation of stem cell-derived cardiomyocytes.

[0015] In one aspect, the invention relates to a kit comprising one or more antiarrhythmic agents and stem cell-derived cardiomyocytes.

[0016] In another aspect, the invention relates to an antiarrhythmic agent for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes.

[0017] A further aspect of the present invention relates to a composition comprising stem cell-derived cardiomyocytes, one or more antiarrhythmic agents, and optionally a biomaterial, for use in a method for the treatment of heart failure.

[0018] Without being bound by any theory, the inventors believe that the arrhythmias observed in hosts transplanted with stem cell-derived cardiomyocytes are a symptom caused by the stem cells not yet fully integrated into the myocardium. The inventors have now demonstrated approaches to prevent or reduce arrhythmias and / or treat heart failure. One approach involves obtaining an antiarrhythmic cardiomyocyte population by contacting stem cell-derived cardiomyocytes in vitro with an antiarrhythmic drug that alters the regulation of gene expression of key genes related to the ability of cardiomyocytes to connect and contract synchronously. This is believed to promote the integration of the antiarrhythmic cardiomyocyte population into the host myocardium after transplantation, thereby preventing or reducing the aforementioned antiarrhythmic effects and providing treatment for heart failure due to the suppression of the ability of stem cell-derived cardiomyocytes to contract and beat independently. Another approach is to co-administer one or more antiarrhythmic drugs in vivo during or after transplantation of stem cell-derived cardiomyocytes into patients. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows the gene expression pattern of CACNA1G in stem cell-derived cardiomyocytes after 5 days of exposure to 0.1 μM and 1 μM amiodarone (day 23 after differentiation induction). The negative and positive controls refer to immature (day 9) and mature (day 42) cardiomyocytes, respectively. [Figure 2] Figure 2 shows the gene expression pattern of GJA5 after 5 days of exposure to 0.1 μM and 1 μM amiodarone in stem cell-derived cardiomyocytes (day 23 after differentiation induction). The negative and positive controls refer to immature (day 9) and mature (day 42) cardiomyocytes, respectively. [Figure 3] Figure 3 shows the gene expression pattern of NPPA after 5 days of exposure to 0.1 μM and 1 μM amiodarone in stem cell-derived cardiomyocytes (day 23 after differentiation induction). The negative and positive controls refer to immature (day 9) and mature (day 42) cardiomyocytes, respectively. [Figure 4]Figure 4 shows the gene expression pattern of NPPB after 5 days of exposure to 0.1 μM and 1 μM amiodarone in stem cell-derived cardiomyocytes (day 23 after differentiation induction). The negative and positive controls refer to immature (day 9) and mature (day 42) cardiomyocytes, respectively. [Figure 5] Figure 5 shows the gene expression pattern of NKX2-5 after 5 days of exposure to 0.1 μM and 1 μM amiodarone in stem cell-derived cardiomyocytes (day 23 after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 6] Figure 6 shows the gene expression pattern of TNNT2 in stem cell-derived cardiomyocytes after 5 days of exposure to 0.1 μM and 1 μM amiodarone (23 days after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 7] Figure 7 shows the gene expression pattern of ACTA2 after 5 days of exposure to 0.1 μM and 1 μM amiodarone in stem cell-derived cardiomyocytes (day 23 after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 8] Figure 8 shows the gene expression pattern of SCN5A in stem cell-derived cardiomyocytes after 5 days of exposure to 0.1 μM and 1 μM amiodarone (day 23 after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 9] Figure 9 shows the gene expression pattern of NPPA after 5 days of exposure to 1 μM, 10 μM, and 100 μM lidocaine in stem cell-derived cardiomyocytes (day 23 after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 10] Figure 10 shows the gene expression pattern of NPPB after 5 days of exposure to 1 μM, 10 μM, and 100 μM lidocaine in stem cell-derived cardiomyocytes (23 days after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 11] Figure 11 shows the gene expression pattern of NKX2-5 in stem cell-derived cardiomyocytes after 5 days of exposure to 1 μM, 10 μM, and 100 μM lidocaine (23 days after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 12] Figure 12 shows the gene expression pattern of TNNT2 in stem cell-derived cardiomyocytes after 5 days of exposure to 1 μM, 10 μM, and 100 μM lidocaine (23 days after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 13] Figure 13 shows the gene expression pattern of SCN5A in stem cell-derived cardiomyocytes after 5 days of exposure to 0.1 μM and 1 μM lidocaine (23 days after differentiation induction). The negative control refers to immature (day 9) cardiomyocytes. [Figure 14] Figure 14 shows gene expression of CACNA1G, GJA5, NPPA, and NPPB in 21-day-old stem cell-derived cardiomyocytes after 5 days of exposure to 10 μM amiodarone followed by a 2-day recovery period in the absence of the drug. Untreated cardiomyocytes are shown as controls. Cardiomyocytes were maintained as three-dimensional suspension clusters with diameters ranging from approximately 150 μm to 300 μm throughout the experiment. [Figure 15] Figure 15 shows the beat-to-beat coefficient of variation (CV) of stem cell-derived cardiomyocytes after exposure to antiarrhythmic drugs at the indicated concentrations at baseline (left) and after stimulation with the arrhythmogenic agent 200 nM moxifloxacin (right panel). Bars represent the mean + standard error of the mean. N=12 and N=18 for compound-treated conditions and controls, respectively. Asterisks indicate statistical significance (p<0.05) based on a Kruskal-Wallis test comparing all compounds to the control treatment. [Figure 16] Figure 16 shows the beat-to-beat coefficient of variation (CV) of stem cell-derived cardiomyocytes after exposure to antiarrhythmic drug combinations applied at concentrations of 1 μM sotalol, 0.1 μM amiodarone, 0.1 μM metoprolol, 1 μM mexiletine, 1 μM propranolol, and comparisons with single agents. All conditions were measured after stimulation with 200 nM moxifloxacin. Bar graphs represent the mean + standard error of the mean. N = 70 and N = 12 for single-compound-treated conditions and compound combinations, respectively. Asterisks indicate statistical significance (p < 0.05) based on a Kruskal-Wallis test comparing each compound-treated group to the single-compound control. DETAILED DESCRIPTION OF THE INVENTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology, which are known to those skilled in the art.

[0021] Please note that all headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0022] The use of any and all examples or exemplary phrases (e.g., "such as") presented herein is intended merely to further clarify the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0023] Throughout this application, the terms "method" and "protocol" are used interchangeably.

[0024] Throughout this application, the terms "cultivating," "contacting," and "exposing" are used interchangeably.

[0025] Throughout this application, the terms "human subject," "patient," and "host" are used interchangeably.

[0026] As used herein, "a" or "an" or "the" may mean one or more. Unless otherwise indicated herein, terms provided in the singular also include plural references.

[0027] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). Furthermore, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted.

[0028] As used herein when referring to a measurable value such as an amount of cells, compounds, or agents of the invention, a dose, a temperature, etc., the term "about" is intended to encompass a variation of 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0029] As used herein, the term "day" in relation to a protocol refers to a specific time for carrying out a specific step.Generally, unless otherwise stated, "day 0" refers to the start of the protocol, by plating stem cells, or transferring stem cells to an incubator, or contacting stem cells in the current cell culture medium with a compound before transplanting stem cells.Typically, the start of the protocol is by transferring undifferentiated stem cells to a different cell culture medium and / or container, plating or incubating, and / or first contacting undifferentiated stem cells with a compound that affects undifferentiated stem cells in such a way that the differentiation process begins.

[0030] When referring to "day x," such as day 1, day 2, etc., this is relative to the start of the protocol on day 0. One of skill in the art will recognize that the exact date and time for performing a step may vary unless otherwise specified. Thus, "day x" is intended to encompass a range of time, such as + / - 10 hours, + / - 8 hours, + / - 6 hours, + / - 4 hours, + / - 2 hours, or + / - 1 hour. Alternatively, durations or times for performing steps of methods according to the invention are described in "hours."

[0031] In the following, the method according to the invention will be described in more detail by means of non-limiting embodiments and examples.

[0032] As used herein, the term "arrhythmia" refers to a condition in which the heart beats in an irregular or abnormal rhythm. In macaques, specific cell-induced arrhythmias have been demonstrated to be non-sustained ventricular tachycardia, sustained ventricular tachycardia, and sustained accelerated idiopathic ventricular rhythm.

[0033] Thus, in one embodiment, the arrhythmia treatment is non-sustained ventricular tachycardia, sustained ventricular tachycardia, and / or sustained accelerated idiopathic ventricular rhythm.

[0034] As used herein, the terms "antiarrhythmic agent" or "antiarrhythmic drugs" or "antiarrhythmic compound" or "antiarrhythmic drug" refer to one or more pharmaceutical agents classified into different drug classes (Vaughan Williams classes) depending on their mechanism of action. Class I drugs primarily block sodium channels, Class II drugs block beta-receptors, Class III drugs block potassium channels, and Class IV drugs affect calcium channels. It is important to note that a drug class may affect multiple ion channel types, but is classified by its primary function.

[0035] In one embodiment, the antiarrhythmic agent is selected from the list of Class I, Class II, Class III, Class IV, and Class V antiarrhythmic agents. In a further embodiment, the antiarrhythmic agent is selected from the list of Class I, Class II, and Class III antiarrhythmic agents. In one embodiment, the antiarrhythmic agent is a Class III antiarrhythmic agent.

[0036] As used herein, the term "antiarrhythmic cardiomyocyte population" should be understood as cardiomyocytes obtained by the methods of the present invention that have modified properties that result in reduced beat-to-beat variability and / or reduced susceptibility to arrhythmias and / or other arrhythmia-like events.

[0037] As used herein, the term "biomaterial" refers to any chemical, synthetic or natural, intended to interact with cellular products. Such biomaterials include a group of natural and / or synthetic polymeric materials, including, but not limited to, alginate, chitosan, cellulose, agarose, gelatin, hyaluronic acid, silk fibroin, fibrin and / or collagen, poly-urethane, poly-vinyl alcohol, poly-hydroxyester, poly-propylene fumarate, and other synthetic, biodegradable and / or stimuli-sensitive hydrogels, bioactive glasses.

[0038] The terms "cardiac muscle cell," "cardiomyocyte," "myocardiocyte," and "cardiac myocyte" may be used interchangeably and refer to the muscle cells that make up the myocardium (heart muscle). Each cardiomyocyte contains myofibrils, specialized organelles made up of long chains of sarcomeres, the basic contractile units of muscle cells.

[0039] As used herein, the term "heart failure" refers to the heart's inability to keep up with demands, specifically, the heart's inability to pump blood with normal efficiency. When this occurs, the heart cannot provide sufficient blood flow to other organs, such as the brain, liver, and kidneys. Heart failure can be due to failure of the right ventricle, left ventricle, or both ventricles. Specifically, heart failure can be myocardial infarction, commonly known as a heart attack, which occurs when blood flow to a part of the heart is reduced or stopped, causing damage to the heart muscle. Prolonged impaired blood flow to the heart triggers an ischemic cascade, and cardiac cells in the area of the blocked coronary artery die, primarily by necrosis (infarction), and never return.

[0040] "Stem cells" should be understood as undifferentiated cells that have differentiation and proliferation capabilities (especially self-renewal capabilities) but maintain differentiation potential. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells according to their differentiation potential. Pluripotent stem cells refer to stem cells that can be cultured in vitro and have the potential to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and / or extraembryonic tissues (pluripotency). Pluripotent stem cells refer to stem cells that have the ability to differentiate into multiple, but not all, types of tissues or cells. Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues or cells. Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, stem cells in tissues, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Pluripotent stem cells also include Muse cells (multilineage differentiation stress-resistant cells) obtained from mesenchymal stem cells (MSCs) and GS cells produced from germ cells (e.g., testes). Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. By introducing the products of a specific set of pluripotency-related genes, adult cells can be converted into pluripotent stem cells. Embryonic stem cells can be produced by culturing the inner cell mass obtained without destroying the embryo. Embryonic stem cells are available from a given tissue and are commercially available.

[0041] As used herein, the term "stem cell-derived cardiomyocytes" refers to cardiomyocytes at various developmental stages induced through in vitro protocols to obtain non-natural stem cell products similar to human cardiac myocytes. In one embodiment, stem cell-derived cardiomyocytes are derived from human pluripotent stem cells, such as human embryonic stem cells. In one embodiment, stem cell-derived cardiomyocytes are derived from induced pluripotent stem cells. In one embodiment, stem cell-derived cardiomyocytes are derived from other sources, such as the transdifferentiation of somatic cells into cardiomyocytes (Masaki Ieda et al., Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors, Volume 142, Issue 3, P375-386, August 6, 2010). Those skilled in the art will be able to provide stem cell-derived cardiomyocytes. One available method for use in the present invention has been described (Kempf H et al. Bulk cell density and Wnt / TGFbeta signaling regulate mesendodermal patterning of human pluripotent stem cells. Nat Commun. 2016;7:13602). In one embodiment, stem cell-derived cardiomyocytes include their precursor or progenitor cells.

[0042] Stem cell-derived cardiomyocyte populations are typically characterized by the expression of at least three of the markers selected from NKX2.5, TNNT2, ACTN2, MYH6 and / or MYH7, MYL2 and / or MYL7, TNNI1 and / or TNNI3. Depending on the maturation state of the stem cell-derived cardiomyocytes, the stem cell-derived cardiomyocytes or their progenitor or precursor cells may include cells expressing ISL1, GATA4, MEF2C, SSEA-1, PDGFRA, MESP1, and / or combinations thereof.

[0043] As referred to herein, the terms "transplantation" and "engraftment" are used interchangeably and refer to the process of transplanting viable stem cell-derived cardiomyocytes or antiarrhythmic cardiomyocyte populations obtained according to the methods of the present invention into or near the heart of a human subject or patient.

[0044] As referred to herein, the terms "graft" and "transplant" refer to stem cell-derived cardiomyocytes or antiarrhythmic cardiomyocyte populations obtained according to the methods of the present invention that are transplanted into a human subject or patient via the aforementioned procedures.

[0045] Aspects of the present invention relate to antiarrhythmic agents for use in methods for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes.

[0046] In one embodiment according to this aspect, the stem cell-derived cardiomyocytes are transplanted into a patient.

[0047] In one embodiment according to this aspect, the one or more antiarrhythmic agents are for co-administration with transplantation of stem cell-derived cardiomyocytes in vivo.

[0048] In one embodiment according to this aspect, one or more antiarrhythmic agents are co-administered during transplantation of stem cell-derived cardiomyocytes.

[0049] In one embodiment according to this aspect, one or more antiarrhythmic agents are co-administered after transplantation of the stem cell-derived cardiomyocytes.

[0050] This coadministration of stem cell-derived cardiomyocyte transplantation and antiarrhythmic drugs can be further improved by combining the cells and drugs with biomaterials such as hydrogels to enable long-term, locally restricted release of the drug at the target site (Jianyu Li and David J. Mooney, "Designing hydrogels for controlled drug delivery," Nat Rev Mater. 2016 Dec;1(12):16071, published online October 18, 2016), for example, by incorporating the antiarrhythmic drug into biodegradable hydrogel particles (Radhika Narayanaswamy and Vladimir P Torchilin, "Hydrogels and Their Applications in Targeted Drug Delivery," Molecules. 2019 Feb;24(3):603, published online February 8, 2019).

[0051] In one aspect, the present invention relates to a method for obtaining a population of antiarrhythmic cardiomyocytes, comprising culturing stem cell-derived cardiomyocytes in a medium comprising one or more antiarrhythmic agents.

[0052] In one embodiment, the present invention relates to a method for obtaining an antiarrhythmic cardiomyocyte population, comprising culturing stem cell-derived cardiomyocytes or their precursors or progenitor cells in vitro in a medium comprising one or more antiarrhythmic agents.

[0053] In one embodiment, the stem cell-derived cardiomyocytes are cultured in a medium containing an antiarrhythmic agent for less than 24 hours, at least 24 hours, 24-48 hours, at least 48 hours, 48-72 hours, at least 72 hours, 72-96 hours, or at least 96 hours.

[0054] In one embodiment, stem cell-derived cardiomyocytes are cultured with 1 nM to 100 nM, about 1 nM, about 10 nM, about 20 nM, about 40 nM, about 60 nM, about 80 nM, about 100 nM, or 0.1 to 100 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 100 μM of a Class I, Class II, and / or Class III antiarrhythmic agent.

[0055] In one embodiment according to this aspect, the population of anti-arrhythmic cardiomyocytes is implanted into the patient.

[0056] Another aspect of the present invention relates to an antiarrhythmic cardiomyocyte population for use as a medicament. In one embodiment according to this aspect, the invention relates to an antiarrhythmic cardiomyocyte population for use in a method for the treatment of heart failure. In one embodiment according to this aspect, the invention relates to an antiarrhythmic cardiomyocyte population for use in preventing or reducing arrhythmias resulting from transplantation of stem cell-derived cardiomyocytes. In one embodiment, the invention relates to preventing or reducing arrhythmia induction.

[0057] The advantageous benefits of transplanting antiarrhythmic cardiomyocyte populations in patients are that it avoids the typical risks of antiarrhythmic drugs (e.g., bradycardia, AV block, QT interval prolongation) (D.P. Zipes, Proarrhythmic Effects of Antiarrhythmic Drugs, 1987 Apr 30;59(11):26E-31E) and other side effects including, but not limited to, interstitial pulmonary fibrosis, hypo- and hyperthyroidism, hepatotoxicity, hypotension, tremor, dizziness, mild fever, photosensitivity, neuropathy, and muscle weakness (Thomas W. Nygaard et al, Adverse Reactions to Antiarrhythmic Drugs During Therapy for Ventricular Arrhythmias, JAMA. 1986;256(1):55-57); Janice B. Schwartz et al, Adverse Effects of Antiarrhythmic Drugs, Drugs volume 21, pages 23-45(1981) to reduce arrhythmia induction.

[0058] The advantage of antiarrhythmic cardiomyocyte populations is that they are likely to exhibit superior engraftment into the host myocardium compared to stem cell-derived cardiomyocytes because their reduced arrhythmogenic potential allows for synchronized beating behavior, promotes rapid and / or correct integration of the cells, and allows for a more rapid maturation process.

[0059] An additional advantage of the present invention is that in vitro exposure of stem cell-derived cardiomyocytes to antiarrhythmic drugs can result in higher concentration levels that are at least 10, 100, 1000, or even 10,000 times higher than typical plasma concentrations in vivo, i.e., 2-6 μg / ml for lidocaine, 0.6-1.7 μg / ml for mexiletine, 2.1-300 ng / ml for propranolol (Plasma concentrations of propranolol and 4-hydroxypropranolol during chronic oral propranolol therapy, Br J Clin Pharmacol. 1979 Aug;8(2):163-167), and 100-140 ng / ml for metoprolol (Plasma levels and effects of metoprolol on blood pressure and heart rate in hypertensive patients after an acute dose and between two doses during long-term treatment, Clinical pharmacology and therapeutics, first published: April 1999). 1975), allowing exposure at 0.5–2.5 μg / ml for amiodarone and 1–3 μg / ml for sotalol, thereby increasing the chances of success in obtaining an antiarrhythmic cardiomyocyte population compared to in vivo treatment.

[0060] Overall, antiarrhythmic cardiomyocyte populations are more likely to successfully integrate into the host myocardium compared with typical stem cell-derived cardiomyocytes, thereby improving cell transplant outcomes by increasing the overall pump function of the recipient heart.

[0061] In a further embodiment, the anti-arrhythmic cardiomyocyte population has at least a 50% reduction in coefficient of variation (CV) or beat-to-beat variability when compared to stem cell-derived cardiomyocytes.

[0062] Antiarrhythmic agents such as amiodarone can be administered as solutions, tablets, hydrogel encapsulations, etc., and by various routes of administration, such as intravenous, oral, or endocardial. This has been shown in patients (Garcia JR et al., Minimally invasive delivery of hydrogel-encapsulated amiodarone to the epicardium reduces atrial fibrillation). In one embodiment, the Class III antiarrhythmic agent is sotalol. As used herein, "sotalol" refers to CAS number 3930-20-9, which has the formula C12H20N2O3S. In one embodiment, the concentration of sotalol is 100 nM.

[0063] In one embodiment of the present invention, the antiarrhythmic cardiomyocyte population is obtained in vitro by culturing stem cell-derived cardiomyocytes in a medium containing one or more antiarrhythmic agents.

[0064] One embodiment of the present invention relates to an antiarrhythmic agent for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes.

[0065] In one embodiment, the Class III antiarrhythmic agent is amiodarone. As used herein, "amiodarone" refers to CAS number 1951-25-3, which has the formula C25H29I2NO3. Amiodarone has been found to specifically increase the expression of gap junctions (GJA5), cell membrane structures that allow cardiomyocytes to connect and contract in a synchronized manner. This is an important part of how cardiomyocytes work together to ensure the normal propagation of electromechanical impulses that ensure proper contraction of the heart. This finding supports the belief that amiodarone increases the engraftment and integration of stem cell-derived cardiomyocytes into host tissue and helps ensure synchronized contraction of cardiomyocytes.

[0066] Furthermore, amiodarone was found to increase CACNA1G expression. Calcium handling is a crucial part of cellular contractility and action potential generation. Therefore, this finding supports the drug's stabilizing effect on cellular rhythm and contraction.

[0067] Amiodarone has also been found to suppress ANP and BNP expression. This may indicate a possible suppression of hypertrophy response, or may simply be an indicator of better functioning cardiomyocytes. ANP and BNP are known to increase as heart failure worsens, so lower ANP and BNP are a sign of better functioning cardiomyocytes and the heart. This finding supports the idea that amiodarone improves the functionality of stem cell-derived cardiomyocytes or antiarrhythmic cardiomyocyte populations. In one embodiment, the concentration of amiodarone is 10 nM.

[0068] In another embodiment, the antiarrhythmic agent is a Class I antiarrhythmic agent. In one embodiment, the Class I antiarrhythmic agent is lidocaine. As used herein, "lidocaine" refers to CAS No. 137-58-6, which has the formula C14H22N2O. In one embodiment, the concentration of lidocaine is 100 nM. In one embodiment, the Class I antiarrhythmic agent is mexiletine. As used herein, "mexiletine" refers to CAS No. 31828-71-4, which has the formula C11H17NO. In one embodiment, the concentration of mexiletine is 100 nM.

[0069] In an alternative embodiment, the antiarrhythmic agent is a Class II antiarrhythmic agent. In one embodiment, the Class II antiarrhythmic agent is metoprolol. As used herein, "metoprolol" refers to CAS No. 51384-51-1, which has the chemical formula C158H25NO3. In one embodiment, the concentration of metoprolol is 10 nM.

[0070] In one embodiment, the Class II antiarrhythmic agent is propranolol. As used herein, "propranolol" refers to CAS number 525-66-6, which has the formula C16H21NO2. In one embodiment, the concentration of propranolol is 100 nM.

[0071] In one embodiment, the antiarrhythmic agent is understood to be a single compound only. In one embodiment, the antiarrhythmic agent is a pharmaceutical preparation. In one embodiment, the antiarrhythmic agent is a combination of one or more antiarrhythmic agents selected from the same or different classes.

[0072] As described herein, treatment of patients receiving stem cell-derived cardiomyocyte transplants can involve the administration of one or more antiarrhythmic agents by any suitable means. Antiarrhythmic agents can be formulated in any suitable manner for administration, including, but not limited to, intravenous injection using an infusion device or ingestion as a tablet. An infusion device refers to a medical system intended to deliver the cell product in the respective formulation to a recipient. Infusion devices are preferentially suitable for pericardial, epicardial, and / or endocardial delivery. Infusion devices can include, but are not limited to, needle-tipped syringes, needleless syringes, and infusion catheter systems suitable for delivery near the myocardial infarction region. Infusion devices include, but are not limited to, devices intended for intracoronary, endocardial, and / or epicardial injection.

[0073] When the antiarrhythmic agent comprises more than one antiarrhythmic compound, they may or may not be co-formulated and may be administered together or separately and / or on different dosing regimens and / or at different time intervals.

[0074] In a preferred embodiment, the antiarrhythmic agent is a combination of at least two classes of antiarrhythmic agents. In one embodiment, the antiarrhythmic agent includes a Class III antiarrhythmic agent and a Class I antiarrhythmic agent. In one embodiment, the antiarrhythmic agent includes amiodarone and a Class I antiarrhythmic agent. In one embodiment, the antiarrhythmic agent includes sotalol and a Class I antiarrhythmic agent. In one embodiment, the Class I antiarrhythmic agent is lidocaine. In one embodiment, the Class I antiarrhythmic agent is mexiletine.

[0075] In one embodiment, the antiarrhythmic agents include amiodarone and lidocaine.

[0076] In one embodiment, the antiarrhythmic agents include amiodarone and mexiletine.

[0077] In one embodiment, the antiarrhythmic agents include sotalol and lidocaine.

[0078] In one embodiment, the antiarrhythmic agents include sotalol and mexiletine.

[0079] In one embodiment, the antiarrhythmic agents comprise 1 μM sotalol and 1 μM mexiletine.

[0080] In one embodiment, the antiarrhythmic agent includes a Class III antiarrhythmic agent and a Class II antiarrhythmic agent. In one embodiment, the antiarrhythmic agent includes amiodarone and a Class II antiarrhythmic agent. In one embodiment, the antiarrhythmic agent includes sotalol and a Class II antiarrhythmic agent. In one embodiment, the Class II antiarrhythmic agent is metoprolol. In one embodiment, the Class II antiarrhythmic agent is propranolol.

[0081] In one embodiment, the antiarrhythmic agents include amiodarone and metoprolol.

[0082] In one embodiment, the antiarrhythmic agents comprise amiodarone and propranolol. In one embodiment, the antiarrhythmic agents comprise 0.1 μM amiodarone and 1 μM propranolol.

[0083] In one embodiment, the antiarrhythmic agent comprises sotalol and metoprolol.

[0084] In one embodiment, the antiarrhythmic agents comprise 0.1 μM metoprolol and 1 μM sotalol.

[0085] In one embodiment, the antiarrhythmic agents include sotalol and propranolol.

[0086] In one embodiment, the antiarrhythmic agent includes a Class I antiarrhythmic agent and a Class II antiarrhythmic agent.

[0087] In one embodiment, the antiarrhythmic agents include lidocaine and metoprolol.

[0088] In one embodiment, the antiarrhythmic agents include lidocaine and propranolol.

[0089] In one embodiment, the antiarrhythmic agents include mexiletine and metoprolol.

[0090] In one embodiment, the antiarrhythmic agents include 0.1 μM metoprolol and 1 μM mexiletine. In one embodiment, the antiarrhythmic agents include mexiletine and propranolol.

[0091] In one embodiment, the antiarrhythmic agent comprises a combination of two antiarrhythmic agents from the same class. In one embodiment, the antiarrhythmic agent comprises two agents from Class I. In one embodiment, the antiarrhythmic agent comprises lidocaine and mexiletine.

[0092] In one embodiment, the antiarrhythmic agent comprises two agents from Class II: hi one embodiment, the antiarrhythmic agent comprises metoprolol and propranolol.

[0093] In one embodiment, the antiarrhythmic agent comprises two agents from Class III: hi one embodiment, the antiarrhythmic agent comprises amiodarone and sotalol.

[0094] In one embodiment, the antiarrhythmic agent comprises a combination of three classes of antiarrhythmic agents: hi one embodiment, the antiarrhythmic agent comprises a combination of Class I, Class II, and Class III antiarrhythmic agents.

[0095] The effects of drugs are potentiated when two or more drugs are combined, which only supports the finding that drugs have related effects on heart rate / frequency and that drug combinations are more effective than single drug treatments.

[0096] The inventors have found that, overall, with increasing concentration, all drugs affect heartbeat, i.e., at low concentrations, cells beat and the beating frequency slows down; at medium concentrations, cells stop beating; and at high concentrations, drugs are known to be toxic, so if the concentration becomes too high, cells are expected to die.This confirms that the drug has a relevant antiarrhythmic effect, with a dose-dependent effect on beating frequency.Therefore, this result confirms that the drug improves beating and rhythm, and that arrhythmia is unlikely to occur.

[0097] Another aspect of the present invention relates to an antiarrhythmic agent for use in a method for treating or preventing arrhythmia caused by transplantation of stem cell-derived cardiomyocytes. In certain embodiments, the arrhythmia is caused by a method for treating heart failure by transplantation of stem cell-derived cardiomyocytes.

[0098] In one embodiment, the method of treatment is to obtain a high probability of successful transplantation outcome of the transplanted stem cell-derived cardiomyocytes or transplanted anti-arrhythmic cardiomyocyte population.

[0099] In another embodiment, the therapeutic method is to promote safer engraftment of transplanted stem cell-derived cardiomyocytes or transplanted anti-arrhythmic cardiomyocyte populations into the host myocardium.

[0100] In further embodiments thereof, the therapeutic method is for improving the heart rate and / or rhythm of transplanted stem cell-derived cardiomyocytes or transplanted antiarrhythmic cardiomyocyte populations. In further embodiments thereof, the method is for reducing alterations in the heart rate and / or rhythm of transplanted stem cell-derived cardiomyocytes or transplanted antiarrhythmic cardiomyocyte populations.

[0101] In another aspect of the present invention, antiarrhythmic agents are used in methods for preventing graft rejection after transplantation of an antiarrhythmic cardiomyocyte population by altering gene expression in the engrafted cells after exposure to the antiarrhythmic agent in vitro. Thus, the inventors have demonstrated that antiarrhythmic agents directly affect stem cell-derived cardiomyocytes.

[0102] Another aspect of the present invention relates to a composition comprising stem cell-derived cardiomyocytes, one or more antiarrhythmic agents, and optionally a biomaterial for use in a method for treating heart failure by transplantation of stem cell-derived cardiomyocytes. In one embodiment, the stem cell-derived cardiomyocytes are single cells, cell clusters, or cell patches. In one embodiment of the composition, the class I antiarrhythmic agent is amiodarone and the class III antiarrhythmic agent is lidocaine.

[0103] Another aspect of the present invention relates to a kit comprising an antiarrhythmic agent and stem cell-derived cardiomyocytes. In one embodiment, the kit is for use in a method for treating heart failure, preferably by transplantation of stem cell-derived cardiomyocytes. In one embodiment, the antiarrhythmic agent is selected from the list of Class I, Class II, Class III, Class IV, and Class V antiarrhythmic agents, or a combination thereof. In a preferred embodiment, the antiarrhythmic agent is selected from the list of Class I, Class II, and Class III antiarrhythmic agents, or a combination thereof. In a further embodiment, the Class I antiarrhythmic agent is lidocaine, the Class II antiarrhythmic agent is metoprolol, and / or the Class III antiarrhythmic agent is amiodarone, or a combination thereof. In a preferred embodiment, the kit comprises amiodarone and lidocaine.

[0104] Another aspect of the present invention relates to a method for obtaining an antiarrhythmic cardiomyocyte population with a high probability of successful transplantation outcome, comprising the step of regulating the expression of a gene selected from the list of GJA5, CACNA1G, NPPA, and / or NPPB.

[0105] In a preferred embodiment, the step of regulating gene expression is carried out by contacting the stem cell-derived cardiomyocytes with an antiarrhythmic agent in vitro.

[0106] In one embodiment, the gene CACNA1G is upregulated by more than about 1.5 times, for example, more than about 2 times. In one embodiment, the gene GJA5 is upregulated by more than about 2 times. In one embodiment, the gene NPPA is downregulated by more than about 2 times. In one embodiment, the gene PPB is downregulated by more than about 2 times, for example, more than about 3 times, preferably more than about 4 times. In one embodiment, the regulation of gene expression is in vitro. As used in this context, "in vitro" refers to a cell population outside the human body, for example, contained in a suitable container.

[0107] Therefore, according to the method of the above-mentioned aspect, obtain a unique cell population, that is, an antiarrhythmic cardiomyocyte population.Therefore, another aspect of the present invention relates to an antiarrhythmic cardiomyocyte population, wherein at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of cardiomyocytes have regulated gene expression, and CACNA1G is upregulated at least about 1.5 times, and / or GJA5 is upregulated at least about 2 times, and / or NPPA is downregulated at least about 2 times, and / or NPPB is downregulated at least about 2 times.In one embodiment, the antiarrhythmic cardiomyocyte population is obtained in vitro.

[0108] In another embodiment, the anti-arrhythmic cardiomyocyte population is used in in vitro assays, including, but not limited to, drug screening, toxicity testing, and / or disease modeling.

[0109] Another aspect of the present invention relates to a method for treating heart failure, comprising the steps of: a) obtaining in vitro stem cell-derived cardiomyocytes; b) transplanting the stem cell-derived cardiomyocytes into a patient; and c) co-administering an antiarrhythmic agent to the patient in vivo during or after transplantation. In a preferred embodiment, the antiarrhythmic agent in step c) comprises amiodarone and lidocaine.

[0110] In one embodiment, the method comprises contacting stem cell-derived cardiomyocytes in vitro with an antiarrhythmic agent to obtain a population of antiarrhythmic cardiomyocytes that are transplanted into the patient. myocardium The method includes contacting the cells with amiodarone and lidocaine in vitro.

[0111] Specific Embodiments Aspects of the present invention will now be further described by the following non-limiting embodiments: 1. A method for obtaining an antiarrhythmic cardiomyocyte population, comprising culturing stem cell-derived cardiomyocytes in a medium containing one or more antiarrhythmic agents. 2. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured in the medium for less than 24 hours. 3. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured with one or more antiarrhythmic agents for at least 24 hours. 4. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured with one or more antiarrhythmic agents for 24 to 48 hours. 5. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured with one or more antiarrhythmic agents for at least 48 hours. 6. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured with one or more antiarrhythmic agents for 48 to 72 hours. 7. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured with one or more antiarrhythmic agents for at least 72 hours. 8. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured with one or more antiarrhythmic agents for 72 to 96 hours. 9. The method of embodiment 1, wherein the stem cell-derived cardiomyocytes are cultured with one or more antiarrhythmic agents for at least 96 hours. 10. The method of any of embodiments 1-9, wherein the antiarrhythmic agent is selected from the list of class I, class II, and class III antiarrhythmic agents, or a combination thereof. 11. The method of embodiment 10, wherein the Class I antiarrhythmic agent is lidocaine or mexiletine, the Class II antiarrhythmic agent is metoprolol or propranolol, and / or the Class III antiarrhythmic agent is amiodarone or sotalol, or a combination thereof. 12. The method of embodiment 10, wherein the antiarrhythmic agent is selected from the class III list in combination with class I and / or class II antiarrhythmic agents. 13. The method of embodiment 11, wherein the antiarrhythmic agents are lidocaine and amiodarone. 14. The method of embodiment 11, wherein the antiarrhythmic agents are mexiletine and sotalol. 15. The method of embodiment 11, wherein the antiarrhythmic agents are metoprolol and sotalol. 16. The method of embodiment 11, wherein the antiarrhythmic agents are amiodarone and propranolol. 17. The method of embodiment 11, wherein the antiarrhythmic agents are lidocaine and sotalol. 18. The method of embodiment 11, wherein the antiarrhythmic agents are amiodarone and metoprolol. 19. The method of embodiment 11, wherein the antiarrhythmic agents are amiodarone and mexiletine. 20. The method of embodiment 11, wherein the antiarrhythmic agents are sotalol and propranolol. 21. The method of embodiment 10, wherein the antiarrhythmic agent is selected from the class II list in combination with a class I antiarrhythmic agent. 22. The method of embodiment 11, wherein the antiarrhythmic agents are metoprolol and mexiletine. 23. The method of embodiment 11, wherein the antiarrhythmic agents are lidocaine and metoprolol. 24. The method of embodiment 11, wherein the antiarrhythmic agents are lidocaine and propranolol. 25. The method of embodiment 11, wherein the antiarrhythmic agents are amiodarone and sotalol. 26. The method of embodiment 11, wherein the antiarrhythmic agents are mexiletine and propranolol. 27. The method of any one of the preceding embodiments, wherein the concentration of the antiarrhythmic agent is at least at least 1 nM. 28. The method of any preceding embodiment, wherein the concentration of the antiarrhythmic agent is in the range of 1 nM to 100 nM. 29. The method of embodiment 28, wherein the concentration of the antiarrhythmic agent is about 1 nM. 30. The method of embodiment 28, wherein the concentration of the antiarrhythmic agent is about 10 nM. 31. The method of embodiment 28, wherein the concentration of the antiarrhythmic agent is about 100 nM. 32. The concentration of the antiarrhythmic drug is within the range of 0.1 μM to 100 μM. Embodiment 27. The method according to any one of 1 to 26. 33. The method of embodiment 32, wherein the concentration of the antiarrhythmic agent is about 0.5 μM. 34. The method of embodiment 32, wherein the concentration of the antiarrhythmic agent is about 1 μM. 35. The method of embodiment 32, wherein the concentration of the antiarrhythmic agent is about 5 μM. 36. The method of embodiment 32, wherein the concentration of the antiarrhythmic agent is about 10 μM. 37. The method of embodiment 32, wherein the concentration of the antiarrhythmic agent is about 100 μM. 38. An antiarrhythmic cardiomyocyte population for use as a pharmaceutical. 39. Antiarrhythmic cardiomyocyte populations for use in the treatment of heart failure. 40. An antiarrhythmic cardiomyocyte population for use in preventing or reducing arrhythmias. 41. An antiarrhythmic cardiomyocyte population for use in preventing or reducing arrhythmia induction. 42. The antiarrhythmic cardiomyocyte population according to embodiments 38-41, for improving the transplant outcome of transplanted stem cell-derived cardiomyocytes. 43. An antiarrhythmic cardiomyocyte population according to any one of embodiments 38 to 41, having a reduced coefficient of variation (CV) or beat-to-beat variability when compared to stem cell-derived cardiomyocytes. 44. The antiarrhythmic cardiomyocyte population of embodiment 43, having at least a 50% reduction in coefficient of variation (CV) or beat-to-beat variability when compared to stem cell-derived cardiomyocytes. 45. The antiarrhythmic cardiomyocyte population of embodiment 43, having at least a 70% reduction in coefficient of variation (CV) or beat-to-beat variability when compared to stem cell-derived cardiomyocytes. 46. An antiarrhythmic cardiomyocyte population according to any one of embodiments 38 to 45, having regulated expression of a gene selected from the list GJA5, CACNA1G, NPPA and NPPB. 47. The antiarrhythmic cardiomyocyte population according to embodiment 46, having upregulation of GJA5 and / or CACNA1G. 48. The antiarrhythmic cardiomyocyte population according to embodiment 46, having downregulation of NPPA and / or NPPB. 49. The antiarrhythmic cardiomyocyte population according to embodiment 46, having upregulation of GJA5 and / or CACNA1G, and downregulation of NPPA and / or NPPB. 50. An antiarrhythmic cardiomyocyte population according to any one of embodiments 46 to 49, wherein the cell population has at least a 1.5-fold upregulation of GJA5, at least a 2-fold upregulation of CACNA1G, at least a 2-fold downregulation of NPPA, and / or at least a 4-fold downregulation of NPPB when compared to stem cell-derived cardiomyocytes. 51. The antiarrhythmic cardiomyocyte population of embodiment 50, wherein at least 10% of the cardiomyocytes have at least a 1.5-fold upregulation of GJA5, at least a 2-fold upregulation of CACNA1G, at least a 2-fold downregulation of NPPA, and / or at least a 4-fold downregulation of NPPB, when compared to stem cell-derived cardiomyocytes. 52. The antiarrhythmic cardiomyocyte population of embodiment 50, wherein at least 20% of the cardiomyocytes have at least a 1.5-fold upregulation of GJA5, at least a 2-fold upregulation of CACNA1G, at least a 2-fold downregulation of NPPA, and / or at least a 4-fold downregulation of NPPB, when compared to stem cell-derived cardiomyocytes. 53. The antiarrhythmic cardiomyocyte population of embodiment 50, wherein at least 40% of the cardiomyocytes have at least a 1.5-fold upregulation of GJA5, at least a 2-fold upregulation of CACNA1G, at least a 2-fold downregulation of NPPA, and / or at least a 4-fold downregulation of NPPB, when compared to stem cell-derived cardiomyocytes. 54. A kit comprising an antiarrhythmic agent and stem cell-derived cardiomyocytes. 55. A kit according to embodiment 54, for use in a method for the treatment of heart failure, preferably by transplantation of stem cell-derived cardiomyocytes. 56. The kit according to any one of embodiments 54-55, wherein the antiarrhythmic agent is selected from the list of class I, class II, class III, class IV, and class V antiarrhythmic agents, or combinations thereof. 57. The kit of embodiment 56, wherein the antiarrhythmic agent is selected from the list of class I, class II, and class III antiarrhythmic agents, or a combination thereof. 58. The kit of embodiment 56, wherein the class I antiarrhythmic agent is lidocaine or mexiletine, the class II antiarrhythmic agent is metoprolol or propranolol, and / or the class III antiarrhythmic agent is amiodarone or sotalol, or a combination thereof. 59. The kit according to embodiment 58, comprising amiodarone and lidocaine. 60. The kit according to embodiment 58, comprising mexiletine and sotalol. 61. The kit according to embodiment 58, comprising metoprolol and sotalol. 62. The kit according to embodiment 58, comprising metoprolol and mexiletine. 63. The kit according to embodiment 58, comprising amiodarone and propranolol. 64. A composition comprising stem cell-derived cardiomyocytes, one or more antiarrhythmic agents, and optionally a biomaterial for use in treating heart failure. 65. An antiarrhythmic agent for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 66. The antiarrhythmic agent according to embodiment 65, wherein the antiarrhythmic agent is selected from the list of class I, class II, class III, class IV, and class V antiarrhythmic agents. 67. The antiarrhythmic agent according to embodiment 65, wherein the antiarrhythmic agent is selected from the list of class I, class II, and class III antiarrhythmic agents. 68. The antiarrhythmic agent according to any one of embodiments 65-67, wherein the antiarrhythmic agent is a class III antiarrhythmic agent. 69. The antiarrhythmic agent according to embodiment 68, wherein the class III antiarrhythmic agent is amiodarone. 70. The antiarrhythmic agent according to embodiment 68, wherein the class III antiarrhythmic agent is sotalol. 71. Amiodarone for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 72. Sotalol for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 73. The antiarrhythmic agent according to any one of embodiments 65-67, wherein the antiarrhythmic agent is a class II antiarrhythmic agent. 74. The antiarrhythmic agent according to embodiment 73, wherein the class II antiarrhythmic agent is metoprolol. 75. The antiarrhythmic agent according to embodiment 73, wherein the class II antiarrhythmic agent is propranolol. 76. Metoprolol for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 77. Propranolol for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 78. The antiarrhythmic agent according to any one of embodiments 65-67, wherein the antiarrhythmic agent is a class I antiarrhythmic agent. 79. The antiarrhythmic agent according to embodiment 78, wherein the class I antiarrhythmic agent is lidocaine. 80. The antiarrhythmic agent according to embodiment 78, wherein the class I antiarrhythmic agent is mexiletine. 81. Lidocaine for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 82. Mexiletine for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 83. The antiarrhythmic agent according to any one of embodiments 65-67, wherein the antiarrhythmic agent is a combination comprising an antiarrhythmic agent of class III and class I. 84. The antiarrhythmic agent according to embodiment 83, wherein the antiarrhythmic agent is a combination comprising amiodarone and a class I antiarrhythmic agent. 85. The antiarrhythmic agent according to embodiment 83, wherein the antiarrhythmic agent comprises amiodarone and lidocaine. 86. The antiarrhythmic agent of embodiment 83, wherein the antiarrhythmic agent comprises amiodarone and mexiletine. 87. The antiarrhythmic agent according to embodiment 83, wherein the antiarrhythmic agent is a combination comprising sotalol and a class I antiarrhythmic agent. 88. The antiarrhythmic agent of embodiment 83, wherein the antiarrhythmic agent comprises sotalol and mexiletine. 89. The antiarrhythmic agent according to embodiment 83, wherein the antiarrhythmic agent comprises sotalol and lidocaine. 90. An antiarrhythmic agent according to any one of embodiments 65 to 89, for obtaining a high probability of successful transplantation outcome of transplanted stem cell-derived cardiomyocytes. 91. An antiarrhythmic agent according to any one of embodiments 65 to 89, for promoting the integration of transplanted stem cell-derived cardiomyocytes into the host myocardium. 92. An antiarrhythmic agent according to any one of embodiments 65 to 89, for improving the pulsation and / or rhythm of transplanted stem cell-derived cardiomyocytes. 93. The antiarrhythmic agent according to any one of embodiments 65 to 89, wherein the antiarrhythmic agent regulates the expression of a gene selected from the list: GJA5, CACNA1G, NPPA, NPPB. 94. An antiarrhythmic agent for use in a method for treating or preventing arrhythmia caused by transplantation of stem cell-derived cardiomyocytes. 95. The antiarrhythmic agent according to embodiment 94, wherein the arrhythmia is non-sustained ventricular tachycardia, sustained ventricular tachycardia, and sustained accelerated idiopathic ventricular rhythm. 96. An antiarrhythmic agent for use in a method for the prevention of graft rejection after transplantation of stem cell-derived cardiomyocytes. 97. The antiarrhythmic agent according to embodiment 94, wherein the arrhythmia is caused by a method for treating heart failure by transplantation of stem cell-derived cardiomyocytes. 98. A composition comprising a Class I antiarrhythmic agent and a Class III antiarrhythmic agent for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 99. The composition of embodiment 98, wherein the class I antiarrhythmic agent is amiodarone and the class III antiarrhythmic agent is lidocaine. 100. Use of an antiarrhythmic agent in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 101. An antiarrhythmic agent and stem cell-derived cardiomyocytes for use in a method for the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 102. An antiarrhythmic agent and stem cell-derived cardiomyocytes for use in a method for preventing arrhythmias in the treatment of heart failure by transplantation of stem cell-derived cardiomyocytes. 103. A method of controlling gene expression in stem cell-derived cardiomyocytes to obtain a high likelihood of successful transplantation outcome, comprising contacting the stem cell-derived cardiomyocytes with an antiarrhythmic agent. 104. The method of embodiment 103, wherein the antiarrhythmic agent is selected from the list of class I, class II, class III, class IV, and class V antiarrhythmic agents, or combinations thereof. 105. The method of embodiment 104, wherein the antiarrhythmic agent is selected from the list of class I, class II, and class III antiarrhythmic agents, or a combination thereof. 106. The method of embodiment 105, wherein the class I antiarrhythmic agent is lidocaine, the class II antiarrhythmic agent is metoprolol, and / or the class III antiarrhythmic agent is amiodarone, or a combination thereof. 107. A method for obtaining stem cell-derived cardiomyocytes with a high probability of successful transplantation outcome, comprising the step of regulating the expression of a gene selected from the list of GJA5, CACNA1G, NPPA, and NPPB. 108. The method of embodiment 107, wherein the gene GJA5 is upregulated at least 1.5-fold, and / or the gene CACNA1G is upregulated at least 2-fold, and / or the gene NPPA is downregulated at least 2-fold, and / or the gene NPPB is downregulated at least 4-fold. 109. The method of any one of embodiments 107-108, wherein the step of controlling the expression of the gene is carried out by contacting the stem cell-derived cardiomyocytes with an antiarrhythmic agent. 110. The method of any one of embodiments 107-108, wherein the regulation of gene expression is in vitro. 111. The antiarrhythmic agent, composition, use, kit, or method of any one of the preceding embodiments, wherein the stem cell-derived cardiomyocytes are derived from human pluripotent stem cells, such as human embryonic stem cells. 112. A method for the treatment of heart failure, comprising: a) obtaining stem cell-derived cardiomyocytes in vitro; b) transplanting the stem cell-derived cardiomyocytes into the patient; c) co-administering an antiarrhythmic agent to the patient. 113. The method of embodiment 112, further comprising contacting the stem cell-derived cardiomyocytes obtained in step a) with an antiarrhythmic agent in vitro to obtain a population of antiarrhythmic cardiomyocytes, and transplanting the population of antiarrhythmic cardiomyocytes into a patient. 114. The method of any one of embodiments 112 and 113, wherein the antiarrhythmic agents comprise amiodarone and lidocaine. 115. The method of any one of embodiments 112 and 113, wherein the antiarrhythmic agents include mexiletine and sotalol. 116. The method of any one of embodiments 112 and 113, wherein the antiarrhythmic agent comprises metoprolol and sotalol. 117. The method of any one of embodiments 112 and 113, wherein the antiarrhythmic agent comprises metoprolol and mexiletine. 118. The method of any one of embodiments 112 and 113, wherein the antiarrhythmic agents include amiodarone and propranolol. 119. An antiarrhythmic agent for use in the method according to any one of embodiments 112-118. [Example]

[0112] Example 1 To determine the effects of antiarrhythmic drugs on stem cell-derived cardiomyocytes beyond the well-established immediate changes in electrophysiological responses due to modulation of ion channel activity, we analyzed changes in gene expression resulting from long-term exposure (>24 h) of cardiomyocytes to antiarrhythmic drugs. For this purpose, we used amiodarone, Lidocaine We evaluated the effects of commonly used antiarrhythmic drugs such as on genes related to electrical signaling, regulation of cardiac hypertrophy, calcium handling, and cardiomyocyte maturation.

[0113] Experimental procedure Human embryonic stem cells (hESCs) were maintained under feeder-free conditions in LN521 (BioLamina) in iPSBrew (Miltenyi). Cells were passaged every 3–4 days using Accutase (Innovative Cell Technology) to a concentration of 1.6–2.4 × 10 4 cells / cm 2 Cells were seeded in iPSBrew supplemented with 10 μM Y-27632 (Sigma) at 100°C. Cell lines tested negative for mycoplasma contamination and karyotypic abnormalities throughout the study.

[0114] Cells were differentiated towards cardiomyocytes in an adapted 3D suspension protocol (Kempf H et al. Bulk cell density and Wnt / TGFbeta signaling regulate mesendodermal patterning of human pluripotent stem cells. Nat Commun. 2016;7:13602). Briefly, cells were seeded into 6-well suspension plates (Greiner) at 0.16 × 10 in iPSBrew supplemented with 10 μM Y-27632. 6 Aggregates were formed at 1000 cells / mL. After 2 days, differentiation was induced using 4-8 μM CHIR99021 (Tocris) for 24 hours, followed by 2 μM Wnt-C59 (Tocris) for 48 hours in RPMI 1640 medium (Life Technologies) supplemented with 2% B27 (Life Technologies) without insulin, or RPMI 1640 medium supplemented with 0.5 mg / mL human recombinant albumin (ScienceCell) and 0.2 mg / mL L-ascorbic acid 2-phosphate (Sigma). Cells were maintained in RPMI 1640 supplemented with 2% B27 from day 5 onwards.

[0115] The obtained cardiomyocytes were dissociated into single cells after 10–15 days of differentiation using a STEMdiff cardiomyocyte dissociation kit (Stem Cell Technologies) according to the manufacturer's instructions for further characterization, functional analysis, and transplantation experiments.

[0116] Evaluation of antiarrhythmic drugs Dissociated cardiomyocytes were plated at 1 × 10 in RPMI 1640 medium supplemented with 2% B27 and 0.1% penicillin / streptomycin (Gibco) on laminin-521 or geltrex (Life Technologies) coated plates. 5 / cm 2Cells were seeded at a cell density of 1 μM, 10 μM, and 100 μM. After 4 days, cardiomyocytes were exposed to antiarrhythmic drugs for at least 72 hours: amiodarone at 1 μM, 10 μM, and 100 μM; metoprolol at 0.1 μM, 1 μM, and 10 μM; and lidocaine at 0.1 μM, 1 μM, and 10 μM (all from Sigma), as well as combinations of these drugs at each concentration. Blank medium, vehicle addition, and cardiomyocytes cultured for 9 days and 42 days were used as controls. Cell beating was assessed at 48, 72, and 96 hours. Cells were harvested after 10 minutes of incubation in RLTplus buffer (Qiagen). Changes in gene expression were determined using a custom NanoString gene panel (NanoString Technologies) according to the manufacturer's instructions for the following target sequences, including the seven housekeeping genes listed in Table 1: [Table 1]

[0117] result To study the direct effects of antiarrhythmic drugs on the properties of hESC-derived cardiomyocytes, we analyzed changes in the expression levels of selected genes related to action potential formation (HCN1, HCN4, KCNA5, KCNE4, KCNH7, KCNJ3, KCNJ5, SCN1B, SCN5A), electrical signaling (GJA1, GJA5, GJD3), calcium handling (CACNA1C, CACNA1D, CACNA1G, RYR2, PLN), cardiac maturation (HOPX, MYH7, MYL2, TNNI3), and cardiac hypertrophy (NPPA, NPPB), as well as pan-cardiomyocyte markers (NKX2-5, TNNT2, ACTA2).

[0118] Surprisingly, treatment of hES-derived cardiomyocytes with 0.1 μM and 1 μM amiodarone for 5 days induced a two-fold increase in the expression of the T-type voltage-gated calcium channel α-subunit 1G CACNA1G compared to untreated controls at day 23 and early (immature) cardiomyocytes at day 9 (Figure 1). 2+The channel is expressed in developing fetal ventricular myocytes (Cribbs LL et al, Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart. Circ Res. 2001;88(4):403-7), and transports Ca from internal stores. 2+ Control of second messenger Ca influx 2+ CACNA1G plays a key role in regulating the intracellular distribution of Ca2+ channels. The channel thereby regulates various cellular processes, including cardiac muscle cell beating. More specifically, CACNA1G controls cardiac electrical and pacing activity. Importantly, channel dysfunction has been associated with both atrial and ventricular arrhythmias, particularly in heart failure (Perez-Reyes E. Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev. 2003;83(1):117-61) (Vassort G, Talavera K, Alvarez JL. Role of T-type Ca2+ channels in the heart. Cell Calcium. 2006;40(2):205-20). Therefore, the clear upregulation of CACNA1G by amiodarone suggests an increased ability of treated hESC-derived cardiomyocytes to control and prevent arrhythmic responses in their intrinsic electrophysiological capacitance.

[0119] Similarly, amiodarone at 0.1 μM and 1 μM resulted in a >3-fold upregulation of the gene encoding the high-conductance gap junction protein GJA5 (Figure 2). GJA5 is expressed not only in the ventricular conduction system but also in the early ventricles (Delorme B et al., Developmental regulation of connexin 40 gene expression in mouse heart correlates with the differentiation of the conduction system. Dev Dyn. 1995;204(4):358-71), and represents a key player in current conduction across the ventricles (Shekhar A et al., Transcription factor ETV1 is essential for rapid conduction in the heart. J Clin Invest. 2016;126(12):444-59). Several somatic mutations in GJA5 are associated with myocardial arrhythmic properties, including ventricular arrhythmias (Delmar M, Makita N. Cardiac connexins, mutations, and arrhythmias. Curr Opin Cardiol. 2012;27(3):236-41). Consequently, upregulation of GJA5 in ES-derived cardiomyocytes by amiodarone likely accelerates electrical signaling across cell-cell contacts, thereby suppressing arrhythmic behavior, particularly via macro- or microreentry, and thereby reducing the risk of ectopic foci development.

[0120] In contrast to the increased levels of CACNA1G and GJA5, treatment with amiodarone resulted in a three-fold and five-fold reduction in NPPA and NPPB expression, respectively (Figures 3 and 4). NPPA and NPPB encode the secretory hormones ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide), which are secreted primarily by the atria and less prominently by the ventricles of the adult heart in response to mechanical stretch. Quantification of natriuretic peptide levels is routinely used as a tool for diagnosing heart failure (McMurray JJ et al. Guidelines ESCCfP. ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J. 2012;33(14):1787-847). Interestingly, both ANP and BNP are involved in regulating cardiac electrophysiology (Perrin MJ, Gollob MH. The role of atrial natriuretic peptide in modulating cardiac electrophysiology. Heart Rhythm. 2012;9(4):610-5).In particular, elevated BNP levels are associated with increased arrhythmic events in patients with left ventricular dysfunction (Galante O et al., Brain natriuretic peptide (BNP) level predicts long-term ventricular arrhythmias in patients with moderate to severe left ventricular dysfunction. Harefuah. 2012;151(1):20-3,63,2). However, the precise mechanism by which natriuretic peptides regulate electrophysiological actions in humans remains unclear. The peptides are thought to induce action potential shortening, thereby increasing the likelihood of reentry. Furthermore, prolongation of action potential duration by reducing ANP and / or BNP levels reduces the likelihood of tachyarrhythmias. Therefore, reducing NPPA and NPPA using amiodarone in hESC-derived cardiomyocytes reduces the risk of graft-induced arrhythmias and the development of tachycardia and ectopic beat foci from hESC-derived cardiomyocytes after cardiac transplantation.

[0121] Notably, the beating frequency of hESC-derived cardiomyocytes was significantly reduced or inhibited at 1 μM and 10 μM, respectively, reducing the likelihood of tachyarrhythmias. At the same time, amiodarone did not alter the expression of pan-cardiomyocyte genes, such as NKX2-5, TNNT2, or ACTA2 (Figures 5-7), suggesting that overall cardiomyocyte identity was not affected. It also did not affect the expression of other ion channels, including SCN5A (Figure 8), one of its targets that controls the rate of action potential rise in the human heart (Honjo H et al., Block of cardiac sodium channels by amiodarone studied by using Vmax of action potential in single ventricular myocytes. Br J Pharmacol. 1991;102(3):651-6).

[0122] Overall, exposure of hESC-derived cardiomyocytes to amiodarone induces a unique expression profile, including elevated levels of CACNA1G and GJA5, accompanied by decreased levels of NPPA and NPPB. This confers distinct electrophysiological characteristics to amiodarone-treated cardiomyocytes, including an increased ability to control intracellular calcium levels, faster signal conduction across cardiac tissue, and reduced susceptibility to arrhythmic events and tachycardia. As a result, these (modified) antiarrhythmic cardiomyocyte populations provide an excellent cell source for cardiac regeneration by eliminating graft-induced arrhythmias and / or tachycardia.

[0123] Similarly, we found that other classes of antiarrhythmic drugs regulate NPPA and NPPB. Lidocaine, the most relevant class 1b antiarrhythmic agent, reduced both natriuretic peptides in a concentration-dependent manner at 1 μM, 10 μM, and 100 μM (Figures 9 and 10) without affecting the expression of pan-cardiomyocyte markers (NKX2-5 and TNNT2; Figures 11 and 12). Given the overall relevance of NPPA and NPPB in controlling cardiomyocyte electrophysiological behavior, treatment of hESC-derived cardiomyocytes with lidocaine represents a further promising strategy to avoid graft-induced side effects in cardiac transplantation.

[0124] Taken together, our results demonstrate an unexpected effect of antiarrhythmic drugs on gene expression in stem cell-derived cardiomyocytes, resulting in an antiarrhythmic cardiomyocyte population with altered expression patterns of genes related to myocardial hypertrophy, calcium handling, and electrical signaling, all relevant classes of genes involved in the control of cardiomyocyte electrophysiological behavior.

[0125] Example 2 To verify sustained effects and robustness across different culture systems, we tested the effects of antiarrhythmic drugs on the gene expression profile of stem cell-derived cardiomyocytes using 3D suspension aggregates and measured gene expression over a 48-hour period after removal of the antiarrhythmic drug.

[0126] Experimental procedure The experiment was carried out as described in Example 1 with the following modifications: instead of dissociating and seeding stem cell-derived cardiomyocytes into a two-dimensional monolayer, the cells were maintained as three-dimensional suspension aggregates obtained directly 14 days after induction of cardiac differentiation. The aggregates were then cultured at approximately 1.5 x 10 cells in 3 mL of medium. 6 Cells were maintained in 6-well suspension plates on an orbital shaker (75 rpm) at a cell density of 1000 cells / mL. The aggregated cells were exposed to 10 μM amiodarone for approximately 120 hours. They were then maintained in RPMI medium supplemented with 2% B27 + 0.1% P / S for an additional 48 hours. Complete medium changes were performed every 48–72 hours. Cells were then harvested and subjected to RNA expression analysis.

[0127] result The gene expression profile of the aggregates measured 48 hours after treatment with 10 μM amiodarone shows a ∼1.75-fold increase in CACNA1G, a ∼2.7-fold increase in GJA5, and a ∼2-fold and >15-fold decrease in NPPA and NPPB, respectively (Figure 14).

[0128] Therefore, the results confirm the sustained and clear effect of antiarrhythmic agents, such as amiodarone, on gene expression levels associated with the altered electrophysiological properties of stem cell-derived cardiomyocytes, resulting in reduced arrhythmia potentials, as shown in Examples 3 and 4. Furthermore, the results show that the effect is independent of culture mode, e.g., induced not only in three-dimensional suspension cultures, which are closer to in vivo tissues, but also in two-dimensional monolayer cultures. Therefore, the effect of antiarrhythmic agents is expected to be reflected in in vivo applications.

[0129] Example 3 To determine the lasting effects on the electrophysiological properties of stem cell-derived cardiomyocytes after exposure to antiarrhythmic drugs, we used Ca 2+Functional cardiomyocyte testing using field potential recordings was performed to determine the arrhythmogenic potential of stem cell-derived cardiomyocyte populations after exposure to antiarrhythmic drugs. Beat-to-beat variability was used as an in vitro surrogate readout for the in vivo (pro)arrhythmic potential of stem cell-derived cardiomyocytes (Rosanne Varkevisser et al., Beat-to-beat variability of repolarization as a new biomarker for proarrhythmia in vivo, Heart Rhythm Volume 9, Issue 10, October 2012, Pages 1718-1726); (Kazuto Yamazaki et al., Beat-to-Beat Variability in Field Potential Duration in Human Embryonic Stem Cell-Derived Cardiomyocyte Clusters for Assessment of Arrhythmogenic Risk, and a Case Study of Its Application, Pharmacology & Pharmacy, Vol. 5 No. 1, 2014, pp. 117-128).

[0130] Notably, all Ca 2+ Recordings were performed at least 24 hours after exposure to exclude known direct effects of drugs via ion channel modulation.

[0131] Experimental procedure Ca 2+ - Recordings were performed on human induced pluripotent stem cell-derived cardiomyocytes (stem cell-derived cardiomyocytes) from Fujifilm Cellular Dynamics, USA (FCDI; iCell2 cardiomyocytes, donor number 01434, lot number 105170).

[0132] Cells were delivered in frozen vials and stored in liquid nitrogen until use. All culture media required for thawing and culturing were provided by the cell supplier. Thawing, plating, and culturing procedures were performed according to the manufacturer's protocol. Cells were plated directly onto fibronectin-coated 384-well Greiner μClear plates. The plating density was 17,500 cells / well in a final volume of 50 μl. Medium was changed (90%) one day after plating, every two days thereafter, and 3 hours before experiments in pilot studies. For compound experiments, compounds were added at in vitro (DIV) 2, followed by a compound-containing medium change (90%) at DIV 4. Treatment was terminated by replacing the medium with standard culture medium at DIV 6. Recordings were performed at DIV 7. Experiments were performed with a minimum of n = 10 for each compound concentration.

[0133] Ca 2+ For imaging experiments, the medium was replaced with HEPES-buffered recording solution for recordings. 2+ The indicator (Cal-520-AM) was applied at a concentration of 2 μM and allowed to accumulate in the cells for 30 min, after which the buffer was replaced with dye-free buffer again. The cells were allowed to recover for 10 min at 37°C in a Hamamatsu FDSS recording system. All experiments were performed at 37°C. The camera frame rate was set to a minimum of 35 Hz for recording, with binning at 4 × 4. To assess whether the quality was sufficient for the experiment, the regularity of the beating, Ca 2+ Several parameters were verified (by eye), including the shape and amplitude of the signal, and the variability of these parameters between different wells. No electrical stimulation was applied, as the cells were spontaneously active.

[0134] Cells were recorded for 5 minutes before compound application. 200 μM moxifloxacin was applied in a single concentration / well format after a baseline phase, followed by a 5-minute washout period. Fluorescence activity was then recorded for an additional 5 minutes. Moxifloxacin without compound preincubation was included as a control (n=18) and distributed across multiple plates.

[0135] Continuous Ca transients within each recording period were recorded using FDSSv3.4 offline. 2+ Beat-to-beat variability, measured as the coefficient of variation (CV) of the time interval between transients, was analyzed, and plots were subsequently further analyzed and edited using Igor Pro 8.0.4.2 (Wavemetrics, USA).

[0136] The coefficient of variation is calculated as follows:

number

[0137] In the formula, σ represents the standard deviation and μ represents the mean.

[0138] SEM is calculated as the ratio between the square root of the standard deviation and the number of experiments,

number

[0139] result The coefficient of variation (CV), which indicates the beat-to-beat variability of the antiarrhythmic cardiomyocyte population at the indicated concentrations, was compared with control treatment. The results show a clear reduction in beat-to-beat variability, as reflected by the coefficient of variation (CV), under baseline conditions and after induction of proarrhythmic conditions using moxifloxacin for all tested compounds, including three different classes of antiarrhythmic agents: Class I (e.g., lidocaine or mexiletine), Class II (e.g., propranolol, metoprolol), and Class III (e.g., amiodarone, sotalol) (Figure 15). The CV was reduced by 80.8% under baseline conditions and 84.7% under proarrhythmic conditions after exposure to 100 nM lidocaine compared with the respective control conditions without exposure to the antiarrhythmic agent. Similarly, under baseline and proarrhythmic conditions, 10 nM amiodarone reduced CV by 76.5% and 83.3%, 10 nM metoprolol by 79.2% and 83.2%, 100 nM mexiletine by 76.79% and 77.5%, 100 nM sotalol by 79.4% and 84.9%, and 100 nM propranolol by 73.7% and 88.5%. Notably, this apparent reduction in beat-to-beat variability was observed 24 hours after cessation of the compounds and, therefore, was not dependent on the continued presence of the drug.

[0140] Overall, the results were Class I, II, or II I The results clearly demonstrated a reduction in beat-to-beat variability after treatment with antiarrhythmic drugs, with an overall reduction in CV of 70-80% at the concentrations tested. Notably, this reduction was observed under both baseline (non-arrhythmic) and proarrhythmic conditions. Taken together, these findings suggest that treatment with antiarrhythmic drugs induces an antiarrhythmic cell population, reducing the cells' susceptibility to arrhythmias. This antiarrhythmic cell population makes the acquired cell population highly attractive for cardiac cell therapy and reduces the previously reported risk of arrhythmias after cell transplantation.

[0141] Furthermore, the data suggest that changes in cardiomyocyte function related to the electrophysiological properties of the cells are associated with sustained changes in gene expression, including CACNA1G, GJA5, NPPA, and / or NPPB. Importantly, the altered properties of stem cell-derived cardiomyocytes are induced (directly or indirectly) at the gene expression level by exposure to antiarrhythmic drugs, but not necessarily through a common mechanism of action related to direct effects through modulation of ion channel activity.

[0142] Example 4 To test whether a combination of antiarrhythmic drugs could further reduce the arrhythmic potential of stem cell-derived cardiomyocytes, stem cell-derived cardiomyocytes were treated with the same Ca2+ / Ca ... 2+ -recording assays were performed applying various combinations of class I, II, and / or III antiarrhythmic drugs and comparing beat-to-beat variability with single compound treatments.

[0143] Experimental procedure Induced pluripotent stem cell-derived cardiomyocytes were treated with a combination of antiarrhythmic drugs for 72 hours, followed by a 24-hour recovery period. Drugs were applied at concentrations of 1 μM sotalol, 0.1 μM amiodarone, 0.1 μM metoprolol, and 1 μM mexiletine. All measurements were performed under proarrhythmic conditions after moxiflocacin treatment. Results show that combinations of Class III drugs with either Class I and / or Class II drugs are more efficient at reducing proarrhythmic potential than either drug alone, with reductions of 46.4% or 31.4% and 46.4%, respectively (Figure 16). Similarly, combinations of Class I and Class II drugs showed a 48.3% reduction compared to single-compound treatment.

[0144] These results indicate that combining antiarrhythmic agents further reduces beat-to-beat variability, resulting in a population of cardiomyocytes with reduced arrhythmic potential compared to single agents alone, further reducing the risk of inducing arrhythmias in vivo.

[0145] Overall, the results of the above examples demonstrate that exposure of stem cell-derived cardiomyocytes to antiarrhythmic drugs induces a significant and sustained reduction in arrhythmic potential, thereby obtaining a cell population with antiarrhythmic properties.

[0146] Antiarrhythmic cardiomyocyte populations represent an excellent cell source for transplantation by reducing the risk of graft-induced arrhythmias and / or tachycardias.

[0147] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. 1. A method for obtaining an antiarrhythmic cardiomyocyte population, comprising culturing stem cell-derived cardiomyocytes in a medium containing one or more antiarrhythmic agents, wherein the cardiomyocyte population exhibits reduced beat-to-beat variability compared to stem cell-derived cardiomyocytes, and wherein the antiarrhythmic agent is lidocaine, metoprolol, amiodarone, or sotalol.

2. 10. The method of claim 1, wherein the one or more antiarrhythmic agents are lidocaine and amiodarone, mexiletine and sotalol, metoprolol and sotalol, amiodarone and propranolol, lidocaine and sotalol, amiodarone and metoprolol, amiodarone and mexiletine, and / or sotalol and propranolol.

3. 10. The method of claim 1, wherein the concentration of the antiarrhythmic agent is in the range of 1 nM to 100 nM.

4. 10. The method of claim 1, wherein the concentration of the antiarrhythmic agent is in the range of 0.1 μM to 100 μM.

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