Application of antiarrhythmic agents to stem cell derived cardiomyocytes and uses thereof

Abstract

To provide treatment of arrythmia caused by taking of myocardial cells derived from stem cell in methods for treating heart failure.SOLUTION: The invention relates to an antiarrhythmic agent, an antiarrhythmic myocardial cell population, a method for obtaining an antiarrhythmic myocardial cell population by in vitro exposure of stem cell-derived myocardial cells to an antiarrhythmic agent, and / or medical use of antiarrhythmic myocardial cell population in the prevention of arrythmia and in the treatment of heart failure.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to the field of stem cells, more particularly to an antiarrhythmic cardiomyocyte cell population, a method for obtaining the antiarrhythmic cardiomyocyte cell population, and its medical use in the treatment of heart failure, preventing or reducing arrhythmias resulting from transplantation of stem cell-derived cardiomyocytes. 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 least regenerative organs in the body, and as a result, when cardiac injury occurs, cardiomyocytes die, leaving behind scarred areas that cannot contract. This leads to reduced pumping power, heart failure, and increased morbidity and mortality. Heart disease is the 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 into non-human primate hearts from the endocardial side, several types of arrhythmias were detected during the first 4–6 weeks after engraftment. These arrhythmias were sustained ventricular tachycardia, nonsustained ventricular tachycardia, and accelerated idiopathic ventricular rhythm.

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

[0006] It has been described in monkeys that the arrhythmias occurring after cell injection are of a different type and origin (from ectopic lesions) than the conventional arrhythmias occurring after acute myocardial infarction and heart failure (reentry mechanism). This means that it is clearly defined that cell transplantation-induced arrhythmias need to be treated and that if the cells had not been injected, the arrhythmias would not have occurred. Therefore, this type of arrhythmia can be considered a new type of condition caused by cell transplantation, and a specific treatment for this type of arrhythmia does not yet exist.

[0007] To regenerate cardiac muscle tissue in vivo after heart injury, various strategies are explored, including various types of pluripotent stem cell-derived cardiac lineage cells, e.g., early cardiovascular progenitor cells, immature beating cardiomyocytes, as well as more mature, e.g., heterotypic tissue engineered cardiac constructs. In general, all approaches to generate such cells in vitro give rise to cardiomyocytes of a relatively immature phenotype that resembles fetal-like cells from the first and second trimesters of pregnancy in terms of their gene expression profile, cell morphology, sarcoma organization, electrophysiological properties, and resulting contractile force. Notably, several of the various strategies have been shown to predominantly produce cells capable of engraftment and maturation after transplantation. However, the overall efficiency has been limited. This is especially because of the limited understanding of the underlying mechanisms mediating engraftment and maturation after transplantation, which calls for further research, especially to identify additional therapeutics for transplanted cardiomyocytes and promote their development and integration.

[0008] The object of the present invention is to address the problem of arrhythmia caused by engraftment of stem cell-derived cardiomyocytes in a method for the treatment of heart failure. In particular, the object of the present invention is 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. Moreover, 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 an anti-arrhythmic cardiomyocyte population, comprising culturing stem cell-derived cardiomyocytes in a medium comprising one or more anti-arrhythmic agents.

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

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

[0014] In one aspect, the invention relates to an anti-arrhythmic cardiomyocyte 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 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 symptoms caused by the stem cells not yet fully integrated into the myocardium. Now, the inventors have presented an approach to prevent or reduce arrhythmias and / or treat heart failure. One of the approaches is to obtain an antiarrhythmic cardiomyocyte population by contacting stem cell-derived cardiomyocytes with an antiarrhythmic agent in vitro that alters the control of gene expression of key genes related to cardiomyocytes being able to connect and contract in a synchronized manner. 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 a 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 agents in vivo during or after transplantation of stem cell-derived cardiomyocytes into a patient. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows the gene expression pattern of CACNA1G after 5 days of exposure to 0.1 μM and 1 μM amiodarone in stem cell-derived cardiomyocytes (day 23 after differentiation induction). Negative and positive controls refer to immature (day 9) and mature (day 42) cardiomyocytes, respectively. [Diagram 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). Negative and positive controls refer to immature (day 9) and mature (day 42) cardiomyocytes, respectively. [Diagram 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). 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). Negative and positive controls refer to immature (day 9) and mature (day 42) cardiomyocytes, respectively. [Diagram 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 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 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 gene expression patterns of SCN5A after 5 days of exposure to 0.1 μM and 1 μM amiodarone in stem cell-derived cardiomyocytes (day 23 after differentiation induction). 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 gene expression patterns 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). Negative control refers to immature (day 9) cardiomyocytes. [Figure 11] Figure 11 shows the gene expression pattern of NKX2-5 after 5 days of exposure to 1 μM, 10 μM, and 100 μM lidocaine in stem cell-derived cardiomyocytes (23 days after differentiation induction). Negative control refers to immature (day 9) cardiomyocytes. [Figure 12] Figure 12 shows gene expression patterns of TNNT2 after 5 days of exposure to 1 μM, 10 μM, and 100 μM lidocaine in stem cell-derived cardiomyocytes (day 23 after differentiation induction). Negative control refers to immature (day 9) cardiomyocytes. [Figure 13] Figure 13 shows gene expression patterns of SCN5A after exposure to 0.1 μM and 1 μM lidocaine for 5 days in stem cell-derived cardiomyocytes (23 days after differentiation induction). 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. Respective untreated cardiomyocytes are shown as controls. Cardiomyocytes were maintained as three-dimensional suspension clusters with diameter sizes of 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). Bar graphs 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] FIG. 16 shows the beat-to-beat coefficient of variation (CV) of stem cell-derived cardiomyocytes following exposure to combinations of antiarrhythmic drugs applied at concentrations of 1 μM sotalol, 0.1 μM amiodarone, 0.1 μM metoprolol 1 μM mexiletine, 1 μM propranolol, and comparison with single agents. All conditions were measured following 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 PREFERRED EMBODIMENTS

[0020] Unless otherwise specified, 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 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 make the invention more clear 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 contexts.

[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] The term "about" as used herein when referring to measurable values, such as amounts of cells, compounds, or agents of the invention, dosages, temperatures, and the like, is intended to encompass variations 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 certain 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 transplantation of 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 by first contacting undifferentiated stem cells with a compound that affects undifferentiated stem cells in such a way that the differentiation process is started.

[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 the methods according to the invention are described in "hours".

[0031] In the following, the method according to the invention is 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 shown to be nonsustained 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 term "antiarrhythmic agent" or "antiarrhythmic drugs" or "antiarrhythmic compound" or "antiarrhythmic drug" refers to one or more pharmaceutical agents classified into different drug classes (Vaughan Williams classes) depending on the 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 also 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 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" means the heart cannot keep up with its demands, specifically, the heart cannot 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, endoderm) and / or extraembryonic tissues (pluripotency). Pluripotent stem cells refer to stem cells that have the potential to differentiate into multiple types of tissues or cells, but not all types. Unipotent stem cells refer to stem cells that have the potential to differentiate into specific tissues or cells. Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), induced pluripotent stem cells (iPS cells), etc. 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" should be understood as cardiomyocytes at various developmental stages induced through in vitro protocols to obtain non-natural stem cell products similar to human cardiac muscle cells. 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 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 06, 2010). A person 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 signalling 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 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 progenitors or precursors 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 anti-arrhythmic 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 anti-arrhythmic 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 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 the stem cell-derived cardiomyocytes.

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

[0050] This co-administration 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 allow for a prolonged and locally restricted release of the drug at its target site (Jianyu Li and David J. Mooney, Designing hydrogels for controlled drug delivery, Nat Rev Mater. 2016 Dec;1(12):16071. Published online on October 18, 2016), e.g., by incorporation of the antiarrhythmic drug into biodegradable hydrogel particles (Radhika Narayanaswamy, Vladimir P Torchilin, Hydrogels and Their Applications in Targeted Drug Delivery, Molecules. 2019 Feb;24(3):603. Published online on February 8, 2019).

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

[0052] In one embodiment, the present invention relates to a method for obtaining an antiarrhythmic cardiomyocyte population, comprising culturing in vitro stem cell-derived cardiomyocytes or their precursors or progenitors 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, between 24 and 48 hours, at least 48 hours, between 48 and 72 hours, at least 72 hours, between 72 and 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 invention relates to an anti-arrhythmic cardiomyocyte population for use as a medicament. In one embodiment according to this aspect, the invention relates to an anti-arrhythmic 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 anti-arrhythmic cardiomyocyte population for use in the prevention or reduction of arrhythmia resulting from transplantation of stem cell-derived cardiomyocytes. In one embodiment, the invention relates to the prevention or reduction of arrhythmia induction.

[0057] The favorable advantages of transplantation of antiarrhythmic cardiomyocyte populations in patients are that it avoids the typical risks of antiarrhythmic drugs (e.g. bradycardia, AV block, QT interval prolongation) (DP 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 an antiarrhythmic cardiomyocyte population is that they are likely to exhibit superior engraftment into the host myocardium compared to stem cell-derived cardiomyocytes, since 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 provides at least 10, 100, 1000, or even 10000 times higher concentration levels 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 for 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 transplantation outcomes by increasing the overall pump function of the recipient heart.

[0061] In further embodiments, 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 encapsulation, etc., and by various routes of administration, such as intravenous, oral, endocardial, etc. 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 with the formula C12H20N2O3S. In one embodiment, the concentration of sotalol is 100 nM.

[0063] In one embodiment of the present invention, the anti-arrhythmic cardiomyocyte population is obtained in vitro by culturing stem cell-derived cardiomyocytes in a medium containing one or more anti-arrhythmic 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 with the formula C25H29I2NO3. Amiodarone is specifically found to increase the expression of gap junctions (GJA5), which are cell membrane structures that allow cardiomyocytes to connect and contract in a synchronized manner. This is a key part of how cardiomyocytes work together to ensure the normal propagation of electromechanical impulses that ensure the proper contraction of the heart. This finding supports that amiodarone helps to increase the engraftment and integration of stem cell-derived cardiomyocytes into host tissue and ensures synchronized contraction of cardiomyocytes.

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

[0067] Amiodarone is also found to suppress ANP and BNP expression. This may indicate the possibility of suppressing hypertrophy response or is simply an indicator of better functioning cardiomyocytes. ANP and BNP are known to rise with worsening heart failure, so if ANP and BNP are low, this is a sign of better functioning cardiomyocytes and heart. This finding supports 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 number 137-58-6 with 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 number 31828-71-4 with 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 number 51384-51-1 with 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 with 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, the treatment of patients receiving transplantation of stem cell-derived cardiomyocytes may be by administration of one or more antiarrhythmic agents by any suitable means. Antiarrhythmic agents may be formulated in any suitable manner for administration, including but not limited to, by intravenous injection using an infusion device or by ingestion as a tablet. An infusion device refers to a medical system intended for delivery of the cell product in the respective formulation to a recipient. An infusion device is preferentially suitable for pericardial, epicardial, and / or endocardial delivery. An infusion device may include, but is not limited to, a needle-tipped syringe, a needle-less syringe, an infusion catheter system suitable for delivery in the vicinity of the myocardial infarction region. An infusion device includes, but is not limited to, a device intended for intracoronary, endocardial, and / or epicardial injection.

[0073] When the antiarrhythmic agent comprises more than one antiarrhythmic compound, it may or may not be co-formulated and may be administered together or separately and / or in 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, 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 include amiodarone and propranolol.In one embodiment, the antiarrhythmic agents include 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 include 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, 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. In one embodiment, the antiarrhythmic agent comprises metoprolol and propranolol.

[0093] In one embodiment, the antiarrhythmic agent comprises two agents from class III. In 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 the drugs are enhanced when two or more drugs are combined, which only supports the finding that drugs have related effects on beat / frequency and that drug combinations are more effective than single drug treatments.

[0096] The inventors found that, overall, with increasing concentration, all drugs affect beating, i.e., at low concentrations, cells beat and the beating frequency slows down, at medium concentrations, cells stop beating, and at high concentrations, the drugs are known to be toxic, so if the concentration becomes too high, cells are expected to die.This confirms that the drugs have a relevant antiarrhythmic effect with a dose-dependent effect on beating frequency.The results therefore confirm that beating and rhythm are improved by the drugs, and 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 therapeutic method 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 heartbeat and / or rhythm of the transplanted stem cell derived cardiomyocytes or transplanted anti-arrhythmic cardiomyocyte population. In further embodiments thereof, the method is for reducing alterations in the heartbeat and / or rhythm of the transplanted stem cell derived cardiomyocytes or transplanted anti-arrhythmic cardiomyocyte population.

[0101] In another aspect of the present invention, antiarrhythmic drugs are used in methods for the prevention of graft rejection after transplantation of antiarrhythmic cardiomyocyte populations by altered gene expression in the engrafted cells after exposure to antiarrhythmic drugs in vitro. Thus, the inventors have shown that antiarrhythmic drugs have a direct effect on 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 biomaterials for use in a method for treating heart failure by transplanting 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 the treatment of 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 combinations thereof. In a preferred embodiment, the antiarrhythmic agent is selected from the list of class I, class II, and class III antiarrhythmic agents, or combinations 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 combinations 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 anti-arrhythmic cardiomyocyte population with a high probability of successful transplantation outcome comprising the step of controlling the expression of a gene selected from the list: GJA5, CACNA1G, NPPA, and / or NPPB.

[0105] In a preferred embodiment, the step of controlling expression of the gene 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] Thus, according to the method of the above-mentioned aspect, a unique cell population, i.e., an antiarrhythmic cardiomyocyte population, is obtained.Therefore, another aspect of the present invention relates to an antiarrhythmic cardiomyocyte population, in which at least 40%, 50%, 60%, 70%, 80, 90%, 95% or 99% of cardiomyocytes have regulated gene expression, 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 the treatment of 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 in vivo to the patient 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 the stem cell-derived cardiomyocytes in vitro with an antiarrhythmic agent to obtain an antiarrhythmic cardiomyocyte population that is transplanted into the patient. In a preferred embodiment, the method comprises contacting the stem cell-derived cardiomyocytes in vitro with amiodarone and lidocaine.

[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 anti-arrhythmic cardiomyocyte population, comprising culturing stem cell-derived cardiomyocytes in a medium containing one or more anti-arrhythmic 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 48-72 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 according to any of the preceding embodiments, 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 agent is 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 agent is 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 agent is 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 of the preceding embodiments, 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 method of any of the preceding embodiments, wherein the concentration of the antiarrhythmic agent is in the range of 0.1 μM to 100 μM. 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 medicine. 39. An antiarrhythmic cardiomyocyte population for use in the treatment of heart failure. 40. An antiarrhythmic cardiomyocyte population for use in preventing or reducing arrhythmia. 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 transplantation outcomes 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 of 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 of embodiment 46, having upregulation of GJA5 and / or CACNA1G, and downregulation of NPPA and / or NPPB. 50. The antiarrhythmic cardiomyocyte population of 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 1.5-fold upregulation of GJA5, at least 2-fold upregulation of CACNA1G, at least 2-fold downregulation of NPPA, and / or at least 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 1.5-fold upregulation of GJA5, at least 2-fold upregulation of CACNA1G, at least 2-fold downregulation of NPPA, and / or at least 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 1.5-fold upregulation of GJA5, at least 2-fold upregulation of CACNA1G, at least 2-fold downregulation of NPPA, and / or at least 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 according to embodiment 56, wherein the antiarrhythmic agent is selected from the list of class I, class II, and class III antiarrhythmic agents, or combinations 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 of 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 an antiarrhythmic agent of class I. 85. The antiarrhythmic agent of 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 an antiarrhythmic agent of class I. 88. The antiarrhythmic agent of embodiment 83, wherein the antiarrhythmic agent comprises sotalol and mexiletine. 89. The antiarrhythmic agent of 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 controls the expression of a gene selected from the list: GJA5, CACNA1G, NPPA, NPPB. 94. An antiarrhythmic agent for use in a method for the treatment or prevention of 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 following transplantation of stem cell-derived cardiomyocytes. 97. The antiarrhythmic agent according to embodiment 94, wherein the arrhythmia is caused by a method for the treatment of 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 the prevention of 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 combinations 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 controlling 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. A method according to any one of embodiments 107 to 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 according to any one of embodiments 107 to 108, wherein the control 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; and 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 an antiarrhythmic cardiomyocyte population, and transplanting the antiarrhythmic cardiomyocyte population into a patient. 114. The method of any one of embodiments 112 and 113, wherein the antiarrhythmic agent comprises amiodarone and lidocaine. 115. The method of any one of embodiments 112 and 113, wherein the antiarrhythmic agent comprises 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 agent comprises amiodarone and propranolol. 119. An antiarrhythmic agent for use in the method according to any one of embodiments 112 to 118. EXAMPLES

[0112] Example 1 To determine the effects of antiarrhythmic drugs on stem cell-derived cardiomyocytes beyond the well-established immediate changes in electrophysiological responses through modulation of ion channel activity, we analyzed changes in gene expression resulting from prolonged exposure (>24 h) of cardiomyocytes to antiarrhythmic drugs. To this end, we evaluated the effects of commonly used antiarrhythmic drugs, such as amiodarone and lidocaine, 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) and cultured at 1.6–2.4 × 10 4 cells / cm 2 Cell lines were seeded at 37 °C in iPSBrew supplemented with 10 μM Y-27632 (Sigma) at 37 °C. Cell lines were 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 signalling regulate mesendodermal patterning of human pluripotent stem cells. Nat Commun. 2016;7:13602). Briefly, cells were seeded in 6-well suspension plates (Greiner) and cultured at 0.16 × 10 6 Aggregates were allowed to form at 1000 cells / mL. After 2 days, differentiation was induced using 4-8 μM CHIR99021 (Tocris) for 24 h followed by 2 μM Wnt-C59 (Tocris) for 48 h in RPMI1640 medium (Life Technologies) supplemented with 2% B27 (Life Technologies) without insulin or RPMI1640 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 RPMI1640 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 2After 4 days, cardiomyocytes were exposed to antiarrhythmic drugs for at least 72 hours at the following concentrations: 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 their combinations at each concentration. Blank medium, addition of solvent, and cardiomyocytes cultured for 9 days and day 42 were used as controls. Cell beating was evaluated after 48 hours, 72 hours, 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 the influx of second messenger Ca 2+ CACNA1G plays a key role in regulating the intracellular distribution of Ca2+. Thereby, the channel regulates various cellular processes, including the beating of cardiomyocytes. More specifically, CACNA1G controls the electrical and pacing activity of the heart. Importantly, dysfunction of the channel is associated with both atrial and ventricular arrhythmias, especially 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). Thus, the clear upregulation of CACNA1G by amiodarone suggests an increased capacity of treated hESC-derived cardiomyocytes in their intrinsic electrophysiological capacitance to control and prevent arrhythmic responses.

[0119] Similarly, 0.1 μM and 1 μM amiodarone led to a more than three-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 the conduction of currents across the ventricles (Shekhar A et al, Transcription factor ETV1 is essential for rapid conduction in the heart. J Clin Invest. 2016; 126(12): 4444-59). 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). As a result, upregulation of GJA5 in ES-derived cardiomyocytes by amiodarone is likely to accelerate electrical signaling across cell-cell contacts, thereby suppressing arrhythmic behaviors, particularly via macro- or micro-reentry, thereby reducing the risk of developing ectopic foci.

[0120] In contrast to the increased levels of CACNA1G and GJA5, treatment with amiodarone resulted in a reduction of NPPA and NPPB expression by approximately 3-fold and 5-fold, respectively (Figures 3 and 4). NPPA and NPPB code for the secretory hormones ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide), which are secreted mainly from the atria and less prominently from the ventricles of the adult heart in response to mechanical stretch. Quantification of natriuretic peptide levels is routinely used as a tool for the diagnosis of 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 exact mechanism of how natriuretic peptides control electrophysiological actions in humans remains unclear. It is believed that the peptides induce shortening of action potentials, thereby increasing the likelihood of reentry. Furthermore, prolongation of action potential duration by reducing ANP and / or BNP levels reduces the likelihood of tachyarrhythmias. Thus, lowering NPPA and NPPA using amiodarone in hESC-derived cardiomyocytes reduces the risk of graft-induced arrhythmias and the development of tachycardia and ectopic beating foci from hESC-derived cardiomyocytes after cardiac transplantation.

[0121] Notably, the beating frequency of hESC-derived cardiomyocytes was obviously reduced or inhibited at 1 μM and 10 μM, respectively, reducing the possibility of tachyarrhythmia. At the same time, amiodarone did not alter the expression of pan-cardiomyocyte genes such as NKX2-5, TNNT2, and ACTA2 (Figures 5-7), suggesting that the 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 (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), which controls the rate of rise of the action potential in human hearts.

[0122] Overall, exposure of hESC-derived cardiomyocytes to amiodarone induces a unique expression profile that includes elevated levels of CACNA1G and GJA5 accompanied by a decrease in 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 regenerating the heart by omitting graft-induced arrhythmias and / or tachycardia.

[0123] Similarly, we found that other classes of antiarrhythmic drugs modulate NPPA and NPPB. Lidocaine, the most relevant class 1b antiarrhythmic drug, reduced both natriuretic peptides at 1 μM, 10 μM, and 100 μM in a concentration-dependent manner (Figures 9, 10) without affecting the expression of pan-cardiomyocyte markers (NKX2-5 and TNNT2; Figures 11, 12). Considering the overall relevance of NPPA and NPPB for 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 validate sustained effects and robustness across different culture systems, we tested the effect of antiarrhythmic drugs on the gene expression profile of stem cell-derived cardiomyocytes using 3D suspension aggregates and measured gene expression over a 48-h period following 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 × 10 6 Cells were maintained in 6-well suspension plates on an orbital shaker (75 rpm) at a cell density of 1000 cells / mL. Cells in the aggregates were exposed to 10 μM amiodarone for approximately 120 h. Cells were then maintained for an additional 48 h in RPMI medium supplemented with 2% B27 + 0.1% P / S. Complete medium changes were performed every 48–72 h. Cells were then harvested and subjected to RNA expression analysis.

[0127] result Gene expression profiles of aggregates measured 48 hours after treatment with 10 μM amiodarone show 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] Thus, the results confirm the sustained and clear effect of antiarrhythmic drugs, such as amiodarone, on gene expression levels related to the altered electrophysiological properties of stem cell-derived cardiomyocytes, which leads to the reduction of arrhythmic potentials as shown in Examples 3 and 4. Furthermore, the results show that the effect is independent of culture mode, e.g., it is 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 drugs is expected to be reflected in in vivo applications.

[0129] Example 3 To determine the sustained effects on the electrophysiological properties of stem cell-derived cardiomyocytes following exposure to antiarrhythmic drugs, we used Ca 2+-recordings were used to perform functional cardiomyocyte testing to determine the arrhythmogenic potential of stem cell-derived cardiomyocyte populations following 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 as frozen vials and stored in liquid nitrogen until use. All culture media required for thawing and culturing were supplied by the cell supplier. Thawing, plating, and culturing procedures were performed according to the protocol provided by the manufacturer. Cells were plated directly into fibronectin-coated 384-well Greiner μClear plates. Plating density was 17.500 cells / well in a final volume of 50 μl. Medium was changed (90%) 1 day after plating, every 2 days thereafter, and 3 h 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 DIV4. Treatment was terminated by replacing the medium at DIV6 with standard culture medium. Recordings were performed at DIV7. 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 before the buffer was exchanged again for dye-free buffer. 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 inspection), including the shape and amplitude of the signal, as well as 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 min before compound application. 200 μM moxifloxacin was applied after a baseline phase in a single concentration / well format, followed by a 5 min wash phase. Fluorescence activity was then recorded for an additional 5 min. Moxifloxacin without compound preincubation was included as a control (n=18) and distributed across multiple plates.

[0135] Using FDSSv3.4 offline, consecutive Ca transients within each recording period were recorded. 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 was 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 to the control treatment. The results show a clear reduction in beat-to-beat variability, 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, namely 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 to the respective control conditions that were not exposed to the antiarrhythmic agent. Similarly, 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% under baseline and proarrhythmic conditions, respectively. Of note, this apparent reduction in beat-to-beat variability was observed 24 hours after cessation of the compounds and is therefore not dependent on the continued presence of the drug.

[0140] Overall, the results clearly demonstrated a reduction in beat-to-beat variability following treatment with class I, II, or III antiarrhythmic agents, 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, our results suggest that treatment with antiarrhythmic agents induces an antiarrhythmic cell population, reducing the susceptibility of cells 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 following cell transplantation.

[0141] Furthermore, 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 a direct effect 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 h, followed by a 24-h recovery period, and then measured. Drugs were applied at the following concentrations: 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. The results show that combinations of class III drugs with either class I and / or class II are more efficient in reducing proarrhythmic potential than the drugs alone, with a reduction of 46,4% or 31,4% and 46,4%, respectively (Figure 16). Similarly, the combination of class I and class II showed a reduction of 48,3% compared to single compound treatment.

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

[0145] Overall, the results of the above examples show 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. A pharmaceutical comprising an antiarrhythmic cardiomyocyte population.

2. A medicament for use in the treatment of heart failure comprising a population of antiarrhythmic cardiomyocytes.

3. The pharmaceutical agent of claim 1 or 2, wherein the antiarrhythmic 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.

4. The pharmaceutical of any one of claims 1 to 3, wherein the antiarrhythmic cardiomyocyte population has regulated expression of a gene selected from the list of GJA5, CACNA1G, NPPA, and NPPB.

5. The pharmaceutical agent of claim 4, wherein the antiarrhythmic cardiomyocyte population has upregulated expression of GJA5 and / or CACNA1G, and downregulated expression of NPPA and / or NPPB.

6. The pharmaceutical agent of claim 5, wherein the antiarrhythmic cardiomyocyte 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.

7. A kit comprising an antiarrhythmic agent and stem cell derived cardiomyocytes.

8. 8. The kit of claim 7, 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.

9. An antiarrhythmic agent for use in a method for treating or preventing heart failure by transplantation of stem cell-derived cardiomyocytes.

10. A composition comprising stem cell derived cardiomyocytes, one or more antiarrhythmic agents, and optionally a biomaterial, for use in the treatment of heart failure.

Images