Compounds and methods for treating and / or alleviating the side effects of myocardial infarction

Abstract

The present invention relates to compounds capable of modulating Zeb1. In particular, the present invention relates to compounds capable of modulating the transcription factor Zeb1 for use in a treatment of the myocardium after myocardial infarction. Furthermore, the present invention provides an ex vivo method of maturing iPS CM, the method comprising, administering to an iPS CM a compound capable of decreasing the levels of Zeb1. Lastly, the present invention relates to a method of treatment of the myocardium in a subject having experienced myocardial infarction.

Description

[0001] COMPOUNDS AND METHODS FOR TREATING AND / OR ALLEVIATING THE SIDE EFFECTS OF MYOCARDIAL INFARCTION

[0002] Technical field of the invention

[0003] The present invention relates to compounds capable of modulating Zebl. In particular, the present invention relates to compounds capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction. Furthermore, the present invention provides an ex vivo method of maturing iPS CM, the method comprising, administering to an iPS CM a compound capable of decreasing the levels of Zebl. Lastly, the present invention relates to a method of treatment of the myocardium in a subject having experienced myocardial infarction.

[0004] Background of the invention

[0005] Cardiovascular diseases are among the leading causes of death worldwide, and one major explanation is the inability to regenerate the heart after myocardial infarction (MI), leaving the affected subjects with impaired cardiac function. The mammalian heart forms through cardiomyocyte (CM) proliferation during fetal development, but around birth CMs enter cell cycle arrest. Thus, after MI, CMs are lost, and they are not replenished. As a result, the pump function of the heart is reduced, and the patient may die.

[0006] It is sought for to find strategies on how to replenish the CM number after MI. One strategy is to identify factors capable of inducing proliferation in remaining CMs, to repair the damaged heart after MI.

[0007] Another strategy is to use induced pluripotent stem cell (iPS) derived CMs and transplant to the MI heart. The discovery of somatic cell reprogramming to generate iPS has created great excitement because of the ability to produce, patient-specific iPS-CMs. To generate iPS-CMs, somatic cells can be obtained from patient's or a donor's tissue including, but not limited to hair, blood, skin, fat, urine, or oral mucosa. These cells are then reprogrammed to a pluripotent state by introducing pluripotency-associated genes. The resulting iPS cells are then differentiated into cardiomyocytes by using several strategies. After ~8 to 12 days, clusters of beating CMs typically appear, and after further maturation and selection close to 100% CMs are obtained. It is however well known that these iPS-CMs appear immature when comparing to adult CMs, which increases the risk of arrhythmias and limits their translation. Despite this, trials are currently undergoing where iPS-CMs are being tested in the clinic, and in particular grafted to patient hearts having suffered from MI.

[0008] It is thus furthermore sought for to identify factors to be used in maturing iPS- CMs. Such factors are applied during iPS-CM culture prior to engraftment into the heart, or used in situ to further mature the already engrafted iPS-CMs in the heart that requires further treatment.

[0009] Hence, an improved regeneration of the myocardium after a myocardial infarction would be advantageous, and in particular a more efficient and / or reliable maturation of iPS cardiomyocytes would be advantageous.

[0010] Summary of the invention

[0011] An object of the present invention relates to the provision of compounds and methods for treating and / or alleviating the side effects of MI by replenishing the CM number after MI.

[0012] The present inventors have surprisingly found that Zebl is involved in the regulation of the cell cycle of CMs. The inventors have made several interesting findings that find use in the two strategies described above. Firstly, the inventors identified Zebl as a novel regulator of G2 / M progression and CM proliferation before birth in mice and found that Zebl overexpression after birth leads to increased cell cycle activity with CM endoreplication and high CM ploidy. Thus, to increase the numbers and / or the workload of endogenous CMs, Zebl should be overexpressed in CMs. These surprising findings are presented in examples 3-8. Secondly, the inventors identified Zebl expression to be highest in human iPS- CMs around the time of beating initiation, and then declines as CMs become more mature. Thus, in the beginning of iPS CM culture Zebl should be high, but then Zebl should be downregulated to promote maturation of iPS-CMs during iPS-CMs culture. These surprising findings are presented in examples 9-11.

[0013] Essentially, the aspects relating to iPS CM maturation should resemble these findings, initially the Zebl should be present to drive CM differentiation and then later in differentiation be decreased, thus effectively resembling the findings from these mice studies, that at E16.5 Zebl is high and after birth at days Pl and P5 Zebl is low.

[0014] Thus, by studying the molecular mechanisms behind the cell cycle regulation of CMs, the inventors have realized ways of treating side effects of MI, either by increasing the level of Zebl in endogenous CMs residing in the heart tissue, or by manipulating Zebl levels during iPS-CM culture.

[0015] Several specific compounds are exemplified in the examples. Specific oligonucleotides are presented in examples 8-11 which can increase and decrease the levels of Zebl, and small molecules are identified in example 12, which also increase and decrease the levels of Zebl and hereby either promote iPS-CM maturation or lowers it. The latter is presented in examples 13-14.

[0016] In particular, it is thus an object of the present invention to provide compounds capable of modulating Zebl. It is preferred that the modulation is to decrease or increase the protein levels of Zebl.

[0017] Thus, one aspect of the invention relates to a compound capable of modulating the transcription factor Zebl for use as a medicament.

[0018] Thus, a more specific aspect of the invention relates to a compound capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction.

[0019] In another related aspect, the invention relates to a compound capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction:

[0020] • wherein the compound is a compound capable of decreasing the levels of Zebl in an iPS cardiomyocyte (CM) and treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool by engrafting said iPS CM into a subject having experienced myocardial infarction; or • wherein the compound is a compound capable of increasing the levels of Zebl in a cardiomyocyte residing in a subject having experienced myocardial infarction and treatment of the myocardium is conceived by reestablishing the cardiomyocyte pool by treating said endogenous cardiomyocytes.

[0021] Another aspect of the present invention relates to an ex vivo method of maturing iPS CM, the method comprising, administering to an iPS CM a compound capable of decreasing the levels of Zebl. A specific embodiment of this method is described in example 1 and examples 9-11.

[0022] Yet another aspect of the present invention is to provide an iPS cardiomyocyte matured by the method as described above. A closely related aspect is thus a container comprising the iPS CM by the method as described above.

[0023] Yet another aspect of the present invention is to provide an iPS cardiomyocyte matured by the method as described above, for use in the treatment of the myocardium after myocardial infarction.

[0024] Another aspect of the present invention relates to a method of inducing proliferation and / or CM polyploidy in cardiomyocytes, the method comprising, delivering to a cardiomyocyte a compound capable of increasing the level of Zebl in said cardiomyocyte. A specific embodiment of this method is described in example 1 and examples 7-8.

[0025] Still another aspect of the present invention is to provide a method of treatment of the myocardium in a subject having experienced myocardial infarction, the method comprising administering to the subject an effective amount of a compound capable of modulating Zebl.

[0026] Brief description of the figures

[0027] Figure 1 shows enriched set of transcription factors (TFs) specific for CM

[0028] G2 / M progression, a, UMAP plot of 2n- and 4n CMs at E16.5, Pl, and P5 with cell cycle phase identity visualized, b, UMAP plot of extracted G2 / M, 4n CMs from E16.5 and P5 developmental stage from the two Cell division clusters as indicated in (a), c, Heatmap of expression of top genes with increased expression in E16.5 CMs compared to P5 CMs in (b). d, cnetplot of selected cell-cycle related GO terms enriched among the genes in (c). e, Enriched TFs (oPOSSUM) for genes with increased expression in E16.5 CMs compared to P5 CMs in (b).

[0029] Figure 2 shows cell cycle related genes in mouse CMs regulated by Zebl. The number of cell cycle related genes affected is designated for Zebl (Blue), whereas the size of the dot indicates the frequency of a gene across the cell cycle related GO terms indicated. Genes are shown on the X-axis.

[0030] Figure 3 shows ZEB1 expression in mouse heart, a, UMAP plot of E16.5-4n CMs extracted from UMAP plot of all six conditions with blue color indicating CMs expressing Zebl mRNA. b, Dot plot of Zebl expression for all six conditions with size indicating percentage of CMs expressing Zebl and color indicating the average expressing level in Zebl-expressing CMs.c, Violin plots illustrating Zebl expression levels in CMs at each development stage and ploidy to emphasize expression dynamics.

[0031] Figure 4 shows ZEB1 knockdown reduce CM proliferation before birth, a, Schematic of the study design and workflow. Embryonic hearts were isolated at E16.5 and cells were cultured for 24 h before adenoviral transduction with either Ad-GFP-shRNA (control; scrambled shRNA) or Ad-GFP-shRNA-Zebl. The insert depicts the viral construct containing an eGFP reporter. Cells were fixed 96 h after transduction and EdU pulsing, and analyzed by flow cytometry and immunocytochemistry (ICC), b, Normalized mRNA level of ZEB1 in Ad-GFP-shRNA and Ad-GFP-shRNA-Zebl transduced cells (normalized against B2m and Gapdh; Paired t-test, n = 3, *P < 0.05). c, EdU positive CMs transduced with Ad-GFP- shRNA or Ad-GFP-shRNA-Zebl quantified by flow cytometry (Paired t-test, n = 5, **P < 0.01). d, CM ploidy of Ad-GFP-shRNA and Ad-GFP-shRNA-Zebl transduced, EdU positive cells quantified by flow cytometry based on Hoechst intensity (Paired t-test, n = 5, **P < 0.01; ****P < 0.0001).

[0032] Figure 5 shows ZEB1 knockdown reduce CM proliferation before birth, a,

[0033] Normalized mRNA levels of Ccndl, Ccne2, Ccng2, Ccnbl, and Ccnd3 (normalized against B2m and Gapdh; Paired t-test, n = 3, * P < 0.05; **P < 0.01). b, Normalized mRNA levels of Cdkl, Cdk4, Cdknla, and Cdknlb (normalized against B2m and Gapdh; Paired t-test, n = 3, *P < 0.05). c, Normalized mRNA levels of Cenpe, Cenpf, Aurkb, Gmnn, and Aurka (normalized against B2m and Gapdh; Paired t-test, n = 3, *P < 0.05; **P < 0.01).

[0034] Figure 6 shows ZEB1 increases CM cell cycling while decreasing cell size, a, Schematic of the study design and workflow. Neonatal hearts were isolated at P0 and cultured for 24 h before transduction with adenovirus (Ad). The insert depicts the viral construct, note the eGFP reporter in both Ads. Cells were fixed after 72 h transduction and EdU subjection and analyzed by flow cytometry, b, Zebl expression in CMs after 72 h Ad-GFP or Ad-GFP-Zebl transduction as quantified by qRT-PCR (normalized against B2m and Rpl_13A; Unpaired t-test, n = 6-7, **P < 0.01). c, Incorporation of EdU was observed at both 50, 100, and 150 MOI after 72 h Ad-GFP and Ad-GFP-Zebl transduction (Unpaired t-test, n = 3, *P < 0.05, ***p < 0.001). d, Flow cytometric assessment of CM size 72 h after Ad-GFP-Zebl transduction using the geometric mean of CMs and non-myocytes (NMs) (Two-way ANOVA, n = 9, ****P < 0.0001). e, Percentages of CMs of the total cell number after 72 h in culture (non-transduced), or after 72 h Ad-GFP or Ad-GFP-Zebl transduction (One-way ANOVA followed by Tukey's multiple comparisons test, n = 5).

[0035] Figure 7 shows ZEB1 increase CM endoreplication after birth, a, Normalized mRNA levels of Ccndl, Ccne2, Ccng2, Ccnbl, and Ccnd3 (normalized against B2m and Rpl_13A; Unpaired t-test or Mann-Whitney based on normality, n = 6-7, **P < 0.01). b, Normalized mRNA levels of Cdkl, Cdk4, Cdknla, and Cdknlb (normalized against B2m and Rpl_13A; Unpaired t-test or Mann-Whitney based on normality, n = 6-7, **P < 0.01). c, Normalized mRNA levels of Mstn, and Myh6 (normalized against B2m and Rpl_13A; Unpaired t-test or Mann-Whitney based on normality, n = 6-7, **P < 0.01). d-e, Neonatal pups (P0) were injected with Ad- GFP or Ad-GFP-Zebl through the superficial temporal vein, followed by two subcutaneous injections of EdU at P4 and P6. Hearts were collected at P8 for ventricular dissociation followed by flow cytometry, d, Percentages of EdU+GFP+CMs of GFP+CMs at P8 after Ad-GFP injection at P0 compared to Ad-GFP-Zebl injection (Paired t-test, n = 7-9, **P < 0.01). e, Percentage-wise distribution of ploidy at P8 after Ad-GFP or Ad-GFP-Zebl injection at P0 (Paired t-test, n = 7-9, *P < 0.05).

[0036] Figure 8 shows Zebl overexpression by AAV9 CM transduction and the effect on CM cell cycle activity, a, Schematic of the study design and workflow. Neonatal heart cells were isolated at P0 and cultured for 24h, before transductions with AAV9-cTnT-Zebl or control. Insert depicts as modified from Addgene. EdU incorporation into mCherry+and mCherry CMs together with CM- (MYH1+) and ploidy- (Hoechst) identity were then assessed by flow cytometry, b, mRNA levels of Zebl were quantified by qRT-PCR including normalization against stably expressed endogenous controls B2m and Gapdh) after 72h of AAV9-cTnT-Zebl CM transduction, c, The percentage of EdU+mCherry+CMs was compared to that of empty vector transduced CMs (AAV9-cTnT) (T-test, ****P < 0.0001). d, Flow cytometric assessment of the percentage of diploid- (2n), tetrapioid- (4n) and polyploid- (>4n) transduced and non-transduced CMs as indicated.

[0037] Figure 9 shows Zeb expression differs across iPSC-CM differentiation. iPSCs were cultured as in general and CM differentiation initiated at day 0 (x- axis). RNA was isolated at day 0-30, and mRNA transcript levels quantified. Graphs depict normalized mRNA levels of pluripotency genes OCT4 and SOX2), CM genes TNNT2, MYH6, MYH7), and CM maturation genes (SIRPA, FABP3, SCN5A, CMLC2) (normalized against B2M and GAPDH, and tested by ONE-WAY ANOVA (Kruskal-Wallis test): p<0.01 for all).

[0038] Figure 10 shows Zebl KO in iPS-CMs. iPSCs were cultured as in general and CM differentiation initiated at day 0. At day 8, Zebl was knocked down by adenovirus mediated delivery of shRNA against ZEB1. At day 11, iPS-CMs were harvested and analyzed by scRNAseq (a-c) and qRT-PCR (d). a, b, ScRNAseq data reveal that Zebl is expressed in Troponin T+ CMs at day 11. Transcription factors such as Zebl are only low detected by scRNAseq, but still a high fraction of CMs are found to be Zebl + . Moreover, shRNA-Zebl reduce ZEB1 expression as both tested by scRNAseq and qRT-PCR. For the latter data was normalized against GAPDH and RPL13A that were stably expressed. Figure 11 shows Zebl KO in iPS-CMs. iPSCs were cultured as in general and CM differentiation initiated at day 0. At day 8, Zebl was knocked down by adenovirus mediated delivery of shRNA against ZEB1 or shRNA-control. At day 11, iPS-CMs were harvested and analyzed by scRNAseq. ScRNAseq data comparing differential gene expression between shRNA-Zebl and shRNA-control reveal that gene ontology terms related to cardiac muscle is higher in shRNA-control treated iPS-CMs (Shown) as compared to shRNA-Zebl treated iPS-CMs.

[0039] Figure 12 shows the Connectivity platform by Broad Institute used to identify compounds that dictate Zebl levels and which can be used to manipulate Zebl levels in vitro and in vivo to either increase or decrease Zebl levels.

[0040] Figure 13 shows Zebl lowering in iPS-CMs by compounds predicted in Figure 12. iPSCs were cultured as in general and CM differentiation initiated at day 0. At day 25, cells were treated with compounds (Rigosertib and Nocodazole shown) or vehicle. At day 27 (48h) and 30 (120h), iPS-CMs were harvested and analyzed by qRT-PCR for ZEB1 (a, c) and MYL2 (b,d). Data reveal that Zebl levels are significantly lowered after 48hours of compound treatment, and that this already after 48h significantly increases MYL2 expression, and that this is much further substantiated at day 30 (120h). This shows that compounds lowering Zebl in the late stage of derivation significantly increases maturation. QRT-PCR data was normalized against GAPDH, B2M and PGK1 that were stably expressed. One- WAY ANOVA with post-test (two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli) was performed to test statistical significance. Exact P-value is indicated for each comparison.

[0041] Figure 14 show that Zebl levels in iPS-CMs may be increased by compounds predicted in Figure 12. iPSCs were cultured as in general and CM differentiation initiated at day 0. At day 25, cells were treated with compounds (Anisomycin shown) or vehicle. At day 27 (48h) iPS-CMs were harvested and analyzed by qRT-PCR for ZEB1 (a) and MYL2 (b). Data reveal that Zebl levels are significantly increased after 48 hours of compound treatment, and that this already after 48h significantly decreases MYL2 expression. This shows that compounds may be used to increase Zebl levels when needed for instance during early iPS-CM derivation. QRT-PCR data was normalized against GAPDH, B2M and PGK1 that were stably expressed. One-WAY ANOVA with post-test (two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli) was performed to test statistical significance. Exact P-value is indicated for each comparison.

[0042] The present invention will now be described in more detail in the following.

[0043] Detailed description of the invention

[0044] Definitions

[0045] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0046] Myocardial infarction

[0047] In the present context, the term "myocardial infarction" or "MI" refers to a heart attack which is a condition wherein blood flow of at least one of the hearts coronary arteries is blocked or lower than normal.

[0048] Zebl

[0049] In the present context, the term "Zebl", or "Zinc finger E-box-binding homeobox 1" refers to the transcription factor Zebl. Zebl is mainly known for its role in epithelial to mesenchymal transition where it seems to promote sternness in cancer by enhanced DNA damage response (DDR) and DNA repair capacity, selfrenewal, and chemoresistance.

[0050] Zebl is in the present context understood as the protein encoded by the ZEB1 gene. In some embodiments, Zebl has the sequence according to SEQ ID NO: 1 or a variant thereof having at least 80 % sequence identity to the sequence according to SEQ ID NO: 1. Human Zebl is for example also identified in the uniprot database, where it can be found under the accession number P37275.

[0051] Cardiomyocyte

[0052] In the present context, the term "cardiomyocyte", or "CM" is understood as the cell responsible for the contraction of the heart. Cardiac muscle cells, i.e. cardiomyocytes, are the contractile myocytes of the cardiac muscle. The cells are surrounded by an extracellular matrix produced by supporting fibroblast cells. Specialised modified cardiomyocytes known as pacemaker cells, set the rhythm of the heart contractions. Endogenous CM are understood as the subject's own cells located or residing in the cardiac tissue, such as before a ML iPS cardiomyocytes

[0053] In the present context, the term "induced pluripotent stem cell", "iPS", and "iPSCs", is understood as a previous somatic cell that have undergone reprogramming to generate a cell type that can be differentiated into a specific cell type. More specifically "iPS CM" is thus understood as an iPS, where differentiation has been initiated with the aim of developing a CM. Differentiation initiation may be defined as when B27-medium (or an equivalent thereto capable of initiating CM differentiation) is added. Example 1 provides a typical protocol for differentiating iPS CM, however variations to such a protocol exist, which still produces an iPS CM. The protocol in example 1 steers the differentiation over app. 30 days, however the different stages of maturation is preferably monitored as described below.

[0054] Without being bound by theory, a rough schematic of the iPS CM culture can be viewed as:

[0055] - D0-D8 - is a mesoderm differentiation where a variety of different cell types can be found in the culture,

[0056] - D8 app. - 60-70 % of the culture can be identified as iPS CM, and beating is observed,

[0057] - D8-D18 - iPS CM specification and differentiation,

[0058] - D18-D22 - iPS CM selection by removal of glucose, and selected iPS CM should be able to primarily grow on free fatty acid metabolism - the end result is app. more than 98% iPS CM, and

[0059] - D24 and onwards is primarily additional final maturation.

[0060] Maturation of iPS CM may be monitored by observing beating initiation, and / or by measuring one or more of the genes that are expressed at different stages of differentiation, such as one or more genes selected from:

[0061] - pluripotency genes such as OCT4 and / or SOX2;

[0062] - CM genes such as TNNT2, MYH6, and / or MYH7 as a sign of iPS-CM differentiation; and CM maturation such as SIRPA, FABP3, SCN5A, and / or CMLC2, and / or preferably MYL2.

[0063] Proliferation

[0064] In the present context, the term "proliferation" or "cell proliferation" is the process by which a cell grows and divides to produce two daughter cells. Cell proliferation leads to an exponential increase in cell number and is therefore a rapid mechanism of tissue growth. Cell proliferation requires both cell growth and cell division to occur at the same time, such that the average size of cells remains constant in the population. Cell proliferation may be monitored by the observance of an increase in cell number over time.

[0065] Endoreplication

[0066] Endoreplication is understood as replication of the nuclear genome in the absence of mitosis, which leads to elevated nuclear gene content and polyploidy. An effect of the increase in ploidy is that the workload of a polyploidic cell is larger.

[0067] CM ploidy will increase as a result of increased endoreplication, and thus result in CM polyploidy.

[0068] Whether endoreplication has occurred can for example be studied by tissue cytometry, FACS, and other suitable methods of counting of individual nuclei or an increase in DNA content of individual cells. Expectantly, an increase in cyclin E is a sign of endoreplication, whereas a decrease in cyclin B reflects that the cell does not undergo division.

[0069] Spheroid

[0070] In the present context, the term "spheroid" refers to a three-dimensional (3D) cell aggregate that can mimic tissues. Spheroids can be prepared with cardiomyocytes, such as iPS CM, ex vivo and transplanted to subjects. Preparation of cardiomyocytes into spheroids can be done to improve engraftment. Subject

[0071] In the present context, the term "subject" comprises humans of all ages, other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals in general, including commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, mice, rats, rabbits, cats and dogs, as well as birds. Preferred subjects are humans.

[0072] Pericardium

[0073] In the present context, the term "pericardium" refers to the pericardial sac enclosing the heart. The sac comprises an outer fibrous pericardium layer and an inner serous pericardium layer ("the epicardium"). Between the two layers is the pericardial cavity.

[0074] Pericardial fluid

[0075] In the present context, the term "pericardial fluid" refers to the fluid in the pericardial cavity.

[0076] Intracardiac administration

[0077] In the present context, the term "intracardiac administration" (I-CARDI) refers to administration into the heart muscle or circulatory system.

[0078] Intrapericardial fluid administration

[0079] In the present context, the term "intrapericardial fluid administration" refers to an intrapericardial administration (I-PERICARD) which is an administration within the pericardium, preferably to the intrapericardial fluid.

[0080] Homologue or homolog

[0081] In the present context, a "homologous gene", or "homolog" is a gene inherited in two species from a common ancestor or the protein encoded by said gene. While homologous genes can be similar in sequence, similar sequences are not necessarily homologous. Sequence identity

[0082] In the context of the present invention, the term "sequence identity" or "homologue" indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences. If the two sequences to be compared are not of equal length, they must be aligned to give the best possible fit, allowing the insertion of gaps or, alternatively, truncation at the ends of the polypeptide sequences or nucleotide sequences. The sequence (Nref-Ndlf)100 N ref identity can be calculated asJ, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif=2 and Nref=8).

[0083] With respect to all embodiments of the invention relating to amino acid sequences or nucleotide sequences, the percentage of sequence identity between one or more sequences may also be based on alignments using the clustalW software (http: / www. ebi.ac.uk / clustalW / index.html) with default settings. For nucleotide sequence alignments these settings are: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, DNA weight matrix: identity (IUB). For amino acid sequence alignments the settings are as follows: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, Protein weight matrix: Gonnet.

[0084] Alternatively, nucleotide sequences may be analysed using programme DNASIS Max and the comparison of the sequences may be done at http: / / www.paralign.org / . This service is based on the two comparison algorithms called Smith-Waterman (SW) and ParAlign. The first algorithm was published by Smith and Waterman (1981) and is a well-established method that finds the optimal local alignment of two sequences. The other algorithm, ParAlign, is a heuristic method for sequence alignment; details on the method are published in Rognes (2001). Default settings for score matrix and Gap penalties as well as E- values were used. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0085] Compounds and methods for treating and / or alleviating the side effects of MI by replenishing the CM number after MI.

[0086] As outlined above, the present invention relates to compounds and methods for treating and / or alleviating the side effects of MI by replenishing the CM number after ML The goal of these compounds is to modulate the level of Zebl. One strategy is to induce proliferation in remaining CMs, to repair the damaged heart after MI, achieved by using a compound that increases the level of Zebl. Another strategy is to use iPS CM and engraft them to the MI heart, the iPS CM either treated with a compound that decreases the level of Zebl prior to and / or after engraftment. These compounds are described further below, and generally finds use in the methods described herein.

[0087] Firstly, however, the compounds are described in view of their use in a treatment of the myocardium after myocardial infarction.

[0088] Thus, an aspect of the invention relates to a compound capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction.

[0089] As further detailed below, another aspect of the invention relates to a compound capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction:

[0090] • wherein the compound is a compound capable of decreasing the levels of Zebl in an iPS cardiomyocyte (CM) and treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool by engrafting said iPS CM into a subject having experienced myocardial infarction; or

[0091] • wherein the compound is a compound capable of increasing the levels of Zebl in a cardiomyocyte residing in a subject having experienced myocardial infarction and treatment of the myocardium is conceived by reestablishing the cardiomyocyte pool by treating said endogenous cardiomyocytes. The damages incurred on the tissue after a MI infarction can have severe longterm effects on the patient. However, these damages are often defined in various ways, and can therefore be referred to by various definitions such as repairing the cardiac tissue, recovering cardiac function, re-establishing the cardiomyocyte pool. Thus, in one embodiment of the present disclosure, treatment of the myocardium is a repair of the cardiac tissue. In another embodiment of the present disclosure, treatment of the myocardium results in recovering cardiac function. In a further embodiment of the present disclosure, treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool reestablishing the cardiomyocyte pool.

[0092] Evidently, since the compounds of the present invention finds use in treatment of the myocardium after myocardial infarction, these compounds are medicaments in general, and as such another aspect of the invention relates to a compound capable of modulating the transcription factor Zebl for use as a medicament.

[0093] Treatment by targeting iPS cells, and compounds therefore

[0094] As presented above, one strategy of treating the myocardium is by the usage of iPS cells. The iPS CM can either be engrafted to the patient and further matured inside the patient, the cells can be matured ex vivo and engrafted later in the process (see further below), or a combination may be used.

[0095] Thus, in one embodiment of the present disclosure, treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool, such as by engrafting iPS cardiomyocytes (CM). In another embodiment of the present disclosure, the treatment comprises engrafting iPS CM into a subject having experienced myocardial infarction. In a further embodiment of the present disclosure, the cardiomyocyte pool is re-established by maturing engrafted iPS CM. In one embodiment of the present disclosure, the compound is a compound capable of decreasing the levels of Zebl in an iPS cardiomyocyte (CM).

[0096] One advantage of using iPS CM is that a large number of cells can be produced, having the same genome as the patient suffered from MI. Thus, in one embodiment of the present disclosure, the iPS CM are derived from the subject having experienced a myocardial infarction. On the opposite, it may be preferable to use cells that are not derived from the subject having experienced a myocardial infarction, in this way "off-the shelf" products can be prepared in advance, and routinely kept at stages where CM can be engrafted to patients. Thus, in one embodiment of the present disclosure, the iPS CM are not derived from the subject having experienced a myocardial infarction. In one embodiment of the present disclosure, the iPS CM are allogenic.

[0097] As identified by the present invention, for an iPS CM to further mature, the levels of Zebl should be decreased. In one embodiment of the present disclosure, the compound is a compound capable of decreasing the levels of Zebl. In one embodiment of the present disclosure, the compound is a compound capable of decreasing the levels of Zebl in an iPS CM engrafted into a subject having experienced myocardial infarction. These surprising findings are presented in examples 9-11.

[0098] An iPS CM can be engrafted at different stages of maturation, and thus the specific timepoint at which the compound capable of decreasing the levels of Zebl should be administered, can be dependent thereon. Preferably, the compound is administered when the iPS CM is administered. iPS CM maturation should resemble findings from the mice studies disclosed herein, initially Zebl should be present to drive CM differentiation and then later in differentiation be decreased, thus effectively resembling the findings from the mice studies disclosed herein, that at E16.5 Zebl is high and after birth at days Pl and P5 Zebl is low.

[0099] The later time point of administration shall allow the iPS CM to grow and differentiate, and thus maturation can be initiated at a later stage. In one embodiment of the present disclosure, the compound is administered to reduce the levels of Zebl when selection is initiated, such as after day 18. In another embodiment of the present disclosure, the compound is administered to reduce the levels of Zebl to finally mature the CM after which they have been selected, such as after day 24. In one embodiment of the present disclosure, the compound is administered to reduce the levels of Zebl after day 18, such as in the days prior to or after day 24, such as at day 22, such as at day 23, such as at day 24, such as at day 25, such as at day 26, preferably at day 24.

[0100] Several ways exist to lower levels of proteins inside cells, one strategy is to use oligos, and another is to use small molecules. In one embodiment of the present disclosure, the compound capable of decreasing the levels of Zebl is an antisense RIMA (asRNA), antisense DNA (asDNA), or a silencing RNA, such as shRNA or siRNA. In a specific embodiment of the present disclosure, the shRNA is the shRNA according to SEQ ID NO: 2. Small molecules can be used to target another compound inside the cell, and through that interaction decrease the levels of Zebl. In another embodiment of the present disclosure, the compound capable of decreasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of Sa-792574, BAS-09104376, Lysylphenylalanyl-tyrosine, ofloxacin, lopinavir, tacrolimus, nocodazole, CAY- 10618, Mirin, and ON-01910.

[0101] As seen from example 13 these compounds decrease the levels of Zebl, and increases the CM maturation gene, MYL2. In a specific embodiment of the present disclosure, the compound capable of decreasing the levels of Zebl is nocodazole. In another embodiment of the present disclosure, the compound capable of decreasing the levels of Zebl is Rigosertib / ON-01910. Rigosertib is the tradename used for marketing the compound ON-01910.

[0102] Treatment by targeting endogenous cardiomyocytes, and compounds therefore

[0103] The other main strategy is to induce proliferation and / or CM polyploidy in remaining CM, to repair the damaged heart after MI. Thus, these endogenous CM can be targeted, and activated so that the heart is repaired. Thus, in one embodiment of the present disclosure, treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool, such as by treating endogenous cardiomyocytes.

[0104] As identified by the present invention, to induce a CM to proliferate or undergo endoreplication, the levels of Zebl should be increased. In one embodiment of the present disclosure, the compound is a compound capable of increasing the levels of Zebl. In one embodiment of the present disclosure, the compound is a compound capable of increasing the levels of Zebl in a cardiomyocyte residing in the subject having experienced myocardial infarction. As presented in the examples, overexpression in CM after birth, primarily led to CM polyploidy, thus to eventually result in proliferation, the treatment described herein might need combination with an additional factor that induces division of the cell thus leading to proliferation. These surprising findings are presented in examples 3-8.

[0105] In one embodiment of the present disclosure, the cardiomyocyte pool is reestablished by inducing proliferation and / or increasing CM polyploidy of the endogenous cardiomyocyte pool. In one embodiment of the present disclosure, increasing the levels of Zebl induces proliferation and / or increases CM polyploidy in the cardiomyocytes.

[0106] Several ways exist to increase levels of proteins inside cells, one strategy is through molecular biological tools, such as nucleic acids, and another is to use small molecules. In one embodiment of the present disclosure, the compound capable of increasing the levels of Zebl is a nucleic acid encoding Zebl, or a plasmid encoding Zebl. Small molecules can be used to target another compound inside the cell, and through that interaction increase the levels of Zebl. In one embodiment of the present disclosure, the compound capable of increasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of mesalazine, anisomycin, tegaserod, otenzepad, Sarmentogenin, CS-110266, Naringenin, Strophanthidin, Cephaeline, Olopatadine. As seen from example 14 these compounds increase the levels of Zebl, and lowers the CM maturation gene, MYL2. In a specific embodiment of the present disclosure, the compound capable of increasing the levels of Zebl is anisomycin.

[0107] As presented in the examples, overexpression in CM after birth, primarily led to CM polyploidy, thus to eventually result in proliferation, the treatment described herein might need combination with an additional factor that induces division of the cell thus leading to proliferation. Thus, in one embodiment, the treatment may be supplemented by a treatment that induces division, such as by activating or increasing one or more of MycN, MycC or other known cell cycle factors such as Cyclins. However, the treatment may not be dependent upon actual proliferation, and thus can be used seperately to only result in CM polyploidy.

[0108] Delivery and variants of Zebl

[0109] It should now be obvious how the skilled person can choose among a variety of compounds, and that these compounds can be used to treat subjects having experienced ML When nucleic acids are used, specific ways exist to deliver the nucleic acids. In one embodiment of the present disclosure, the compound is administered by a vector, such as a plasmid, such as a viral vector, such as adenovirus, adeno-associated virus, or lentivirus.

[0110] The compound may also be administered locally, thus in one embodiment of the present disclosure, the compound is administered by intracardiac-, or intrapericardial fluid administration.

[0111] Preferably, the compound is modulating the subject's or the iPS cell's endogenous levels of Zebl, thus in one embodiment of the present disclosure, the compound is capable of modulating the endogenous levels of Zebl. In a preferred embodiment, the present invention is aimed at treating humans, thus evidently the compound modulates human Zebl. In one embodiment of the present disclosure, Zebl is human Zebl or a homologue thereto such as mouse Zebl.

[0112] In one embodiment of the present disclosure, Zebl is human Zebl according to SEQ ID NO: 1, or a variant thereof having 80 % sequence identity to the sequence according to SEQ ID NO: 1. In another embodiment, the Zebl variant is a variant having at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity, such as 99% sequence identity with the amino acid according to SEQ ID NO: 1.

[0113] Indications for therapy, and period of administration

[0114] After MI, the cardiac tissue is under severe stress, and several processes are undergoing at a molecular level. A well studied example thereof is apoptosis. It is recognized that many CMs suffer from the stress to a degree in which apoptosis is activated in the cells, and the cells are removed from the cardiac tissue.

[0115] Evidently, this apoptosis activity is one of the first reactions after MI, and removes a large amount of cells from the cardiac tissue. Apoptosis can be found as early as 30 minutes after ML In one embodiment of the invention, this phenomenom is advantagously exploited in a way where treatment is initated after this initial increase in apoptosis has declined, and thus only remaining cells are treated. In one embodiment of the present disclosure, the treatment is initiated after apoptosis has declined, such as after 48 hours of the MI has been removed.

[0116] Since the compound also can be administered when these processes are undergoing, advantageously the compound can also be administered in combination with treatments against these stresses. In one embodiment of the present disclosure, the compound is administered in combination with other treatments, such as a treatment targeting inhibition of fibrotic tissue formation, such as anti-apoptotic treatment, such as a treatment that increases vascularization.

[0117] In one embodiment of the present disclosure, the treatment is initiated at least 1 day after the myocardial infarction, such as at least 2 days after the myocardial infarction, such as at least 3 days after myocardial infarction.

[0118] As the onset of MI can be difficult to clinically assess, due to a subject's reactions to initial symptoms, the days after MI may advantageously be defined as counting from after the cause of the MI has been removed, such as through medication or surgical treatment. In one embodiment of the present disclosure, the treatment is initiated at least 1 day after the cause of the myocardial infarction has been removed, such as at least 2 days after the myocardial infarction, such as at least 3 days after myocardial infarction. In one embodiment of the present disclosure, the cause of the myocardial infarction has been removed by medical treatment, such as via treatment with at least one of the medicaments selected from aspirin, clopidogrel, heparin, or other anticlotting agents, thrombolytic agents, or nitroglycerin. In one embodiment, the cause is thus removed by the intake of such treatment. In another embodiment of the present disclosure, the cause of the myocardial infarction has been removed by heart surgery, such as via angioplasty, laser, atherectomy, bypass surgery, or placement of a stent. In one embodiment, the cause is thus removed by the closure of the surgical wound. As described above, the compound can be administered together with other compound for treating the damaged heart. Thus in an overall aspect, the present invention also relates to a combination comprising the compound for use as described herein and a compound for targeting inhibition of fibrotic tissue formation, compound for anti-apoptotic treatment, or a compound that increases vascularization.

[0119] Method of maturing iPS CMs

[0120] Due to the inventors' realization of the effects of Zebl on the maturation of iPS CM the invention also finds use in maturing cells ex vivo. This is especially useful when iPS cells are differentiated into CM, since these cells often do not mature properly.

[0121] Thus, in another aspect, the present invention relates to an ex vivo method of maturing iPS CM, the method comprising, administering to an iPS CM a compound capable of decreasing the levels of Zebl. Said compound is defined further above.

[0122] In one embodiment of the present disclosure, the compound is administered by transfection, such as by a vector, such as a plasmid, such as a viral vector, such as adenovirus, adeno-associated virus, or lentivirus.

[0123] Since the iPS CM differentiation process is a result of a large variety of cellular process, the time at which the compound is added to the iPS CM can differ. In one embodiment of the present disclosure, the days are counted as the days following DO, where differentiation is initiated by addition of B27-medium.

[0124] It is possible to follow the maturation in the culture dishes by different techniques. The cells are routinely observed by their size and morphological appearance, and especially regarding iPS CM, the beating initiation as well as a particular subset of genes can be monitored to classify the stage of differentiation. In one embodiment of the present disclosure, the maturation of said iPS CM is monitored by observing beating initiation, and / or by measuring one or more of the genes that are expressed at different stages of differentiation, such as one or more genes selected from:

[0125] - pluripotency genes such as OCT4 and / or SOX2; CM genes such as TNNT2, MYH6, and / or MYH7 as a sign of iPS-CM differentiation; and

[0126] CM maturation such as SIRPA, FABP3, SCN5A, and / or CMLC2.

[0127] MYL2 may also preferably be used, thus in some embodiments CM maturation genes may be selected from the group consisting of MYL2, SIRPA, FABP3, SCN5A, and / or CMLC2.

[0128] As visualized by figure 9, Zebl displays a type of parabolic expression from DO to D30, and as such different timepoints can be utilized, at when expression should be decreased.

[0129] Due to the parabolic appearance of the Zebl expression in iPS cultures, compounds for increasing Zebl expression may advantageously be used in the initial phases of CM differentiation, and then later compounds for decreasing the levels of Zebl are added to fully mature CM.

[0130] Thus, in one embodiment, a compound capable of increasing the levels of Zebl is administered in the initial phases, such as until beating initiation, preferably until the selection phase, and followed by administration of a compound capable of decreasing the levels of Zebl. iPS CM maturation should resemble findings from the mice studies disclosed herein, initially Zebl should be present to drive CM differentiation and then later in differentiation be decreased, thus effectively resembling the findings from the mice studies disclosed herein, that at E16.5 Zebl is high and after birth at days Pl and P5 Zebl is low.

[0131] The later time point of administration shall allow the iPS CM to grow and differentiate, and thus maturation can be initiated at a later stage. In one embodiment of the present disclosure, the compound is administered to reduce the levels of Zebl when selection is initiated, such as after day 18. In another embodiment of the present disclosure, the compound is administered to reduce the levels of Zebl 24 to finally mature the CM after which they have been selected, such as after day 24. In one embodiment of the present disclosure, the compound is administered to reduce the levels of Zebl after day 18, such as in the days prior to or after day 24, such as at day 22, such as at day 23, such as at day 24, such as at day 25, such as at day 26, preferably at day 24. The selection phase referred to here, is the phase at which iPS CM are grown in low glucose environment, as described in example 1, and thus are selected upon the metabolic phenotype. Thus, In one embodiment of the present disclosure, the method comprises a step of selecting CM based on their metabolic phenotype.

[0132] Preferably, Zebl levels are kept at a decreased level once the maturation has been initated, thus in one embodiment of the present disclosure, the compound is administered so that Zebl is reduced from the first day of administration until the end of the iPS procedure, such as at day 30 or whenever the procedure has reach its endpoint, such as until iPS engraftment. Depending on the compound, this might require daily addition. In one embodiment of the present disclosure, the compound is administered each day following the first day of administration.

[0133] The method may also be an ex vivo method or a method for research purposes, i.e. not for use in a clinical setting. Thus, in one embodiment of the present disclosure, the method is performed on a non-human subject.

[0134] When the method is applied for general research as well as when the resulting iPS CM are to be engrafted to a subject, the iPS CM can be prepared as spheroids, this improves the 3-D environment of the cell and often improve engraftment. In one embodiment of the present disclosure, the iPS CM are cultured as a spheroid.

[0135] Sometimes when the method is applied for use in a clinical setting, as described above, it may be advantageous to employ a subject's own cells, thus in one embodiment of the present disclosure, the iPS CM are derived from a subject having experienced a myocardial infarction. However, as described in more detail above, allogenic engraftment of iPS CM is favourably applied. Thus, in one embodiment of the present disclosure, the iPS CM are not derived from the subject having experienced a myocardial infarction. In one embodiment of the present disclosure, the iPS CM are allogenic. iPS CM matured as such and use thereof

[0136] As previously described the above method can find use for research purposes and the resulting iPS CM can also be used for engraftment, such as in clinical settings where a heart of a subject needs to be repaired, such as after ML Thus, iPS CM derived from such a method are important assets. iPS CM are routinely used for toxicological screening of novel compounds, and thus iPS CM that are as close as possible to natural CM is sought for. Additionally, iPS CM derived from a specific subject can be used to screen for that specific subject's reaction to a given medication, and thus asses how likely the subject is to respond to a treatment.

[0137] This in one aspect, the disclosure provides use of the iPS CM for toxicological screening.

[0138] This in one aspect, the disclosure provides use of the iPS CM for medicament screening.

[0139] Thus, another aspect of the invention relates to an iPS cardiomyocyte matured by the method as described above. A related aspect is thus a container comprising the iPS CM matured by the method as described above.

[0140] Such an iPS CM can be used the same treatments as described in this disclosure, and thus another aspect relates to an iPS cardiomyocyte matured by the method as described above, for use in the treatment of the myocardium after myocardial infarction. In one embodiment of the present disclosure, the iPS cardiomyocyte is engrafted into a subject, such as by ultrasound guidance.

[0141] Methods of treatment and manufacturing

[0142] The inventors' realizations can also be employed on cardiomyocytes. Such methods can both be performed in vivo and ex vivo.

[0143] Thus, in another aspect the invention relates to a method of inducing proliferation and / or CM polyploidy in cardiomyocytes, the method comprising, delivering to a cardiomyocyte a compound capable of increasing the level of Zebl in said cardiomyocyte.

[0144] Said compound is defined further above and may in particular be delivered as described in the following embodiments. In one embodiment of the present disclosure, the compound is delivered by transfection, or by a vector, such as a plasmid, such as a viral vector, such as adenovirus, adeno-associated virus, or lentivirus. In one embodiment of the present disclosure, the method is performed on a subject, such as a mammal, such as a mouse, a primate, preferably a human.

[0145] The method may also be an ex vivo method or a method for research purposes, i.e. not for use in a clinical setting. Thus, in one embodiment of the present disclosure, the method is performed on a non-human subject.

[0146] A related aspect is a method of treatment of the myocardium in a subject having experienced myocardial infarction, the method comprising administering to the subject an effective amount of a compound capable of modulating Zebl.

[0147] Said compound is defined further above, and may in one embodiment of the present disclosure be:

[0148] - a compound capable of downregulating Zebl in an iPS CM, and wherein said iPS CM have been engrafted to the myocardium of the subject, or

[0149] - a compound capable of overexpressing Zebl in cardiomyocytes.

[0150] In one embodiment of the present disclosure, the subject is a mammal, such as a mouse, a primate, preferably a human.

[0151] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0152] The invention will now be described in further details in the following non-limiting examples.

[0153] Examples

[0154] EXAMPLE 1 — Test subjects

[0155] CMs from Mice

[0156] C57BL / 6J mice were obtained from Taconic Europe, housed with a 12 / 12 hour light / dark cycle, and fed ad libitum. For scRNA-seq, mice were plug bred, and litters for each of the three timepoints were obtained from different breeding pairs. Plug was checked in the morning and evening. For E16.5 primary cultures mice were plug bred as well, whereas for primary P0 cultures continuous breeding was used. All animal experiments were approved by the Danish Council for Supervision with Experimental Animals (#2016-15-0201-00941 and #2021-15- 0201-01026).

[0157] CMs for scRNA-seq

[0158] For scRNA-seq, the left heart ventricle from mice was dissected under a stereomicroscope at E16.5, Pl, and P5 (n = 3 litters, each counting 4-8 pups), and enzymatically dissociated using the semiautomatic GentleMACS tissue dissociator system (MACS Miltenyi Biotec; Neonatal Heart Dissociation Kit 130-098-373) according to the manufacturer's recommendations. Following viable cell counting (NC-200, ChemoMetec), dissociated cells were stained with a fixable viability stain (Fixable viability stain 570; BD Biosciences, 564995) prior to fixing in methanol for 15 min followed by rehydration to reverse the RNA to its original state. During rehydration, the RNase inhibitor, RNasin Plus (Promega; N2615), was added to prevent RNA degradation and included in all subsequent steps. After rehydration, samples were stored at -80°C until analysis.

[0159] Fixed cardiac cells were stained for the CM marker MYH1 (Mouse IgG2b,k; 1:300; MF20-c; DSHB) and visualized by donkey anti-mouse IgG Alexa Fluor 488 (1:200; Invitrogen, A21202), whereas Hoechst 33342 (Sigma) was added 5 min before sorting (FACSArialll, BD Biosciences). Prior to FACS, cells were filtered (Falcon, 352235) to avoid cell clumps. Strict RNase free conditions as described above including new tubing were prioritized throughout the procedure. Analysis and sorting gating strategy included hierarchical gating using the FACSDiva software v8.0.1 (BD Biosciences) based on FSC / SSC, viability Alexa 570, and MYHl-Alexa 488, and Hoechst 33342. For each developmental stage (E16.5, Pl, P5), three independent sortings (n=3, each consisting of cells from one litter) were performed.

[0160] E16.5 CM cultures

[0161] On embryonic day 16.5 (E16.5), the pregnant mice were sacrificed by cervical dislocation and the hearts from the pups were quickly removed and placed in a cardioplegic buffer (MIB; 1.2mM KH2PO4 (pH 7.4); 0.25g / l Na2COs; 6.44g / l NaCI; 2.6 mM KCI; 1.2 mM Mg2SO4; 11 mM glucose) supplemented with 1% Bovine Serum Albumin (BSA; MIB / 1%BSA). The heart ventricles were dissected under a stereomicroscope before enzymatically dissociation into a single cell suspension using the semiautomatic GentleMACS tissue dissociator system as described by the manufacturer. Dissociated cells were counted (NC-200; ChemoMetec), plated on extracellular matrix (ECM) at a density of approx. 118,500 cells / cm2, and cultured in growth medium (79.5% DMEM (supplemented with 1% PenStrep (PS)), 19.5% Medium 199 (supplemented with 1% PS), and 1% newborn calf serum).

[0162] Neonatal CM cultures

[0163] Neonatal (P0) mouse pups from each litter were sacrificed by decapitation, whereafter the hearts were quickly removed, and the ventricles dissected under a stereomicroscope. Dissected ventricles were pooled in a tube of MIB / 1%BSA before enzymatic dissociation into a single cell suspension using the semiautomatic GentleMACS tissue dissociator system as described by the manufacturer. Dissociated cells were resuspended in growth medium and the number of cells were counted (NC-200; ChemoMetec). Cells were seeded in 12- well plates pre-coated with ECM at a density of approx. 236,500 cells / cm2and placed in an incubator (37°C, 5% CO2). iPSC culture and cardiomyocyte derivation

[0164] Undifferentiated iPSCs (AICS-0060-027, Coriell Institute for Medical Research) were maintained on approximately 10 pg / cm2 growth factor reduced Matrigel (356231, Corning)-coated surfaces at 37°C and 5% CO2 in mTeSR+ medium (100-0274, Stem Cell Technologies) and passaged with Accutase (A11105-01, Thermo Fisher). All passages were done with lOpM Rock inhibitor (Ri) Y27632 2HCI (S1049, Selleckchem) added to the medium and a subsequent replenish of media without Ri after 24 h.

[0165] For CM derivation, 20,000 iPSC / cm2 were plated in mTeSR+ / Ri three days prior (derivation day -3 (D-3)) to derivation start (DO). To initiate differentiation, mTeSR+ medium was changed to B27-medium, consisting of RPMI (61870-010, Thermo Fisher) supplemented with B27 without insulin (A18956-01, Thermo Fisher). From DO to D2, B27-medium was supplemented with 7.5 pM CHIR99021 (13122, Cayman Chemical Company) and from D2 to D4, B27- medium was supplemented with 5pM XAV939 (13596, Cayman Chemical Company) and 5pM IWP-2 (A3512, APExBIO). From D8, cultures were maintained in B27+ medium, consisting of RPMI supplemented with B27 containing insulin (17504-044, Thermo Fisher), which was replenished on D12. The first beating is visible on D8. On D16, cultures were split 1:2 by trypsin / EDTA (0.05%) (T4049, Thermo Fisher), dilution in RPMI media containing 10% FBS and replating in B27+ containing Ri on Matrigel coated plates. On D17 the Ri was replaced with B27+ medium. Selection was initiated on D18 with selection medium (SO) consisting of RPMI without glucose (11879-020, Thermo Fisher), B27+ supplement, 30pM Linoleic Acid (90150, Cayman Chemical Company), 30pM Oleic acid (90260, Cayman Chemical Company), 30pM Fatty Acid free BSA (A-421-500, GoldBio), O.lpM T3 (T2877, Sigma), 0,01pM R3-IGF-1 (100-11R3, PeproTech), lOOpM Wy 14643 (70730, Cayman Chemical Company) and 2000pM L-Carnitine (21489, Cayman Chemical Company). SO was replenished on D20. Selection was ended on D22 where SO was replaced with fatty acid media (FAM) consisting of RPMI containing B27+ supplement, 30pM Linoleic Acid, 30pM Oleic acid, 30pM Fatty Acid free BSA and 2000pM L-Carnitine. Cells were passaged on D24 as on D16, but were seeded at a density of 50,000 cells / cm2 on Matrigel in FAM / Ri. FAM media was replenished on D25 and D28 before cells were used for experiments on D30.

[0166] EXAMPLE 2 — methods and analysis

[0167] ScRNA-seq of mouse cells

[0168] For scRNA-seq, cells originating from three independent FACS were pooled (14-20 pups / sample) to account for biological diversity in the scRNA-seq analysis. Single Cell 3' RNA-Seq libraries were prepared using Chromium Single Cell 3' Reagent Kits v2 (lOx Genomics) according to the user guide. In brief, cellular suspensions of approx. 1,200 cells / pl were mixed with master mix reagents and loaded on a Single Cell A Chip (lOx Genomics) together with Single Cell 3' Gel Beads (lOx Genomics) and partitioning oil to generate single cell gel beads-in-emulsion (GEMs). The GEM generation took place in a Chromium Controller (lOx Genomics). Single cell reverse transcription was performed in a standard thermal cycler, and the GEMs were subsequently broken using Recovery Agent (lOx Genomics). The resulting cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific) and SPRIselect Reagent Beads (Beckman Coulter), and then amplified by PCR using Single Cell 3' Reagent Kit v2 (No. of cycles: 8). After another cDNA clean-up with SPRIselect Beads, the fragment sizes and concentrations were measured using QIAxcel DNA High Resolution Kit (1200) (Qiagen) and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), respectively. Enzymatic fragmentation, end-repair, and A-tailing were performed in one-step using the Single Cell 3' Reagent Kit v2, and fragments of approx. 200 bp were selected by double sided-size selection using SPRIselect Beads. NGS libraries were then constructed by adapter ligation and PCR mediated sample indexing (No. of cycles: 13). After a final double-sided size selection, the NGS library concentrations were measured using Qubit dsDNA Assay Kit. Libraries were sequenced on the Illumina NextSeq 500 platform using NextSeq 500 / 550, high output Reagent Cartridge V2, Illumina Kit (Read 1 = 26 cycles, i7 Index = 8 cycles, Read 2 = 130 cycles), and the second analysis was performed on a Illumina NovaSeq 6000.

[0169] Read alignment and construction of gene expression matrix Base calls were converted to FASTQ format and demultiplexed using the cellranger mkfastq function embedded in the lOx Genomics cellranger software package using default s ett i n g s (https: / / support.10xqenomics.com / sinqle-cell-gene- expression / software / overview / welcome). Single cell gene counts matrices were generated using the cellranger count command. During this step, FASTQ files generated by the cellranger mkfastq step, were aligned to the Mus musculus genome (mmlO / GRCm38) using the splice-aware aligner STAR. Subsequently, STAR used the Mus musculus transcriptome reference (GRCm38.84) to segregate the mapped reads into exonic, intronic and intergenic regions and for assessment of how confidently the reads have been mapped to these regions. Only nonduplicated reads which were confidently mapped to the transcriptome, and which had barcodes and unique molecular identifiers (UMIs) were used for UMI counting. The expression matrices were generated by counting the number of strandspecific UMI for each cell mapping to either the exonic or intronic regions of each gene.

[0170] Clustering and UMAP visualization Using the R package Seurat v 2.3.0 and 3.1.5 dimensionality reduction by principal component analysis (PCA) was performed; subsequently the PCA data analysis was used as input for visualization by Uniform Manifold Approximation and Projection (UMAP) clustering[69, 70]. Cell clustering by expression pattern was performed by first calculating the k-nearest neighbors and constructing the shared nearest neighbor (SNN) and next optimizing the modularity function to determine clusters. Clustering and Heatmaps The Seurat v 2.3.0 and 3.1.5 were used for cluster visualization by UMAP and for differential gene expression of marker genes between clusters.

[0171] For each of the samples, a Seurat object was created, and the cells filtered based on whether they expressed a combination of the CM-specific markers Tnni3, Tnnt2, Actcl, and Tnncl. Each sample was then log-normalized, variable features were identified using "vst" as selection method and 2000 nfeatures, and the data was scaled using nCount_RNA for vars. to. regress. PCA was run and based on jackstraw- and PC elbow plots the optimal number of dimensions was determined (range: 9-14). Moreover, when applied, all samples were merged, integrated using FindlntegrationAnchors and IntegrateData, filtered, normalized, and scaled as described above with generated UMAP plots depicting cell cycle phases, clusters, and original data affiliation as for each individual sample.

[0172] Visualization, clustering, and cell cycle analysis UMAP plots and clusters were generated as described above using PCA as reduction type and resolution = 0.6; based on the top 30 marker genes for each cluster. Subsequent GO term enrichment was evaluated using clusterProfiler: :enrichGO and the "org.Mm.eg.db" library with ont = "BP", pAdjustMethod = "BH", and cutoff values = 0.01. Features witg avg_log2FC > 0.5 were used, where each cluster was named according to biological identity. Finally, each dataset was split into three groups (G1-, S-, or G2 / M-phase) based on the expression of cell cycle markers and each cell was assigned with a cell cycle score using Seurat : :CellCycleScoring.

[0173] Analysis across developmental stages After merging and integration as described above, two clusters of cell cycle active E16.5 and P5 cells, respectively, were subtracted from the data and compared using FindMarkers. The resulting list of features was used for generating cnetplot and TF analysis. Mouse single site analysis was used for TFs (oPOSSUM version 3.0), all genes in current dataset as background, all vertebrate profiles with a minimum specificity of 8 bits, conservation cutoff 0.40, matrix score threshold 85%, up / downstream sequence 5000 / 5000). In addition, oPOSSUM results were supported by GSEA using the Molecular Signature Database and transcription factor targets (TFT).

[0174] Trajectory analysis Data was prepared in Seurat (filtered and cell cycle assigned; since UMI data was used, normalization was avoided in agreement with recommendations by the Monocle platform) in merged pools of either 2n-, 4n-, or all samples, respectively. Subsequently, phenotype data and feature data were extracted from the Seurat object and converted to a Monocle CellDataSet (CDS) object. Next, dispersion estimates for count dataset were obtained using monocle: :estimateDispersions and cells were sorted according to num_genes_expressed (500< num_genes_expressed < 3000). A set of ordering genes was isolated using differentialGeneTest and used to order the CDS by the monocle: :setOderingFilter. Next, the dimensions were reduced and cells were ordered along the trajectory using monocle: :reduceDimension and monocle: :orderCells, respectively. The trajectory was plotted depicting original identity, cell cycle phase, and pseudotime state. The monocle: :BEAM function was utilized in each branch point of the trajectory plots to evaluate branch point dependent gene expression.

[0175] ScRNA-seq of iPS-CMs

[0176] ScRNAseq of iPS-CMs were performed as described above for mouse CMs though without prior FACS. Instead iPS-CMs at D8 were transduced with shRNA-control or shRNA-Zebl and analyzed by scRNAseq at day 11.

[0177] Plasmids and AAV9 packaging

[0178] Generation of plasmids Plasmids harboring the genes of interest were purchased from Origene (Mouse Tagged ORF Clones). The AAV backbone transfer vector was derived through modifications of the plasmid pAAV-EFla-mCherry-IRES-Cre (Addgene plasmid # 55632; http: / / n2t.net / addqei 32;

[0179] RRID:Addgene_55632), allowing simultaneous transcription of mCherry and the gene of interest through the internal ribosome entry site (IRES). Thus, due to the IRES site, transcription of the gene of interest correlates to the level of mCherry. To unify the process of gene insertions, the restriction sites Sgfl and Mlul were inserted into the plasmid. Briefly, the already existing Mlul restriction site was removed by introducing a point mutation in the plasmid by PCR amplification using the following primers: Forward : CGCACGGGTAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGA (SEQ ID NO: 3) and Reverse: AAGCTTACCCGTGCGGCCGCAGGAACCCCTAGTGAT (SEQ ID NO: 4). The Cre site was then removed and the Sgfl and Mlul restriction sites were hereafter inserted by PCR amplification using the primers Forward: TCTGGTGCGATCGCCTAGACGCGTTAGATTCGATATCAAGCTTATCGATAATCAACCTCT (SEQ ID NO: 5) and Reverse: CTAGGCGATCGCACCAGAACCACCATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCC (SEQ ID NO : 6). Finally, a truncated chicken cardiac Troponin T promoter (cTnT promoter; synthesized by GeneArt (Thermo Fisher); the DNA sequence was kindly provided by Professor Brent A. French, University of Virginia, USA) was inserted for CM specificity by PCR amplification in two steps: First, plasmid pAAV-EFla- mCherry-IRES was PCR amplified by primers (Forward : GGAATTCCATATGGGTACCGGATCCGTGAGC (SEQ ID NO: 7)and Reverse : GCTCTAGAAATTCCCACTCCTTTCAAGACCTAG (SEQ ID NO: 8)) containing the Xbal and Ndel restriction sites to excise the EFla promoter. Secondly, the cTnT promoter was inserted between the two restriction sites (Forward : GCTCTAGAGCAGTCTG (SEQ ID NO : 9)and Reverse: GGAATTCCATATGAGGTC (SEQ ID NO : 10)). The resulting pAcTnT-mCherry-IRES plasmid was then sequenced (Eurofins Genomics, Ebersberg, Germany) for validation (Data not shown). Genes (Origene plasmids and Nfya) were inserted into the pAcTnT- mCherry-IRES plasmid between the Sgfl and Mlul restriction sites. Since the Sgfl restriction site was already included in the Egrl sequence, Egrl was amplified by the following primers Forward : AATGGTGGTTCTGGTGCGATCGCATGGCAGCGGCCAAG (SEQ ID NO: l l)and Reverse: TTGATATCGAATCTAACGCGTGCAAATTTCAATTGTC (SEQ ID NO: 12). Next the Egrlsequence was added to pAcTnT-mCherry-IRES by NEBuilder® HiFi DNA Assembly Master Mix (NEB). Proper gene insertions were validated by enzymatic digestion at the respective restriction sites and size determined by gel electrophoresis (Data not shown).

[0180] For plasmid packaging in an AAV9 serotype capsid we used the Rep / Cap plasmid, pAAV2 / 9n, (Addgene plasmid # 112865; http : / / n2t.net / addgene : 112865 ;

[0181] RRID:Addgene_112865) and the helper plasmid pHelper.

[0182] Virus generation Large-scale AAV generation for in vitro use was performed in HEK293T cells (ATCC; CRL-3216) by co-transfection with pAcTnT-mCherry-IRES (empty vector) or pAcTnT-mCherry-IRES harboring the gene of interest, pAAV2 / 9n and pHelper. Transfection efficiency was addressed by mCherry visualization using immunofluorescence microscopy. Five days after transfection, recombinant AAV was isolated by PEG 8000 precipitation and purified by iodixanol gradient ultracentrifugation followed by centrifugation through an Amicon Ultra Centrifugal filter (50K). Recombinant AAV yields were determined by quantitative real-time PCR (qRT-PCR) through a titration of pAcTnT-mCherry-IRES plasmid using the primers Forward : AGTGTTGCATTCCTCTCTGG (SEQ ID NO: 13) and Reverse: AGCGCATGAACTCCTTGAT (SEQ ID NO: 14).

[0183] Adenoviral constructs were generated by Vector Biolabs (PA, USA) using Adenoviral Human Type 5 (dEl / E3) as backbone. For ZEB1 knockdown experiments, a U6 promoter was driving ZEB1 short-hairpin RNA (shRNA) expression of the sequence 5 'CCGGATAGAGGCTACAAGCGCTTTA-CTCGAG- TAAAGCGCTTGTAGCCTCTA- 1 I I I I I G-3' (SEQ ID NO: 15)and a targeting sequence of ATAGAGGCTACAAGCGCTTTA (SEQ ID NO: 16). An eGFP reporter was expressed under a separate CMV promoter. Ad-GFP-U6-scrmb-shRNA (cat. no. 1122N) containing a scrambled shRNA and an eGFP reporter was used as control. For ZEB1 overexpression experiments, the backbone vector contained a CMV promoter to drive expression of the gene of interest. Ad-GFP-Zebl was generated using mouse cDNA (GenBank: BC139768.1) and eGFP, and ZEB1 were expressed under separate CMV promoters. Ad-GFP (cat.no. 1060) was used as empty control.

[0184] Zebl knockdown

[0185] After 24 h of culturing, mouse CMs were transduced with 10 MOI of either Ad- GFP-shRNA or Ad-GFP-shRNA-Zebl. In addition, 10 pM of 5-ethynyl-2'- deoxyuridine (EdU) was added to assess for cell cycle activity. The medium, with or without EdU, was replenished every 24 h, and experiments were terminated as indicated at 96 h after transduction for analysis.

[0186] Zebl overexpression

[0187] After 24 h the number of mouse CMs in each experiment were estimated (NC- 200; ChemoMetec), and cell cultures were transduced with either 750,000 viral genomes (vg) / cell of the desired AAV9 or 50 MOI of adenovirus. For AAV9 experiments, six, 24, and 48 h after viral transduction, medium was refreshed with medium containing 10 pM EdU. For adenovirus, EdU was added together with the virus and replenished every 24 h. All cells for qRT-PCR were replenished with medium without EdU. Cells were either fixed in 2.5% Neutral Buffered Formalin (NBF) diluted in HBSS / 5%FBS / 1%PS for flow cytometry analysis 72 h post transduction fixed in the wells in 10% NFB or 4% Paraformaldehyde (PFA), or the RNA was isolated for qRT-PCR 48 h post transduction for adenovirus or 72 h post transduction for AAV9.

[0188] Flow cytometry

[0189] Fixed cells (mouse CMs or iPS-CMs) were permeabilized with phosphate buffered saline (PBS) containing 1% BSA and 0.1% Triton X-100 (TX100) and stained with primary antibodies in different combinations (mouse anti-MYHl, 1:300, MF20-C, DSHB; rat anti-mCherry, 1:500, M11217, Thermo Fisher; and rabbit anti-GFP, 1:500, ab290, Abeam) for 1 h in the dark on ice while shaking. After washing, cells were incubated with EdU Click-it reaction cocktail according to the manufacturer's protocol (Invitrogen, C10419), and washed before incubation with secondary antibodies in different combinations (488-donkey anti-mouse, 1:200, A21202, Invitrogen; 555-donkey anti-rat, 1:200, Abl50154, Abeam; 555-donkey anti-mouse, 1 :200, A31570, Invitrogen; and 488-donkey anti-rabbit, 1:200, A21206, Invitrogen) for 30 min in the dark on ice while shaking. Three final washes were performed in PBS / 1% BSA / 0.1% TX100 and Hoechst 33342 was added 5 min before flow cytometry using the LSRII flow cytometer (BD Biosciences). Data was analyzed using the FACSDiva software v8.0.1, and initially gated according to the CM marker MYH1 and then sub-fractionated based on the antibody amplified mCherry or GFP signal. Cells positive or negative for a reporter (mCherry or GFP) were gated according to EdU incorporation to determine cell cycle activity. Ploidy was addressed in subpopulations by Hoechst 33342 using gates (2n, 4, and >4n) defined by the entire CM population.

[0190] RNA isolation, RNA integrity, and qRT-PCR

[0191] Briefly, the cells (mouse CMs or human iPS-CMs) were lysed with TriReagent and the RNA was isolated using Polyacryl carrier, l-Bromo-3-Chloro-Propane and 2- propanol. The RNA was rinsed using 75% ice cold ethanol. Finally, the RNA was dissolved in nuclease-free water and the RNA concentration was determined using a nano-drop. For qRT-PCR, cDNA was generated using the High-Capacity cDNA Reverse Transcriptase kit (Applied Biosystems; 4368814) according to the manufacturer's recommendations. Each sample for qRT-PCR contained 2-4 ng cDNA in a total volume of 10 pl and were analyzed in technical triplicates of qRT- PCR using a mixture of Power SYBRGreen PCR Master Mix (Applied Biosystems, 4367659) and appropriate forward and reverse primers. The qRT-PCR was run on a 7900HT Fast Real-time PCR system (Applied Biosystems) under the following conditions: Holding for 10 min at 95°C, hereafter 40 cycles consisting of 15 seconds of denaturation at 94°C, 30 seconds of annealing at 57-60°C and 30 seconds of elongation at 72°C. The obtained data was analyzed by normalization to multiple stably expressed endogenous gene according to the qBase Plus 3.2 platform (Biogazelle).

[0192] Injection of adenovirus in P0 mouse pups

[0193] The litter was gently taken from their home cage. Pups were then anesthetized by induction of hypothermia before 7.60xl014PFU (in a total volume of 20 pl, diluted in sterile PBS) of the desired adenovirus was injected into the superficial temporal vein. Pups were reheated and placed together with their littermates before the litter was gently put back into their home cage. Following the pups received a 50 pl subcutaneous injections of EdU (2.5 mg / ml) at P4 and P6 before they were sacrificed by decapitation at P8. Hearts were dissected and either dissociated for flow cytometry as described above for P0 pups, except cells were strained (100 pm nylon cell strainer, cat.no. 352360) prior to NBF fixation, and each heart was processed individually, or prepared for paraffin embedding (see below).

[0194] Statistics and reproducibility

[0195] All statistics were performed using the GraphPad Prism (v 9.0.0) software and the appropriate tests, number of independent experiments (n) and replicates (n*) are defined in the corresponding figure legends. We used the significance level a = 0.05 for identifying significant results marked by asterisks.

[0196] EXAMPLE 3 — The cell cycling machinery of cycling mouse E16.5 tetrapioid G2 / M CMs is as compared to that in P5 cycle active tetrapioid

[0197] G2 / M CMs defined by Zebl.

[0198] By comparing scRNAseq data between tetrapioid E16.5 CMs and P5 CMs (Figure la-b) a list of marker genes with higher expression in E16.5-4n-G2 / M-CMs as compared to P5-4n-G2 / M-CMs were obtained (Figure lc) and used for subsequent GO term analysis (Figure Id). Several terms related to the cell cycle: "Mitotic nuclear division", "Cell division", "Chromosome condensation", "Regulation of mitotic cell cycle", and "Nuclear division" and associated genes were unraveled by a gene concept network map (Figure Id). Using the oPOSSUM platform, enriched TF binding sites were determined for the gene expression enriched in E16.5-4n- G2 / M CMs and allowed for prediction of a list of TFs potentially regulating G2 / M progression in dividing CMs (Figure le).

[0199] Conclusion: Together, these analyses supported that the identified TFs play a role in regulating G2 / M of dividing CMs and based on the Fisher score ZEB1 was identified as a key regulator of CM proliferation.

[0200] EXAMPLE 4 — Zebl regulates a high number of cell cycle genes in CMs

[0201] Target genes (according to oPOSSUM 3.0) of Zebl were subjected to GO term analysis (p<0.05, Benjamini corrections) and revealed that 13- (ZEB1) out of 13 GO terms related to "Cell cycling / division" (3), "Mitotic division / cytokinesis" (4), "Telomers" (3), and "Regulation of cell cycling" / "Positive regulation of cell proliferation" (3) (Figure 2). When adjusting for gene overlap between the different cell cycle related GO terms, 118 genes related to these cell cycle related GOs were affected by Zebl (Figure 2).

[0202] Conclusion: Overall, these data underscore that the Zinc Finger E-Box Binding Homeobox 1 gene (Zebl) was superior to other TFs identified in regulating the number (118) of target genes associated to cell cycle activity in CMs, and supports that ZEB1 is a novel key player in CM proliferation.

[0203] EXAMPLE 5 — Zebl is expressed in the G2 / M cell cycle phases of mouse

[0204] CMs, particular at E16.5 whereafter the level declines

[0205] Investigating scRNA-seq data it was found that ZEB1 is present in only a fraction of CMs (Figure 3a) preferably in the G2 / M phases (Figure 3b)), but the level declined with developmental stage (Figure 3b-c). Moreover, Zebl was as expected from its nature as a transcription factor mainly found in the nucleus of CMs.

[0206] Conclusion: During normal heart and CM development, Zebl is expressed in CMs at early stages of CM development where proliferation occurs but is then downregulated. EXAMPLE 6 — Knockdown of Zebl leads to inhibition of mouse CM proliferation

[0207] ZEB1 knockdown before birth was performed using adenoviral transduction for administration of validated Zebl shRNA in E16.5 primary heart cultures (Figure 4a). A high transduction efficiency was confirmed, and overall ZEB1 was reduced by 82.3±5.9% (mean, SD, n = 3) (Figure 4b). Using this setup, it was found that cell cycle activity in CMs as reflected by EdU incorporation was decreased significantly in Ad-GFP-shRNA-Zebl treated CMs from 12.2 ± 3.3% to 2.8 ± 0.8% (mean, SD, n = 5) (Figure 4c). Importantly, the diploid status of EdU+ / CMs decreased by Zebl knockdown (Figure 4d) suggesting that not only S-phase progression was inhibited, but also cytokinesis was reduced by Zebl knockdown. This was supported by a significant downregulation, in Ad-GFP-shRNA-Zebl treated cells, of Ccndl (Cyclin DI), Ccnbl (Cyclin Bl) and Ccnd3 (Cyclin D3), while the levels of Ccne2 (Cyclin E2) and Ccng2 (Cyclin G2) associated with endoreplication were unchanged (Figure 5a). Although, the level of the major cyclin dependent kinase, Cdkl (Cyclin-dependent kinase 1), was slightly reduced when Zebl was knocked down no difference was observed for Cdk4 (Cyclin- dependent kinase 4) and the cell cycle inhibitors Cdknla (Cyclin-dependent kinase inhibitor la, p21) and Cdknlb (Cyclin-dependent kinase inhibitor lb, p27 (Figure 5b). With a focus on factors known from the G2 / M phase, where Zebl predominantly is observed (Figure 4b), we found a significant increase in the expression of Cenpe (Centromere protein E), Cenpf (Centromere protein F), Aurkb (Aurora kinase B), and Aurka (Aurora kinase A), but no change in the level of Gmnn (Geminin) (Figure 5c).

[0208] Conclusion: Zebl can be downregulated by virus mediated shRNAs, and results in an inhibition of CM proliferation. These data confirm that ZEB1 mediates the cell cycle program and is required for CM proliferation. EXAMPLE 7 — Overexpression of Zebl after birth leads to mouse CM endoreplication and less maturity

[0209] An adenovirus (Ad) with an eGFP reporter to be expressed separately from ZEB1 was generated and used for inducing ZEB1 expression in P0 mouse CMs (pCMP0) Specific expression of ZEB1 overexpression was verified (Figure 6a). In agreement, with its nature as a TF, the overexpressed ZEB1 protein mainly localized to the nucleus in CMs. EdU pulse chase labelling with empty virus Ad-GFP and Ad-GFP-Zebl showed that the percentage of EdU+CMs increased significantly with ZEB1 overexpression (Figure 6b). Moreover, a massive reduction in CM size was apparent with ZEB1 overexpression, and did not occur in non-CMs (Figure 6c). No difference was observed in the percentage of CMs between nontransduced, Ad-GFP, and Ad-GFP-Zebl cultures at 72 h after transduction (Figure 6d), suggesting that CM death or apoptosis was minimal. ZEB1 overexpression revealed both single-, bi-, and multinucleated CMs at 72 h after transduction, supporting the observed ability to induce CM S-phase progression. This was accompanied by a downregulation of Ccndl with a concomitant upregulation of Ccne2 and Ccng2, while the levels of Ccnbl and Ccnd3 were unchanged (Figure 7a). No change was observed for Cdkl and Cdk4, whereas Cdknla was dramatically reduced. Likewise, Cdknlb was reduced, but to a lesser extent (Figure 7b). Additionally, it was found that the muscle size inhibitor Mstn (Myostatin) were substantially increased in ZEB1 overexpressing CMs, while the Myh6 expression was substantially reduced (Figure 7c), which together with the smaller size of CMs (Figure 6d) suggest that Zebl overexpression results in immaturity of CMs as is expected from cell cycling CMs. Finally, in vivo data from injecting Ad-GFP and Ad-GFP-Zebl into the superficial temporal vein of P0 mice showed that ZEB1 overexpression directly in the heart, resulted in a significant higher percentage of EdU+CMs in Ad-GFP-Zebl transduced CMs as compared to Ad-GFP transduced CMs (Figure 7d) confirming increased cell cycle activity as a result of Zebl. Moreover, 99±1% of the EdU+ / Ad-GFP-Zebl+CMs exhibited a ploidy of >4n, while the remaining 1% were 4n, which was different from EdU+ / Ad-GFP+CMs showing lower ploidy (Figure 7e).

[0210] Conclusion: These data thus demonstrate that Zebl also after birth facilitates CM cell cycle activity, but unlike before birth, favors CM endoreplication and final division of the high ploidy CMs may depend on other factors. Moreover, the data shows that Zebl induces immaturity of CMs and thus should be downregulated as occurring during normal heart and CM development.

[0211] EXAMPLE 8 — Overexpression of Zebl in mouse CMs by adeno associated virus mediated delivery

[0212] The biological effect of the Zebl on CM cell cycle activity was evaluated by overexpressing Zebl in P0 cardiac cells (CMP0). To enable CM specific TF overexpression and identification of transduced CMs, we generated CM specific (cTnT promoter) adeno associated viruses of serotype 9 (AAV9) with a mCherry reporter and an internal ribosome entry site (IRES) for Zebl (denoted AAV9-cTnT- Zebl) (Figure 8a). Virus functionality for AAV9-cTnT-Zebl was validated by mRNA expression (Figure 8b), whereas accumulated cell cycle activity in CMpowas measured 72 h after AAV9-cTnT-Zebl transductions using flow cytometry for EdU / MYHl / mCherry / Hoechst (Figure 8c).

[0213] When compared to empty control transduced mCherry+CMP0(5.3±3.3%), Zebl (26.8±5.6%) overexpression resulted in a significant increase in the percentage of EdU+mCherry+CMs (Figure 8c). Thus, overexpression of Zebl corresponded to 5.1-fold induction in the percentage of EdU+CMs. Thus, Zebl, was able on its own to increase cell cycle activity of CMP0. By assessing ploidy in the transduced CMs a significant change in ploidy for EdU+mCherry+CMs overexpressing Zebl was found (Figure 8d).

[0214] Conclusion: Zebl may be overexpressed using AAVs and favor CM polyploidy at the expense of diploid CMs (Figure 8d).

[0215] EXAMPLE 9 — Expression of Zebl in human iPS-CMs

[0216] Pluripotency genes (OCT4 and SOX2) are expressed early and then declines. At D8 of iPS-CM differentiation and at the time of beating, CM genes (TNNT2, MYH6, MYH7) appear and are relative constant reflecting CM differentiation. Then CM maturation starts and associated genes (SIRPA, FABP3, SCN5A, CMLC2) are expressed and increases reflecting the switch in metabolism and cytoskeletal proteins as well as ion-channels as maturation occur. For comparison ZEB1 starts to be expressed in human iPS-CMs around the time of beating initiation D8, and then declines at late stages as CMs become more mature and lose their ability to proliferate (Figure 9). However, iPS-CMs at D30 still express Zebl at some levels and retain some abilities to cycle / proliferate (Figure 9).

[0217] Conclusion: As seen during development, ZEB1 is expressed in early human iPS- CM and is required for CM specification, but as seen in development above needs to be downregulated at the time of iPS-CM maturation. Zebl levels thus may be further increased during early timepoints to facilitate CM specification / differentiation but then at later timepoints needs to be decreased / removed for maturation to occur.

[0218] EXAMPLE 10 — Zebl knockdown in human iPS-CMs iPSCs cultured as in general and CM differentiation initiated at day 0. Then at day 8, Zebl was knocked down by adenovirus mediated delivery of shRNA against ZEB1 using the sequence SEQ ID NO: 2.

[0219] At day 11, iPS-CMs were harvested and analyzed by scRNAseq and qRT-PCR which confirmed that Zebl is expressed in iPS-CMs (Figure lOa-b) and that shRNA-Zebl mediates Zebl knockdown in iPS-CMs (Figure lOc-d).

[0220] Conclusion: As seen in CMs during heart development in mouse, Zebl is also expressed in iPS-CMs and may be lowered by shRNA molecules.

[0221] EXAMPLE 11 — Zebl knockdown in iPS-CMs reduces proliferation scRNAseq data of iPS-CMs at day 11, three days after Zebl knockdown reveals that iPS-CM specification requires Zebl at this early timepoint. This is confirmed by an a downregulation of Gene Ontology terms related to muscle and cardiac muscles.

[0222] Conclusion: Early Zebl knockdown in iPS-CMs reduces their CM specification and thus Zebl is required during early CM differention / specification. Yet, as seen in Figure 9, Zebl needs at a later points to be downregulated for iPS-CM maturation to occur in order to obtain a phenotype resembling adult CMs. EXAMPLE 12 - Compounds identified to regulate Zebl levels in human cells

[0223] Connectivity mapping is used to identify compounds that mimic gene expression. Connectivity maps were used (Figure 12) to identify that mesalazine, anisomycin, tegaserod, otenzepad, Sarmentogenin, CS-110266, Naringenin, Strophanthidin, Cephaeline, Olopatadine all can be used to increase the level of Zebl, whereas Sa-792574, BAS-09104376, Lysylphenylalanyl-tyrosine, ofloxacin, lopinavir, tacrolimus, nocodazole, CAY-10618, Mirin, ON-01910 can be used to lower Zebl levels.

[0224] Conclusion: Existing small compounds may be used to manipulate the levels of Zebl in vitro or in vivo either to induce CM proliferation or increase CM maturity.

[0225] EXAMPLE 13 — Compounds lowering Zebl levels in iPS-CMs increases maturation iPSCs cultured as in general and CM differentiation initiated at day 0. Then at day 24 after metabolic selection, cells were treated with compounds, as exemplified by Nocodazole and Rigosertib / ON-01910 from the list of compounds predicted in example 12, and assayed by qRT-PCR for levels of Zebl and MYL2 (Figure 13), the latter being the most important marker for ventricular maturation. Rigosertib is the tradename used for marketing the compound ON-01910.

[0226] Data, shown in figure 12, reveal that Zebl levels are significantly lowered after 48 hours of compound treatment, and that this already after 48h significantly increases MYL2 expression, and that this is much further substantiated at day 30 (120h). This shows that compounds lowering Zebl in the late stage of derivation significantly increases maturation.

[0227] Conclusion: Compounds lowering Zebl in iPS-CMs at the late stage increases iPS-CM maturation / differentiation and thus low levels of Zebl is required during late CM differention / maturation to obtain a phenotype resembling adult CMs.

[0228] EXAMPLE 14 — Compounds increasing Zebl levels in iPS-CMs lowers maturation iPSCs cultured as in general and CM differentiation initiated at day 0. Then at day 24 after metabolic selection, Zebl was increased by adding anisomycin to the medium. iPS-CMs were assayed by qRT-PCR for levels of Zebl and MYL2 (Figure 14), the latter being the most important marker for ventricular maturation.

[0229] Data, shown in figure 14, reveal that Zebl levels are significantly increased after 48 hours of compound treatment, and that this already after 48h significantly decreases MYL2 expression. This shows that compounds may be used to increase Zebl levels when needed for instance during early iPS-CM derivation.

[0230] Conclusion: Compounds may be used to increase Zebl levels during early stages of iPS-CM derivation, where maturation is not needed and thus high levels of Zebl should dominate.

[0231] Sequence listing

[0232] SEQ ID NO: 1

[0233] >sp | P37275 | ZEBl_HUMAN Zinc finger E-box-binding homeobox 1 OS=Homo sapiens OX=9606 GN = ZEB1 PE= 1 SV=2

[0234] MADGPRCKRRKQANPRRNNVTNYNTVVETNSDSDDEDKLHIVEEESVTDAADCEGVPEDD LPTDQTVLPGRSSEREGNAKNCWEDDRKEGQEILGPEAQADEAGCTVKDDECESDAENEQ NHDPNVEEFLQQQDTAVIFPEAPEEDQRQGTPEASGHDENGTPDAFSQLLTCPYCDRGYKR FTSLKEHIKYRHEKNEDNFSCSLCSYTFAYRTQLERHMTSHKSGRDQRHVTQSGCNRKFKC TECGKAFKYKHHLKEHLRIHSGEKPYECPNCKKRFSHSGSYSSHISSKKCISLIPVNGRPRTG LKTSQCSSPSLSASPGSPTRPQIRQKIENKPLQEQLSVNQIKTEPVDYEFKPIVVASGINCST PLQNGVFTGGGPLQATSSPQGMVQAVVLPTVGLVSPISINLSDIQNVLKVAVDGNVIRQVLE NNQANLASKEQETINASPIQQGGHSVISAISLPLVDQDGTTKIIINYSLEQPSQLQVVPQNLK KENPVATNSCKSEKLPEDLTVKSEKDKSFEGGVNDSTCLLCDDCPGDINALPELKHYDLKQP TQPPPLPAAEAEKPESSVSSATGDGNLSPSQPPLKNLLSLLKAYYALNAQPSAEELSKIADSV NLPLDVVKKWFEKMQAGQISVQSSEPSSPEPGKVNIPAKNNDQPQSANANEPQDSTVNLQ SPLKMTNSPVLPVGSTTNGSRSSTPSPSPLNLSSSRNTQGYLYTAEGAQEEPQVEPLDLSLP KQQGELLERSTITSVYQNSVYSVQEEPLNLSCAKKEPQKDSCVTDSEPVVNVIPPSANPINIA IPTVTAQLPTIVAIADQNSVPCLRALAANKQTILIPQVAYTYSTTVSPAVQEPPLKVIQPNGNQ DERQDTSSEGVSNVEDQNDSDSTPPKKKMRKTENGMYACDLCDKIFQKSSSLLRHKYEHT GKRPHECGICKKAFKHKHHLIEHMRLHSGEKPYQCDKCGKRFSHSGSYSQHMNHRYSYCK REAEERDSTEQEEAGPEILSNEHVGARASPSQGDSDERESLTREEDEDSEKEEEEEDKEME ELQEEKECEKPQGDEEEEEEEEEVEEEEVEEAENEGEEAKTEGLMKDDRAESQASSLGQKV

[0235] GESSEQVSEEKTNEA

[0236] SEQ ID NO: 2

[0237] CCGGCCTCTCTGAAAGMCACATTACTCGAGTAATGTGTTCTTTCAGAGAGGTTTTT

[0238] SEQ ID NO: 3 CGCACGGGTAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGA SEQ ID NO: 4

[0239] AAGCTTACCCGTGCGGCCGCAGGAACCCCTAGTGAT

[0240] SEQ ID NO: 5 TCTGGTGCGATCGCCTAGACGCGTTAGATTCGATATCAAGCTTATCGATAATCAACCTCT SEQ ID NO: 6

[0241] CTAGGCGATCGCACCAGAACCACCATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCC SEQ ID NO: 7 GGAATTCCATATGGGTACCGGATCCGTGAGC

[0242] SEQ ID NO: 8

[0243] GCTCTAGAAATTCCCACTCCTTTCAAGACCTAG

[0244] SEQ ID NO: 9

[0245] GCTCTAGAGCAGTCTG

[0246] SEQ ID NO: 10

[0247] GGAATTCCATATGAGGTC

[0248] SEQ ID NO: 11

[0249] AATGGTGGTTCTGGTGCGATCGCATGGCAGCGGCCAAG

[0250] SEQ ID NO: 12

[0251] TTGATATCGAATCTAACGCGTGCAAATTTCAATTGTC

[0252] SEQ ID NO: 13

[0253] AGTGTTGCATTCCTCTCTGG

[0254] SEQ ID NO: 14

[0255] AGCGCATGAACTCCTTGAT

[0256] SEQ ID NO: 15

[0257] CCGGATAGAGGCTACAAGCGCTTTACTCGAGTAAAGCGCTTGTAGCCTCTATTTTTTG

[0258] SEQ ID NO: 16

[0259] ATAGAGGCTACAAGCGCTTTA

[0260] Items

[0261] 1. A compound capable of modulating the transcription factor Zebl for use as a medicament.

[0262] 2. A compound capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction.

[0263] 3. The compound for use according to any of the preceding items, wherein treatment of the myocardium is a repair of the cardiac tissue.

[0264] 4. The compound for use according to any of the preceding items, wherein treatment of the myocardium results in recovering cardiac function.

[0265] 5. The compound for use according to any of the preceding items, wherein treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool, such as by engrafting iPS cardiomyocytes (CM).

[0266] 6. The compound for use according to any of the preceding items, wherein the treatment comprises engrafting iPS CM into a subject having experienced myocardial infarction.

[0267] 7. The compound for use according to item 5 or item 6, wherein the cardiomyocyte pool is re-established by maturing engrafted iPS CM.

[0268] 8. The compound for use according to any of the preceding items, wherein the compound is a compound capable of decreasing the levels of Zebl in an iPS CM engrafted into a subject having experienced myocardial infarction.

[0269] 9. The compound for use according to any of items 6-8, wherein the iPS CM are derived from the subject having experienced a myocardial infarction.

[0270] 10. The compound for use according to any of the preceding items, wherein the compound is a compound capable of decreasing the levels of Zebl.

[0271] 11. The compound for use according to item 2, item 8 or item 10, wherein the compound capable of decreasing the levels of Zebl is an antisense RIMA (asRNA), antisense DNA (asDNA), or a silencing RNA, such as shRNA or siRNA.

[0272] 12. The compound for use according to item 11, wherein the shRNA is the shRNA according to SEQ ID NO: 2.

[0273] 13. The compound for use according to item 2, item 8 or item 10, wherein the compound capable of decreasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of Sa-792574, BAS-09104376, Lysylphenylalanyl-tyrosine, ofloxacin, lopinavir, tacrolimus, nocodazole, CAY- 10618, Mirin, and ON-01910. 14. The compound for use according to item 2, wherein treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool, such as by treating endogenous cardiomyocytes.

[0274] 15. The compound for use according to any of items 2 and 14, wherein the compound is a compound capable of increasing the levels of Zebl.

[0275] 16. The compound for use according to item 2, and any of items 14-15, wherein the compound is a compound capable of increasing the levels of Zebl in a cardiomyocyte residing in the subject having experienced myocardial infarction.

[0276] 17. The compound for use according to item 2, and any of items 14-16, wherein the compound capable of increasing the levels of Zebl is a nucleic acid encoding Zebl, or a plasmid encoding Zebl.

[0277] 18. The compound for use according to item 2, and any of items 14-17, wherein the compound capable of increasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of mesalazine, anisomycin, tegaserod, otenzepad, Sarmentogenin, CS-110266, Naringenin, Strophanthidin, Cephaeline, Olopatadine.

[0278] 19. The compound for use according to item 2, and any of items 14-16 wherein the cardiomyocyte pool is re-established by inducing proliferation and / or increasing CM polyploidy of the endogenous cardiomyocyte pool.

[0279] 20. The compound for use according to item 2, and any of items 14-17, wherein increasing the levels of Zebl induces proliferation and / or increases CM polyploidy in the cardiomyocytes.

[0280] 21. The compound for use according to any of items 2-20, wherein the compound is administered by a vector, such as a plasmid, such as a viral vector, such as adenovirus, adeno-associated virus, or lentivirus.

[0281] 22. The compound for use according to any of items 2-21, wherein the compound is capable of modulating the endogenous levels of zeb 1.

[0282] 23. The compound for use according to any of the preceding items, wherein Zebl is human Zebl or a homologue thereto such as mouse Zebl.

[0283] 24. The compound for use according to any of the preceding items, wherein Zebl is human Zebl according to SEQ ID NO: 1, or a variant thereof having 80 % sequence identity to the sequence according to SEQ ID NO: 1.

[0284] 25. The compound for use according to any of items 2-24, wherein the treatment is initiated after apoptosis has declined, such as after 48 hours of the MI has been removed. 26. The compound for use according to any of items 2-25, wherein the treatment is initiated at least 1 day after the cause of the myocardial infarction has been removed, such as at least 2 days after the myocardial infarction, such as at least 3 days after myocardial infarction.

[0285] 27. The compound for use according to any of items 2-26, wherein the treatment is initiated at least 1 day after the myocardial infarction, such as at least 2 days after the myocardial infarction, such as at least 3 days after myocardial infarction.

[0286] 28. The compound for use according to any of items 25-27, wherein the cause of the myocardial infarction has been removed by medical treatment, such as via treatment with at least one of the medicaments selected from aspirin, clopidogrel, heparin, or other anticlotting agents, thrombolytic agents, or nitroglycerin.

[0287] 29. The compound for use according to any of items 25-27, wherein the cause of the myocardial infarction has been removed by heart surgery, such as via angioplasty, laser, atherectomy, bypass surgery, or placement of a stent.

[0288] 30. The compound for use according to any of items 2-29, wherein the compound is administered by intracardiac, or intrapericardial fluid administration.

[0289] 31. The compound for use according to any of items 2-30, wherein the compound is administered in combination with other treatments, such as a treatment targeting inhibition of fibrotic tissue formation, such as anti-apoptotic treatment, such as a treatment that increases vascularization.

[0290] 32. A combination comprising the compound for use according to any of items 1- 31 and a compound for targeting inhibition of fibrotic tissue formation, compound for anti-apoptotic treatment, or a compound that increases vascularization.

[0291] 33. An ex vivo method of maturing iPS CM, the method comprising, administering to an iPS CM a compound capable of decreasing the levels of Zebl.

[0292] 34. The method according to item 33, wherein the compound capable of decreasing the levels of Zebl is an antisense RIMA (asRNA), antisense DNA (asDNA), or a silencing RNA, such as shRNA or siRNA.

[0293] 35. The method according to item 34, wherein the shRNA is the shRNA according to SEQ ID NO: 2.

[0294] 36. The method according to item 34, wherein the compound capable of decreasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of Sa-792574, BAS-09104376, Lysylphenylalanyl-tyrosine, ofloxacin, lopinavir, tacrolimus, nocodazole, CAY- 10618, Mirin, and ON-01910. 37. The method according to any of items 33-35, wherein the compound is administered by transfection, such as by a vector, such as a plasmid, such as a viral vector, such as adenovirus, adeno-associated virus, or lentivirus.

[0295] 38. The method according to any of items 33-37, wherein the compound is administered to reduce the levels of Zebl to finally mature the CM after which they have been selected.

[0296] 39. The method according to any of items 33-37, wherein the compound is administered to reduce the levels of Zebl after day 18, such as in the days prior to or after day 24, such as at day 22, such as at day 23, such as at day 24, such as at day 25, such as at day 26, preferably at day 24.

[0297] 40. The method according to any of items 33-39, wherein the compound is administered each day following the first day of administration.

[0298] 41. The method according to any of items 33-39, wherein the compound is administered so that Zebl is reduced from the first day of administration until day 30.

[0299] 42. The method according to any of items 38-41, wherein the days are counted as the days following DO, where differentiation is initated by addition of B27- medium.

[0300] 43. The method according to any of items 33-42, wherein the maturation of said iPS CM is monitored by observing beating initiation, and / or by measuring one or more of the genes that are expressed at different stages of differentiation, such as one or more genes selected from:

[0301] - pluripotency genes such as OCT4 and / or SOX2;

[0302] - CM genes such as TNNT2, MYH6, and / or MYH7 as a sign of iPS-CM differentiation; and

[0303] - CM maturation such as SIRPA, FABP3, SCN5A, and / or CMLC2.

[0304] 44. The method according to any of items 33-43, wherein the iPS CM are cultured as a spheroid.

[0305] 45. The method according to any of items 33-44, wherein the iPS CM are derived from a subject having experienced a myocardial infarction.

[0306] 46. An iPS cardiomyocyte matured by the method according to any of items 33-

[0307] 45.

[0308] 47. A container comprising the iPS CM according to item 46.

[0309] 48. An iPS cardiomyocyte matured by the method according to any of items 33-

[0310] 45, for use in the treatment of the myocardium after myocardial infarction. 49. The iPS cardiomyocyte for use according to item 48, wherein the iPS cardiomyocyte is engrafted into a subject, such as by ultrasound guidance.

[0311] 50. A method of inducing proliferation and / or CM polyploidy in cardiomyocytes, the method comprising, delivering to a cardiomyocyte a compound capable of increasing the level of Zebl in said cardiomyocyte.

[0312] 51. The method according to item 50, wherein the compound is delivered by transfection, or by a vector, such as a plasmid, such as a viral vector, such as adenovirus, adeno-associated virus, or lentivirus.

[0313] 52. The method of according to item 50 or item 51, wherein the method is performed on a subject, such as a mammal, such as a mouse, a primate, preferably a human.

[0314] 53. The method of according to item 50 or item 51, wherein the method is performed on a non-human subject.

[0315] 54. A method of treatment of the myocardium in a subject having experienced myocardial infarction, the method comprising administering to the subject an effective amount of a compound capable of modulating Zebl.

[0316] 55. The method of treatment according to item 54, wherein the compound is

[0317] - a compound capable of downregulating Zebl in iPS CM, and wherein said iPS CM have been engrafted to the myocardium of the subject, or

[0318] - a compound capable of overexpressing Zebl in cardiomyocytes.

[0319] 56. The method of treatment according to any of items 54-55, wherein the subject is a mammal, such as a mouse, a primate, preferably a human.

[0320] Numbered embodiments

[0321] Embodiment 1. A compound capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction.

[0322] Embodiment 2. The compound for use according to embodiment 1, wherein treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool by engrafting iPS cardiomyocytes (CM) to a subject having experienced myocardial infarction.

[0323] Embodiment 3. The compound for use according to any of the preceding embodiments, wherein the compound is a compound capable of decreasing the levels of Zebl in an iPS CM engrafted into a subject having experienced myocardial infarction. Embodiment 4. The compound for use according to embodiment 3, wherein the compound capable of decreasing the levels of Zebl is a silencing RIMA, such as shRNA or siRN, an antisense RNA (asRNA), or an antisense DNA (asDNA). Embodiment 5. The compound for use according to embodiment 4, wherein the shRNA is the shRNA according to SEQ ID NO: Embodiment 2.

[0324] Embodiment 6. The compound for use according to embodiment 3, wherein the compound capable of decreasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of Sa-792574, BAS-09104376, Lysylphenylalanyl-tyrosine, ofloxacin, lopinavir, tacrolimus, nocodazole, CAY- 10618, Mirin, and ON-0191Embodiment 0.

[0325] Embodiment 7. The compound for use according to embodiment 1, wherein treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool by treating endogenous cardiomyocytes.

[0326] Embodiment 8. The compound for use according to embodiment 1 or embodiment 7, wherein the compound is a compound capable of increasing the levels of Zebl in a cardiomyocyte residing in a subject having experienced myocardial infarction. Embodiment 9. The compound for use according to embodiment 1, or any of embodiments 7-8, wherein the compound capable of increasing the levels of Zebl is a plasmid encoding Zebl, or a nucleic acid encoding Zebl.

[0327] Embodiment 10. The compound for use according to embodiment 1, or any of embodiments 7-8, wherein the compound capable of increasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of mesalazine, anisomycin, tegaserod, otenzepad, Sarmentogenin, CS-110266, Naringenin, Strophanthidin, Cephaeline, Olopatadine.

[0328] Embodiment 11. The compound for use according to any of the preceding embodiments, wherein Zebl is human Zebl according to SEQ ID NO: 1, or a variant thereof having 80 % sequence identity to the sequence according to SEQ ID NO: 1.

[0329] Embodiment 12. An ex vivo method of maturing iPS CM, the method comprising, administering to an iPS CM a compound capable of decreasing the levels of Zebl, such as the shRNA according to SEQ ID NO: 2.

[0330] Embodiment 13. An iPS cardiomyocyte matured by the method according to embodiment 12. Embodiment 14. An iPS cardiomyocyte matured by the method according to embodiment 12, for use in the treatment of the myocardium after myocardial infarction.

[0331] Embodiment 15. A method of inducing proliferation and / or CM polyploidy in cardiomyocytes, the method comprising, delivering to a cardiomyocyte a compound capable of increasing the level of Zebl in said cardiomyocyte, wherein the method is performed on a non-human subject or an ex vivo cell.

Claims

Claims1. A compound capable of modulating the transcription factor Zebl for use in a treatment of the myocardium after myocardial infarction:• wherein the compound is a compound capable of decreasing the levels of Zebl in an iPS cardiomyocyte (CM) and treatment of the myocardium is conceived by re-establishing the cardiomyocyte pool by engrafting said iPS CM into a subject having experienced myocardial infarction; or• wherein the compound is a compound capable of increasing the levels of Zebl in a cardiomyocyte residing in a subject having experienced myocardial infarction and treatment of the myocardium is conceived by reestablishing the cardiomyocyte pool by treating said endogenous cardiomyocytes.

2. The compound for use according to claim 1, wherein the iPS CM is treated with said compound that decreases the level of Zebl prior to engraftment.

3. The compound for use according to claim 2, wherein the compound is administered to decrease the levels of Zebl to finally mature the CM after which they have been selected, such as after day 24.

4. The compound for use according to claim 1, wherein the compound is a compound capable of decreasing the levels of Zebl in an iPS CM after the iPS CM has been engrafted into a subject having experienced myocardial infarction.

5. The compound for use according to any of the preceding claims, wherein the compound capable of decreasing the levels of Zebl is a silencing RIMA, such as shRNA or siRN, an antisense RNA (asRNA), or an antisense DNA (asDNA).

6. The compound for use according to claim 5, wherein the shRNA is the shRNA according to SEQ ID NO: 2.

7. The compound for use according to claim any of claims 1-5, wherein the compound capable of decreasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of Sa-792574, BAS-09104376,Lysylphenylalanyl-tyrosine, ofloxacin, lopinavir, tacrolimus, nocodazole, CAY- 10618, Mirin, and ON-01910, preferably nocodazole, or ON-01910.

8. The compound for use according to claim 1, wherein the compound capable of increasing the levels of Zebl is a plasmid encoding Zebl, or a nucleic acid encoding Zebl.

9. The compound for use according to claim 1, wherein the compound capable of increasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of mesalazine, anisomycin, tegaserod, otenzepad, Sarmentogenin, CS-110266, Naringenin, Strophanthidin, Cephaeline, Olopatadine, preferably anisomycin.

10. The compound for use according to any of the preceding claims, wherein Zebl is human Zebl according to SEQ ID NO: 1, or a variant thereof having 80 % sequence identity to the sequence according to SEQ ID NO: 1.

11. An ex vivo method of maturing iPS CM, the method comprising, administering to an iPS CM a compound capable of decreasing the levels of Zebl, such as the shRNA according to SEQ ID NO: 2.

12. The method according to claim 11, wherein the method comprises a step of selecting CM based on their metabolic phenotype.

13. The method according to claim 11 or claim 12, wherein the compound capable of decreasing the levels of Zebl is a silencing RNA, such as shRNA or siRN, an antisense RNA (asRNA), or an antisense DNA (asDNA).

14. The method according to claim 13, wherein the shRNA is the shRNA according to SEQ ID NO: 2.

15. The method according to claim 11 or claim 12, wherein the compound capable of decreasing the levels of Zebl is a small molecule, such as a small molecule selected from the group consisting of Sa-792574, BAS-09104376,Lysylphenylalanyl-tyrosine, ofloxacin, lopinavir, tacrolimus, nocodazole, CAY- 10618, Mirin, and ON-01910, preferably nocodazole, and / or ON-01910.

16. The method according to any of claims 11-15, wherein the compound is administered to reduce the levels of Zebl after day 18, such as in the days prior to or after day 24, such as at day 22, such as at day 23, such as at day 24, such as at day 25, such as at day 26, preferably at day 24.

17. The method according to any of claims 11-16, wherein the maturation of said iPS CM is monitored by observing beating initiation, and / or by measuring one or more of the genes that are expressed at different stages of differentiation, such as one or more genes selected from:- pluripotency genes such as OCT4 and / or SOX2;- CM genes such as TNNT2, MYH6, and / or MYH7 as a sign of iPS-CM differentiation; and- CM maturation such as SIRPA, FABP3, SCN5A, CMLC2, and / or MYL2.

18. An iPS cardiomyocyte matured by the method according to claim any of claims 11-17.

19. An iPS cardiomyocyte matured by the method according to claim any of claims 11-17, for use in the treatment of the myocardium after myocardial infarction.

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