Decoding the epigenetic and transcriptional basis of direct cardiac reprogramming

Abstract

Heart disease, particularly resulting from myocardial infarction (MI), continues to be a leading cause of mortality, largely due to the limited regenerative capacity of the human heart. Current therapeutic approaches seek to generate new cardiomyocytes from alternative sources. Direct cardiac reprogramming, which converts fibroblasts into induced cardiomyocytes (iCMs), offers a promising alternative by enabling in situ cardiac regeneration and minimizing tumorigenesis concerns. Here we review recent advancements in the understanding of transcriptional and epigenetic mechanisms underlying cardiac reprogramming, with a focus on key early-stage molecular events, including epigenetic barriers and regulatory mechanisms that facilitate reprogramming. Despite substantial progress, human cardiac fibroblast reprogramming and iCM maturation remain areas for further exploration. We also discuss the combinatorial roles of reprogramming factors in governing transcriptional and epigenetic changes. This review consolidates current knowledge and proposes future directions for promoting the translational potential of cardiac reprogramming techniques.

Graphical Abstract

Fibroblast fate is tightly maintained by epigenetic mechanisms, including DNA methylation and chromatin modifications. During direct cardiac reprogramming, this fibroblast gene program is rewritten by transcription factors, epigenetic factors and signaling pathways to establish the path to reprogramming. These changes produce induced cardiomyocytes that closely resemble endogenous cardiomyocytes in both gene expression and phenotype.

Significance statement.

Heart disease remains a primary cause of mortality due to the human heart’s limited ability to regenerate damaged tissue. This review highlights emerging approaches in cardiac reprogramming, specifically the direct conversion of fibroblasts into cardiomyocytes, as a potential method for heart repair. Unlike stem cell-based therapies, direct reprogramming may reduce tumor risks and promote safe, in situ regeneration of heart tissue. By consolidating recent insights into the transcriptional and epigenetic mechanisms of cardiac reprogramming, this review provides a foundation for advancing reprogramming strategies toward clinical translation.

Introduction

Heart disease remains a leading cause of mortality in the United States and worldwide.^1-3 Due to the limited regenerative capacity of cardiomyocytes, ischemic injuries such as MI result in permanent myocardial cell death, followed by fibrosis, scar formation and often chronic heart failure.^4,5 Despite advancements in cardiovascular medical therapies and devices that improve patient survival rates, the core challenge of cardiomyocyte loss remains unaddressed.^6-8 To rebuild the myocardium, 3 promising therapeutic strategies have emerged: inducing preexisting cardiomyocyte proliferation, remuscularizing myocardium via transplantation of stem cell-derived cardiomyocytes, and directly reprogramming non-myocytes into cardiomyocytes.⁹ Among these, direct cardiac reprogramming offers unique advantages, such as avoiding tumorigenesis risks and overcoming low engraftment rates by enabling in situ cardiac tissue regeneration.¹⁰

Direct lineage conversion of fibroblasts into cardiomyocytes has been a fast-developing field for over a decade. In 2010, Ieda et al first demonstrated that retroviral transduction of cardiac transcription factors (TFs) Gata4, Mef2c, and Tbx5 (GMT) could convert mouse postnatal cardiac fibroblasts (CFs) into functional iCMs.¹¹ Compared to the relatively hyperdynamic epigenetic status of iPSC-CMs (induced pluripotent stem cell-derived cardiomyocytes), iCMs exhibit a maturation status that more closely resembles that of adult CMs.¹²

Importantly, direct cardiac reprogramming has been successfully conducted in vivo in murine models of MI. In vivo cardiac reprogramming using viral vectors converted CFs into iCMs that functionally integrated into the myocardium, improved measurements of cardiac function, reduced scar size up to twofold,^13,14 and reduced fibrosis.^13-18 Early on, Qian et al demonstrated that injection of GMT retroviruses into the infarct area of mouse models following left anterior descending artery (LAD) ligation significantly improved ejection fraction, stroke volume, and total cardiac output three months after MI.¹⁴ Using Periostin-Cre and Fsp1-Cre transgenic mice, 2 fibroblast tracing lines, they further demonstrated that GMT generated iCMs from cardiac fibroblasts in the injured areas, rather than promoting proliferation of resident CMs. Scar area was significantly reduced in the GMT group. Recently, Tani et al demonstrated that in vivo cardiac reprogramming using a Tcf21iCre/reporter/MGTH2A transgenic mouse significantly improved myocardial contraction and reduced fibrosis in chronic MI.¹⁹ Furthermore, successful direct cardiac reprogramming has been achieved in vivo using various non-integrating techniques, such as Sendai viral vectors,^16,17 adeno-associated viral vectors,^20,21 and cationic gold nanoparticles²² (Table 1).

Table 1.

Cardiac reprogramming in vivo: selected studies

Delivery	Reporter line	Cocktail	Relevance	Ref
Retrovirus	Fsp1-Cre/LacZ, Tcf21iCre/tdTomato	GMHT	Confirmation of in vivo reprogramming and improved cardiac function in acute MI	13
Retrovirus	Periostin-Cre/LacZ	GMT, GMT + Tβ4	Confirmation of in vivo reprogramming and improved cardiac function in acute MI	14
Tamoxifen induction	Tcf21iCre/Tomato/MGTH	Polycistronic MGTH	Improved cardiac function and reduced fibrosis in chronic MI	19
Sendai virus	Tcf21-iCre/tdTomato	Polycistronic GMT	Improved cardiac function in acute MI without genomic integration	17
Adeno-associated virus	None	GMT, GMT + Tβ4	Reduced fibrotic area in acute MI using Adeno-associated viral vectors	20
Cationic gold nanoparticles	None	GMT	Reduced fibrotic area in acute MI using nanoparticle gene carriers	22

Abbreviations: Fsp1, fibroblast-specific protein; Tcf21, transcription factor 21 (also known as capsulin or epicardin); Tβ4, Thymosin β4.

Recent advances have focused on improving reprogramming efficiency and elucidating reprogramming mechanisms through genome-wide and single-cell assays. Epigenetic regulation and chromatin remodeling during the reprogramming process have emerged as important areas of study, providing crucial mechanistic insight into the fate conversion of CFs into iCMs. Numerous epigenetic mechanisms are involved in direct reprogramming, including histone modification, DNA methylation, and chromatin accessibility (Figure 1).²³ Herein, we will review the major cellular and molecular events associated with direct cardiac reprogramming, particularly highlighting the transcriptional and epigenetic mechanisms coordinating this process. Our goal is to provide an integrative and consolidated overview of the advances in the mechanistic understanding of direct cardiac reprogramming and its impact in the field.

Figure 1.

Model of direct cardiac reprogramming. (A) Fully repressed cardiac loci in fibroblasts. PRC1, PRC2, DNA methylation, histone modifications, low accessibility, and other epigenetic factors (not shown) maintain a repressed chromatin state. (B) Incomplete reprogramming. Epigenetic barriers block gene expression at cardiac loci despite binding of reprogramming factors. (C) Complete reprogramming. Removal of epigenetic barriers and addition of reprogramming boosters facilitate cardiac reprogramming. Additional factors, such as TBX20, increase H3K27ac on cardiac enhancer regions. Epigenetic factors such as PHF7 establish permissive histone modifications and reduce DNA methylation at cardiac loci, leading to gene expression. Abbreviations: PRC1, polycomb repressive complex 1; PRC2, polycomb repressive complex 2; EZH2, enhancer of zeste homolog 2; RING1A/B, ring finger protein 1a/b; BMI1, polycomb ring finger oncogene; PHF7, plant homeodomain finger protein 7; TBX20, T-box transcription factor 20; Me, DNA methylation; H3K27me3, trimethylation of histone H3 at lysine 27 (repressive); H2AK119Ub, ubiquitination of histone H2A at lysine 119 (repressive); H3K27ac, acetylation of histone H3 at lysine 27 (active); H3K4me3, trimethylation of histone H3 at lysine 4 (active).

Epigenetic regulation

Epigenetic regulation of gene expression largely occurs at 3 levels: DNA methylation, histone modification, and chromatin accessibility. DNA methylation involves the transfer of a methyl group to cytosine, forming 5-methylcytosine (5mC).²⁴ This modification generally leads to gene silencing by inhibiting the binding of TFs or recruiting proteins involved in gene repression, although the precise function of DNA methylation is complex and varies with context.^24-27 Mammalian 5mC is typically found in symmetrical CpG (5′-cytosine-phosphate-guanine-3′) sites, where it has a major role in X-chromosome inactivation, genomic imprinting, transposon repression, and transcriptional silencing.²⁵ During direct cardiac reprogramming, myosin heavy chain 6 (Myh6, coding for α-MHC) and natriuretic peptide A (Nppa), two cardiac lineage genes, are significantly activated and accompanied with a stable methylation reduction on their promoter regions.¹¹ The onset of this change is detected as early as day 3.²⁸

Chromatin is organized into nucleosomes, consisting of DNA wrapped around a histone octamer. The histone octamer is composed by two of each histone protein: H2A, H2B, H3, and H4.²⁹ The N-terminal tails of each histone protein are marked by many modifications that function either by remodeling chromatin structure or by recruiting nonhistone proteins.³⁰ Some of the best characterized modifications are acetylation, methylation, phosphorylation, and ubiquitination.³⁰ Histone modifications are frequently enriched near genes, where they are associated with transcriptional activity.²⁹ Histone modification is a particularly important epigenetic mechanism in the context of direct cardiac reprogramming. Histone H3 acetylation at lysine 27 (H3K27ac) marks active enhancers.³¹ Trimethylation of histone H3 at lysine 4 (H3K4me3) is enriched at promoters of transcriptionally active genes, whereas trimethylation of histone H3 at lysine 27 (H3K27me3) is associated with inactive promoters.^32,33 Another modification, ubiquitination of histone H2A at lysine 119 (H2AK119Ub), is frequently linked to gene silencing.³⁴ In addition, combinations of modifications of the same histone can cooperatively regulate transcription.^23,35,36

Chromatin accessibility refers to the physical accessibility of chromatinized DNA to nuclear macromolecules.³⁷ The positioning of nucleosomes alters the availability of binding sites to TFs and the general transcription machinery, thus regulating gene expression.³⁸ Consequently, highly condensed chromatin is generally associated with gene repression, while open or permissive chromatin is generally associated with gene expression. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), a popular method for genome-wide chromatin accessibility mapping, utilizes hyperactive Tn5 transposase to preferentially insert sequencing adapters into accessible regions of chromatin.³⁹ This technique allows for the study of chromatin dynamics during direct cardiac reprogramming.

Phases of cardiac fate conversion

Initial transcriptional and epigenetic changes

The cardiac reprogramming process can be divided into early and late stages with distinct molecular features. Genome-wide assays have revealed that an epigenomic shift towards a cardiomyocyte state occurs rapidly during the early stages of reprogramming.^40-43 Stone et al performed single cell RNA sequencing (scRNA-seq) analysis in day one GMT-transduced cells and identified 3 major trajectories during reprogramming initiation: reprogramming, proliferating, and fibroblast-like.⁴¹ Simultaneously, ATAC-seq of αMHC-GFP⁺ cells was performed throughout the reprogramming process.⁴¹ This analysis revealed extensive chromatin remodeling by day 2,⁴¹ exemplified by a rapid gain of accessibility at early reprogramming marker genes Slc6a6 and Tnnt2. In another study, increased H3K4me3 and reduced H3K27me3 at cardiac promoters were observed in day 3 cells transduced with a single polycistronic vector expressing Mef2c, Gata4, and Tbx5 (MGT), indicating an active epigenetic state.²⁸ All these findings suggest that upregulation of cardiac genes occurs early within 2-3 days in the reprogramming process and corresponds with extensive chromatin remodeling and epigenetic changes (Figure 2).

Figure 2.

Major molecular events occurring during direct cardiac reprogramming. Levels of each event are relative. Abbreviations: mnCF, mouse neonatal cardiac fibroblast; MEF, mouse embryonic fibroblast; ATAC-seq, assay for transposase-accessible chromatin with sequencing; ChIP, chromatin immunoprecipitation; Col1a2, collagen type I alpha 2 chain; Tnnt2, cardiac troponin T2; Fn-EDA, fibronectin extra domain A.

Global changes in protein abundance have been examined using multiplexed quantitative mass spectrometry.⁴⁴ Sauls et al found that chromatin-associated protein networks are predominantly downregulated by day 3, but not on day 2, post-MGT-transduction. This suggests that significant chromatin changes during reprogramming occur by day 3 post-transduction (Figure 2), aligning with previously identified early chromatin alterations during reprogramming.^40-43 Additionally, the proteomics analysis revealed early protein changes, including the upregulation of extracellular matrix protein networks in response to MGT, and a unique reduction of translation factor protein networks at day 2 but not day 3 cells.

In addition to activating cardiac genes, repressing the fibroblast fate is essential for cardiac reprogramming. Notably, changes in fibroblast gene programs were observed to occur later than cardiac gene activation.²⁸ Repressive H3K27me3 at fibroblast marker gene loci did not increase until day 10 of reprogramming, while H3K4me3 at these loci gradually decreased throughout the process²⁸ (Figure 2). Despite efficient gene delivery through polycistronic MGT transduction, which ensures the removal of uninfected fibroblasts, around 50% of MGT-transduced cells retain a fibroblast-like state after two weeks of reprogramming procedure. This suggests that incomplete repression of fibroblast gene programs may hinder the conversion to a cardiac fate. Our scRNA-seq analyses have revealed a branching trajectory in response to MGT, guiding cells either along cardiac reprogramming paths or alternative paths.^41,45 Studies have aimed to identify key events that dictate these trajectories, with potential mechanisms involving cell cycle regulation, inflammation, and autophagy.

Key events in early stage

Cell cycle exit is a key event in cardiac reprogramming. Single cell analysis by Zhou et al revealed that by day 3 of reprogramming, human iCMs (hiCMs) showed decreased expression of cyclin and cyclin-dependent kinase genes, indicating that the proliferative capacity of human fibroblasts is lost early in the reprogramming process.⁴⁵ Additionally, of the MGT + miR133 (MGT-133)-infected cells, none of the cells that gained CM gene signatures were actively proliferating, while 8% of cells retaining fibroblast features remained cell-cycle-active. Similar results have been reported in murine cardiac reprogramming.^46,47 Although MGT expression in murine fibroblasts can generate proliferating iCMs, it leads to a less advanced cardiac state.⁴¹ Collectively, these findings suggest that cell cycle exit is requisite for cardiac reprogramming.

Similarly, inflammatory signaling has been shown to inhibit iCM induction. Stone et al observed a loss of chromatin accessibility at regions associated with the inflammatory response and monocytes during the course of murine cardiac reprogramming.⁴¹ Consistent with this observation, treatment with anti-inflammatory drugs, such as dexamethasone, nabumetone, and diclofenac, significantly improved reprogramming efficiency.⁴⁸ Diclofenac, identified in a screen of 8400 chemical compounds, enhanced reprogramming by inhibiting inflammatory pathways, including cyclooxygenase-2 and interleukin 1β signaling.⁴⁹ Notably, diclofenac’s effects were most pronounced when administered during the first four days of reprogramming, emphasizing the inflammatory inhibition as a key early event.

Autophagy, a process of intracellular self-digestion and remodeling, has been largely unexplored in direct cardiac reprogramming, despite its presumably important role in fate conversion. Wang et al. demonstrated that autophagosome formation is increased in reprogramming cells, and autophagy-related 5 (Atg5)-dependent autophagy is required for human cardiac reprogramming.⁵⁰ Activation of autophagy through inhibition of mammalian target of rapamycin (mTOR) signaling with rapamycin and torin improved reprogramming efficiency. Conversely, shRNA-mediated knockdown of Atg5 significantly reduced reprogramming efficiency, but only when depletion occurred before reprogramming day 8, suggesting that the regulation of Atg5-dependent autophagy is critical during the early initiation of reprogramming.

Insufficient late maturation

Distinct molecular changes occur during the late stage of reprogramming, including silencing of exogenous GMT expression, downregulation of fibroblast markers, and activation of cardiomyocyte maturation genes.^28,40,42 Suppression of fibroblast identity has been observed at both transcriptional and epigenetic levels. In GHMT-infected MEFs, TGF-β signaling and extracellular matrix (ECM) expression are transiently activated during the early stages of reprogramming, but downregulated by day 14⁴² (Figure 2). This is consistent with delayed re-patterning of H3K27me3 and H3K4me3 at promoters of fibroblast genes.²⁸ Additionally, early upregulation of ECM protein networks, observed 2-3 days after MGT transduction through mass spectrometry, further supports this transition.⁴⁴ ChIP-seq analysis of iCMs reprogrammed with GMT, GHMT, or AGHMT revealed that enhancers gained or maintained by day 7 largely overlapped with those active in cardiac precursors and cardiomyocytes during cardiogenesis. These enhancers are highly associated with genes involved in heart or muscle differentiation.⁴⁰ GO terms related to heart and muscle were strongly enriched in GHMT and AGHMT-treated iCMs compared to GMT, spotlighting the role of Hand2 and Akt1 in promoting iCM maturation through activating enhancers unique to postnatal heart.⁴⁰

In murine cardiac reprogramming, about 20% of fibroblasts express cardiac maker genes after one week. However, only around 0.1% of the starting fibroblasts are fully reprogrammed into functional iCMs after 4 weeks,¹¹ indicating the need of a second phase for functional maturation. Key factors for the maturation phase include both intrinsic and extrinsic influences that guide the development of fully functional iCMs. A combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF) has been shown to enhance this late-stage reprogramming by converting partially reprogrammed iCMs into functional iCMs. Notably, this treatment does not increase the quantity of partially reprogrammed cells or the amount of cell proliferation,⁵¹ but acts through p38MAPK and PI3K/AKT pathways. Recent findings indicate that TBX20 plays a critical role in promoting iCM maturation.⁵² Mechanistically, TBX20 activates key cardiac genes, modulates chromatin accessibility at cardiac enhancers, coordinates with other transcription factors, and supports structural and functional maturation. Specifically, TBX20 induces the metabolic shift from glycolysis to oxidative phosphorylation that occurs during cardiomyocyte maturation,⁵³ supporting the high energy demands of mature iCMs.

Environmental and metabolic factors hold great potential in promoting iCM maturation. Culturing iCMs in a soft matrix that mimics the stiffness of the native myocardium has been shown to increase the efficiency and quality of cardiac reprogramming.⁵⁴ In this environment, biophysical cues led to the suppression of YAP/TAZ signaling and the silencing of fibroblast gene programs. Similarly, in chemically induced cardiomyocyte-like cells, approaches such as mitochondrial delivery⁵⁵ and 3-dimensional culture in heart extracellular matrix⁵⁶ have yielded enhanced electrical functionality and sarcomere organization in vitro.

In summary, direct cardiac reprogramming involves early epigenetic changes within the first two days, followed by gradual activation of maturation processes and suppression of the fibroblast identity. However, the transition from the initiation phase to the maturation stage remains a bottleneck. Promoting functional maturation of iCMs through transcriptional, biophysical, metabolic, or other bioengineering means will be crucial for hastening the translational application of direct reprogramming.

Human vs mouse reprogramming process

The main differences between human and mouse direct cardiac reprogramming lie in the efficiency, speed, and complexity of the processes. While mouse cardiac fibroblasts can be reprogrammed into iCMs relatively efficiently, human cardiac reprogramming is significantly slower, less efficient, and requires a more complex cocktail of factors to achieve similar results. Comparative analysis of scRNA-seq data showed that human iCM reprogramming not only progresses at a slower rate but also exhibits lower efficiency and robustness compared to mouse reprogramming.⁴⁵ Unlike mouse iCM reprogramming, where no alternative trajectory occurs, MGT-133 transduced human fibroblasts follow a bifurcated trajectory—either progressing towards iCMs reprogramming or regressing back to their fibroblast identity.⁴⁵ Additionally, the initial phase of cardiac reprogramming in human cells is estimated to be 6 days,⁴⁵ which is longer than the 2 days required in mouse reprogramming.⁴¹ Further, the maturation of human iCMs remains largely unexplored and requires reprogramming boosters such as TBX20⁵² and PHF7⁵⁷ to facilitate the process. These species-specific barriers and regulatory networks influencing cell fate decisions during reprogramming warrant further investigation.

Understanding reprogramming factors

Roles of Gata4, Mef2c, and Tbx5

Gata4, Mef2c, and Tbx5 are highly conserved TFs essential for heart development and play canonical roles in direct cardiac reprogramming.^40,41,58-61 They belong to a group of cardiac TFs including GATA6, ISL1, NKX2-5, SRF, and TBX20 that comprise a “cardiac kernel,” which orchestrates cardiac development through extensive cross-regulatory interactions.⁶¹ The role of these three factors in heart development has been extensively investigated.

Vertebrate GATA TFs contain an N-terminal transactivation domain, a DNA binding domain consisting of two zinc fingers, and a C-terminal domain containing a nuclear localization sequence.⁶² GATA4 is considered a “pioneer” TF capable of interacting with heterochromatin^63,64 and has been shown to interact with numerous histone modification enzymes to coordinate expression of cardiac genes during cardiac differentiation, including histone deacetylase (HDAC) 1 and 2, p300, and polycomb repressive complex 2 (PRC2).^65-67 Contractile gene Myh6 (coding for α-MHC) and natriuretic peptide genes Nppa and Nppb are among GATA4’s downstream targets.^65,68

MEF2C belongs to the myocyte enhancer factor 2 (MEF2) family of TFs that contain MADS and MEF2 DNA-binding domains followed by transactivation domains.⁶⁹ Mef2c is required for activation of a subset of cardiac contractile protein genes and for the development of cardiac structures derived from the secondary heart field.⁶⁰ MEF2C interacts with histone acetyltransferases, histone deacetylases, and p300/CBP to regulate gene expression.⁷⁰

TBX5 belongs to the T-box gene family, containing the highly conserved T-box DNA-binding domain.^71,72 TBX5 is a transcriptional activator of cardiomyocyte maturation genes during early cardiac development and is required for mature cardiomyocyte function and the patterning of the cardiac conduction system in later cardiac development.⁷³ Despite being known as a cardiac transcriptional activator, TBX5 interacts with components of the repressive nucleosome remodeling and deacetylase (NuRD) repressor complex as well as the FACT (facilitates chromatin transcription) complex and members of the TFIID (transcription initiation factor) complex.⁷⁴ The repressive function of TBX5 may prevent ectopic expression of alternative gene programs within the heart, such as a group of neural lineage genes.⁷⁴

Cooperation of reprogramming factors

Understanding the cooperation of GMT is key to dissecting the initiation of direct cardiac reprogramming. Early studies by Wang et al demonstrated that the stoichiometry of the GMT factors is important for efficient reprogramming. Specifically, higher levels of Mef2c and comparatively lower levels of Gata4 and Tbx5 significantly increased direct reprogramming efficiency.⁷⁵ Stone et. al. found that chromatin accessibility changes during direct reprogramming are primarily associated with Mef2c and Tbx5 binding, although all three factors can promote chromatin remodeling individually.⁴¹ Notably, the combinatorial expression of GMT facilitated DNA binding and chromatin opening more effectively than single-factor expression. This cooperative binding, particularly at cardiac loci, was strongly associated with chromatin opening. Likewise, Hashimoto et. al. reported that GMT-driven cardiac reprogramming relies on the cooperative actions of the three factors.⁴⁰ Through ChIP-seq analysis, they identified 24,933 binding sites, many of which were co-occupied by several factors and correlated with sequential transcriptomic changes. In contrast, single-factor responsive enhancers or single-factor binding peaks showed weak signals in MGT-specific enhancers.

Beyond GMT, other factors have been identified to increase reprogramming efficiency in a variety of fibroblasts, as summarized elsewhere.⁷⁶ Among these are GMT + Hand2 (GMHT),¹³ GMHT + Nkx2.5,⁷⁷ MGT-133 + TBX20,⁵² GHMT + Akt1,⁷⁸ micro-RNA combinations,⁷⁹ and small molecules.^80,81 Additional factors such as TBX20, Hand2, and Akt1 have been shown to enhance GMT co-occupancy and activate maturation-related cardiac enhancers.^40,52 Genomic binding peaks of the GMT factors show significant overlap with TBX20 peaks in hiCMs, and addition of TBX20 to the MGT cocktail results in a substantially higher frequency of GMT co-occupancy and a global increase in GMT binding, particularly at cardiac contractility genes.⁵² Similarly, addition of Hand2 to GMT increases co-occupancy in Gata4, Mef2c, and Tbx5 binding peaks, and subsequent addition of Akt1 increases the total number of Gata4 binding and co-binding sites of Gata4-Hand2 and Gata4-Tbx5 pairs.⁴⁰

Intriguingly, Ascl1, a neuron reprogramming factor, has demonstrated cross-lineage potential to induce direct cardiac reprogramming.⁸² The addition of Ascl1 significantly increased the percentage of cTnT⁺ cells, surpassing GMT alone. When overexpressed in MEFs, Ascl1 acts as a pioneer factor, binding to genes involved in muscle and neuronal development. However, co-expression of Ascl1 with Mef2c (A + M) redirects Ascl1 from its neuronal targets to its cardiac targets. Single-cell multi-omics analyses of cells at day 3 post-reprogramming with GMT or A + M induction identified an additional iCM state with increased activation of cardiac sarcomere, ion-channel, and cardiogenic genes, showing the highest expression of Ascl1. There was no analogous cell state in GMT reprogramming, suggesting that the Ascl1 + Mef2c induces more complete reprogramming at day 3 post-transduction.⁸²

Epigenetic factors involved in cardiac reprogramming

Overcoming epigenetic barriers is critical for efficient reprogramming

Recent advances have emphasized the critical role of epigenetic regulation in the efficacy of direct cardiac reprogramming. Key studies have identified various factors that either enhance or inhibit this process regardless of the cocktails or fibroblasts used. The understanding of this epigenetic reconfiguration not only provides novel mechanistic insights but also clarifies the approach for potential clinical translation of cardiac reprogramming.

Among the first, Zhou et al identified Bmi1 as a key epigenetic barrier to murine cardiac reprogramming in a loss-of-function screen of chromatin modifying or remodeling complexes.⁸³ Bmi1 is an essential component of PRC1, which mediates monoubiquitination of histone H2A at lysine 119 (H2AK119ub) together with Ring1A and Ring1B.^84-86 Reducing Bmi1 expression significantly enhances the induction of beating iCMs from neonatal and adult mouse fibroblasts (Table 2), corresponding with a removal of H2AK119ub at key cardiogenic loci, such as Gata4.⁸³ Bmi1 depletion followed by Mef2c + Tbx5 transduction was sufficient to produce spontaneously contracting iCMs with calcium oscillations and sarcomere structures after 2-4 weeks of culture. However, knockdown of Bmi1 was effective in increasing iCM reprogramming efficiency only if performed within the first 3 days of MGT infection, suggesting that its role is in the early stage of the process.⁸³ In addition, an independent study reported that pharmacological inhibition of Bmi1 resulted in a similar enhancement of chemically-inducwed direct cardiac reprogramming.⁸¹

Table 2.

Factors mediating epigenetic changes in direct cardiac reprogramming

Factors	Cell type	Efficiencyb	Mechanism	Ref
GMT + Akt1 + Hand2 + Phf7a	TTF	FC, cTnT+α-MHC-GFP + 18.8%	Cooperation with SWI/SNF complex to increase chromatin accessibility and TF binding at cardiac super enhancers	57
MGT + shBmi1	mnCF	FC, cTnT+α-MHC-GFP+, 21.9%	Removal of repressive mark H2AK119ub from cardiac loci; repression of PRC1	83
MGT-133 + shEzh2	H9F	FC, cTnT+, 32.7%	Removal of repressive mark H3K27me3 from cardiac loci; suppression of PRC2	87
MGT + MM408 (Mll1 inhibitor)	MEF	FC, cTnT+α-MHC-GFP+, 10%	Suppression of alternative lineage gene expression	88
M2TAD + GT	MEF	FC, cTnT+, 4.5%; 30-fold increase in beating cells	Recruitment of p300 and cardiogenic TFs to cardiac loci to induce chromatin remodeling	89
shp63 + H/M	hCF	FC, cTnT+, 14.9%; synchronous contractions in co-culture with neonatal rat cardiomyocytes	Reduced HDAC1 interaction with p63 at promoter sites of cardio-differentiation genes	90
MGT-133 + shTET1 + shTLR3	hCF	FC, cTnT+, 14.8%; partially rescued reduction in efficiency caused by TLR3 knockdown	Reduced methylation rates at cardiac promoters compared to TLR3 knockdown cells	91

^aEpigenetic factors are shown in bold.

^bReprogramming efficiencies vary widely between different labs and reprogramming protocols.

Abbreviations: BMI1, polycomb ring finger oncogene; cTnT, cardiac troponin T; EZH2, enhancer of zeste homolog 2; FC, flow cytometry; H2AK119ub, ubiquitination of histone 2A at lysine 119; H3K27me3, trimethylation of histone 3 at lysine 27; PHF7, plant homeodomain finger protein 7; H9F, H9 human embryonic stem cell line; hCF, human cardiac fibroblast; HDAC1, histone deacetylase 1; MEF, mouse embryonic fibroblast; Mll1, lysine methyltransferase 2A, a H3K4 methyltransferase; mnCF, mouse neonatal cardiac fibroblasts; p300, E1A-associated 300 kDa protein, a major H3K27 acetyltransferase; p63, tumor protein p63; PRC, polycomb repressive complex; TET1, tet methylcytosine dioxygenase 1; TF, transcription factor; TLR3, toll-like receptor 3; TTF, tail-tip fibroblast; α-MHC, myosin heavy chain alpha.

Mll1, a H3K4 methyltransferase, has been identified as another barrier to cardiac reprogramming. Inhibition of Mll1 improved the reprogramming efficiency of MEFs by more than threefold as measured by cTnT and α-MHC-GFP double positive cells (Table 2)⁸⁸ and increased spontaneously contracting loci and calcium oscillations. Similarly to Bmi1, Mll1 inhibition was most effective if performed one day after MGT induction, stressing the importance of early epigenetic repatterning in cardiac reprogramming. Mechanistically, Mll1 inhibition suppressed adipocyte lineage genes such as Ebf1, Fabp4, and Pparg, suggesting that its inhibition promotes cardiac specificity by preventing the activation of non-cardiac lineages.

Human iCM reprogramming requires additional epigenetic remodeling.^{3,76,92,93,94} We found that Tet methylcytosine dioxygenase 1 (TET1), a potent epigenetic factor involved in DNA methylation,^95,91 plays a role in innate immunity gene-regulated events during direct cardiac reprogramming.⁴⁵ Reprogramming efficiency was decreased by knockdown of TLR3, NFKB1, and PTGS2, but could be restored by TET1 depletion, likely due to the reduction of methylation at cardiac promoters. Recently, the same group identified enhancer of zeste homolog 2 (EZH2), a histone methyltransferase that serves as the catalytic subunit of PRC2 which mediates H3K27me3 modification,⁹⁶ as an important epigenetic barrier to human direct cardiac reprogramming.⁸⁷ EZH2 knockdown led to a three- to fourfold increase in cTnT + cells (Table 2). Critically, repressive H3K27me3 marks on cardiac structural genes were significantly decreased with treatment of EZH2 inhibitors, while the same genes are not fully activated in control cells.⁸⁷ These findings demonstrate the need to overcome epigenetic barriers like TET1 and EZH2 to enhance cardiac reprogramming from human fibroblasts, which exhibit additional epigenetic resistance compared to their mouse counterparts.

Interestingly, the tumor suppressor gene p63 has been shown to act as an“anti-plasticity gene” in both iPSC reprogramming⁹⁷ and human cardiac reprogramming.⁹⁰ Pinnamaneni et al. showed that a Hand2/Myocardin (H/M) cocktail along with p63 knockdown improved the reprogramming efficiency of human cardiac fibroblasts⁹⁰ (Table 2). Inhibiting p63 reduced HDAC1 binding at the promoter of cardiogenic genes such as Gata4, Tnnt2, and Myh6, leading to increased H3K27ac and gene activation. The p63 transactivation inhibitory domain (p63-TID), which competitively binds to HDAC1, enhanced reprogramming similarly to p63 knockdown. Approximately 5% of human iCMs generated without p63 exhibited synchronous contraction when cocultured with neonatal rat CMs. This study utilized an alternative strategy of increasing target cell susceptibility to overcome barriers to human cardiac reprogramming.

Critical epigenetic factors facilitate cardiac fate conversion

Certain epigenetic factors facilitate cardiac reprogramming. One such factor is PHF7, which Garry et al detected as a critical regulator of epigenetic remodeling during direct cardiac reprogramming.⁵⁷ PHF7 was identified through an overexpression screen of gene regulatory factors, where it significantly increased reprogramming efficiency. Specifically, PHF7, in combination with Akt1, Gata4, Hand2, Mef2c, Tbx5 (AGHMT), generated ~20% double-positive cTnT⁺ and α-MHC-GFP⁺ iCMs from tail-tip fibroblasts (TTFs), representing a tenfold improvement compared to AGHMT cocktail alone (Table 2). Notably, PHF7 also enhanced the direct reprogramming of adult human cardiac fibroblasts. Mechanistically, PHF7 interacts with the SWI/SNF complex, which is required for the maintenance of lineage specific enhancers,⁹⁸ to increase chromatin accessibility and TF binding at cardiac super enhancers in fibroblasts.⁹⁹ This interaction leads to the upregulation of mature cardiac structural proteins, developmental genes, and all four TFs of GHMT,⁵⁷ positioning PHF7 an epigenetic facilitator of cardiac fate acquisition.

Similarly, the engineered factor M2TAD, which recruits the p300 co-activator, plays a crucial role in cardiac fate conversion and functional maturation.⁸⁹ M2TAD is a transcriptionally activated MEF2C construct that fuses MEF2C isoform 2 (M2) with the MYOD transactivation domain. Replacing wildtype MEF2C with M2TAD increased reprogramming efficiency by approximately twofold (Table 2) and raised the number of beating iCMs by approximately 30-fold after 4 weeks of culture. M2TAD recruited the p300 co-activator specifically to cardiac loci, enhancing H3K27 acetylation and chromatin remodeling. Knockdown of p300 significantly diminished the effects of M2TAD. Furthermore, p300-depleted iCMs displayed immature morphology with disarrayed myofibrils, shorter sarcomeres, and smaller cell sizes, implicating p300’s essential role in iCM maturation.

Conclusion and perspectives

Direct cardiac reprogramming has made significant strides since its inception, with improved reprogramming efficiency across various fibroblast types and an advanced understanding of its fundamental mechanisms. However, several challenges remain, particularly regarding the maturation of iCMs and the transition from basic research to clinical applications.

While early chromatin dynamics and epigenetic remodeling during the first three days of the reprogramming process have been extensively studied, the later stages—particularly the maturation process of iCMs—remain poorly characterized. This gap in knowledge is likely due to the heterogeneity and complexity of the process, which limits our ability to fully understand and optimize iCM maturation. As iCMs must achieve functional integration to be viable for cardiac repair, understanding these maturation steps is essential. Furthermore, many epigenetic factors influencing reprogramming have been studied primarily in murine models. Considering that the ultimate goal of regenerating human myocardium after MI, there is an urgent need to identify epigenetic factors and reprogramming cocktails that enhance the efficiency of adult human fibroblast reprogramming.

The reprogramming factors Mef2c, Gata4, and Tbx5 act in concert with an assortment of proteins and pathways involved in cardiac development, many of which have been further explored in direct cardiac reprogramming. Unsurprisingly, direct cardiac reprogramming appears to share enhancer activation patterns with cardiogenic processes.⁴⁰ A comprehensive understanding of the similarities between cardiac development and cardiac reprogramming could pave the way for more effective iCMs maturation strategies. Moreover, further investigation of broader cellular processes not mentioned in this review, such as translational regulation,¹⁰⁰ signaling pathways,^101,102 and alternative splicing⁴⁷ would provide new insights into the molecular events occurring in the context of cardiac reprogramming.

To date, most epigenetic factors studied in cardiac reprogramming function as barriers, maintaining the fibroblast identity and impeding reprogramming. Thus, through either genetic depletion or pharmacological inhibitors, their inhibition enhances reprogramming efficiency. On the other hand, epigenetic factors like PHF7 facilitate cardiac reprogramming when overexpressed, promoting chromatin accessibility and transcription factor binding at key cardiac loci. Despite these insights, there has been little effort to combine multiple epigenetic modulators to maximize reprogramming efficiency.

Finally, while transcriptional and epigenetic regulation of cardiac reprogramming has been thoroughly investigated, most insights remain limited to in vitro processes. Translating these findings to in vivo reprogramming demands further effort and the development of new technologies. While challenges still lie ahead, we look forward to unraveling these fundamental mechanisms of direct cardiac reprogramming and cell fate determination to develop effective therapies for heart disease.

Author contributions

William G. Peng (Conceptualization, Investigation, Methodology, Writing); Anteneh Getachew (Funding acquisition, Investigation, Writing—original draft, Writing—review & editing); Yang Zhou (Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing—original draft, Writing—review & editing).

Funding

This work was supported by National Institutes of Health grants R01 HL153220 (Y.Z.), American Heart Association Transformational Project Award 969529 (Y.Z.), and Postdoctoral Fellowship 24POST1188989 (A.G.).

Conflicts of interest

The authors declared no potential conflicts of interest.

Data availability

No new data were generated or analyzed in support of this research.