Potential Strategies for Cardiac Diseases: Lineage Reprogramming of Somatic Cells into Induced Cardiomyocytes

Abstract

Lineage reprogramming has become a potential strategy for therapy of cardiac diseases. Somatic cells can be directly converted into the induced cardiomyocytes (iCMs) without passing through an induced pluripotent stem cell stage; this strategy has some advantages such as directional differentiation and preferable security. However, there are still many challenges which need to be further studied, such as identification of safer induced factors, exploration of molecular mechanisms, improvement of the mature level of iCMs and so on. Therefore, the structures of key factors, including transcription factors, microRNAs (miRNAs), epigenetic regulators and small molecules and their functions in the cardiac development and lineage reprogramming, molecular mechanisms underlying lineage conversion, strategies for generating matured iCMs, and major challenges were reviewed to lay the foundation for further applications of iCMs.

Introduction

Cardiovascular disease is one of the main causes leading to the death of people, because cardiomyocytes (CMs) are hardly proliferous, which results in the loss of the number and function of CMs after being damaged. Furthermore, damaged tissues will cause fibrosis, which increases the risk of arrhythmia and subsequent heart failure (Fong et al., 2016). However, effective drugs for promoting the regeneration of myocardium have not been developed.

Embryonic stem cells (ESCs) have the capacity to be induced to differentiate into induced cardiomyocytes (iCMs), but ethical and safety issues faced by them will limit their applications in this field. Since induced pluripotent stem cells (iPSCs) were first reported by Takahashi and Yamanaka (Takahashi et al., 2007), transplantation of exogenous iCMs derived from iPSCs into the damaged site of heart provides a potential strategy for the treatment of heart diseases, increase in the number of CMs, and recovery of certain cardiac functions had been observed in mouse (Masumoto et al., 2014), rat (Iseoka et al., 2018) and pig (Tabei et al., 2019). Especially, the likelihood of immune rejection can also be effectively reduced. But potential tumorigenicity and nondirectional differentiation still exist in this method.

Lineage reprogramming that somatic cells are converted into desired cells with defined factors may be a better choice due to directional differentiation without passing through an induced pluripotent stage (Fig. 1). Ieda et al. (2010) first reported that CMs could be successfully induced from fibroblasts with transcription factors G-binding protein 4 (Gata4), myocyte enhancer factor 5 2C (Mef2c), and T-box containing transcription factor (Tbx5) (GMT). Inspiringly, with the development of lineage reprogramming in heart, searching the novel factors, such as microRNAs (miRNAs), epigenetic regulators, and small molecules, to induce the generation of CMs is still generally a concern. However, iCMs are usually immature, which resemble with fetal CMs (Dhahri et al., 2018; Yang et al., 2014a), but matured CMs are more suitable for clinical applications in regenerative medicine because cardiac diseases mainly occur in adult and aged people.

FIG. 1.

Strategies for reprogramming somatic cells into iCMs. ESCs, embryonic stem cells; iCMs, induced cardiomyocytes; iCPCs, induced cardiac progenitor cells; iPSCs, induced pluripotent stem cells.

Therefore, various induced factors, potential mechanisms for lineage reprogramming, and strategies for improving the functional maturation of iCMs are summarized in this review (Fig. 2), which looks forward to contribute to the applications of iCMs in cell therapy, disease modeling, drug discovery, and so on.

FIG. 2.

Factors involved in cardiac lineage reprogramming. Mean of SCPF, CRFVPTZ, and 9C are same as notes in Table 1. 3D, three-dimensional; BAF, BRG1/BRM-associated factor; CMs, cardiomyocytes; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; ECM, extracellular matrix; ICMs, induced cardiomyocytes; JAK, Janus kinase; LIF, leukemia inhibitory factor; miRNA, microRNA; STAT, signal transducers and activators of transcription.

Cardiac Lineage-Specific Transcription Factors

Gata4

Gata4, a zinc finger transcription factor, including two class IV zinc-finger domains, belongs to GATA family (Evans and Felsenfeld, 1989). Members of this family can bond the consistent HGATAR DNA motif and act as the promoters of many genes (Tsai et al., 1989). Gata4 is a core transcription factor for the formation of early cardiac patterning and vascularization involved in embryogenesis and myocardial differentiation, it is essential for the survival of embryo (Bono et al., 2018; Srivastava, 2006). Expression of Gata4 promoted myocardial regeneration in neonatal mice through inducing the proliferation of CMs and angiogenesis (Mohammadi et al., 2018).

Mutations of Gata4 gene were related with septal defects of heart and reproductive defects (Zhang et al., 2017). Dominant Gata4 mutations caused congenital heart diseases (CHDs), specifically atrial septal defects and atrioventricular septal defects. Gata4 mutants also demonstrated Hedgehog (Hh) signaling defects. Gata4 acted directly upstream of Hh components: Gata4 could activate a cis-regulatory element at Gli1 in vitro and occupy the element in vivo (Zhou et al., 2017b). Meanwhile, Gata4 could broadly co-occupy cardiac enhancer with transcriptional regulator Tbx5, which implicated in the formation of human cardiac septa. Mutation of GATA4-G296S would affect DNA-binding affinity and disrupt the recruitment of Tbx5, and genes related to the phenotypic abnormalities, including cardiac septal defect, were dysregulated (Ang et al., 2016).

Gata4 is considered as a “pioneer” factor to open the structure of chromatin in cardiac loci, allowing other factors to be integrated into their specific target sites and activating the cardiac reprogramming. Enforced expression of GMT mediated by the nanoparticle-based gene carrier could convert mouse fibroblasts into iCMs in vivo after myocardial infarction (MI), resulting in effective recovery of cardiac functions and the decrease of scar area (Chang et al., 2019). The synergy effect of Gata4 and Tbx5 had the ability to activate the atrial natriuretic factor (ANF) promoter, cooperatively activating transcription involved in cardiac development (Garg et al., 2003; Laforest et al., 2018).

Through overexpression of Gata4, Mesp1, Tbx5, Nkx2.5, and Baf60c, fibroblasts could be converted into induced cardiac progenitor cells (iCPCs) which had the capability to further differentiate into CMs, smooth muscle cells (SMCs), and endothelial cells (ECs) (Lalit et al., 2016). If Gata4 was removed, expression of cardiac troponin T (cTnT) would be reduced, which suggested that Gata4 was a critical factor for the differentiation of iCPCs into mature CMs in specific sarcomere (Lalit et al., 2016).

Tbx5

Tbx5 contains an extremely conserved DNA motif (T-domain); this domain can bind to target DNA and then form the homodimers through interacting with major and minor groove of target DNA (Gentsch et al., 2017; Müller and Herrmann, 1997). It has been demonstrated that Tbx5 could efficiently affect the development of four chambers, electrical conduction system, and septum separation in heart (Boogerd and Evans, 2016; Sadahiro et al., 2018).

A study of human Holt/Oram syndrome showed that an amino acid, which nears the aminoterminal end of the T-box was replaced, then the interaction between Tbx5 and the major groove of targeted DNA sequence was suppressed, which resulted in the significant malformations of heart (Vanlerberghe et al., 2019). Holt/Oram syndrome was also related to the murine Brachyury mutations (or T mutations), which resulted from the insertion or deletion of Tbx5 gene (Spiridon et al., 2018). Those results indicated that T-protein in specific tissue had important effects on cardiac size, morphogenesis, and organogenesis. Therefore, distinct nucleotides of Tbx5 are more susceptibility to be mutated, which often results in abnormal structure and function of heart.

Tbx5 and its associated transcriptional cofactors, Gata4 and/or Nkx2.5, could activate cardiac genes during the lineage reprogramming, which resulted in biophysical coactions of specific DNA sequences, then the transcriptional activities of these cofactors were directly or indirectly regulated through altering three-dimensional (3D) structure of Tbx5 which bound to target DNA (Papaioannou, 2014). Combinatorial interactions between Tbx5 and Gata4 are critical for heart development and have been proposed to be the main etiopathogenesis of human CHDs. The rescue of the rhythm defects caused by Tbx5 haploinsufficiency might be mediated specifically by rescue of altered calcium homeostasis. Gata4 modulated a Tbx5-Serca (sarco endoplasmic reticulum Ca²⁺-ATPase) Ca²⁺-dependent pathway to maintain normal atrial rhythm (Laforest et al., 2018).

Overexpression of GMT through retrovirus vectors had been used to efficiently and rapidly reprogram mouse and human cardiac fibroblasts (CFs) into iCMs, which shortened the duration of inducing the generation of beating cells in vitro, improved the cardiac functions, and reduced the fibrosis after MI in vivo (Miyamoto et al., 2018, Wang et al., 2015). The physical interaction between Tbx5 and Gata4 as well as Nkx2.5 could not only coactivate the expression of cardiac genes, such as Cx40, Myh6 in early development of heart, but also facilitate the spontaneous contraction of CMs (Ghosh et al., 2009).

Mef2c

Mef2c belongs to Mef2 transcription factor family, which is involved in cardiac morphogenesis, cardiac myogenesis, and vascular development, but not in the generation of endothelial lineage cells (Leifer et al. 1993; Materna et al. 2019). As Mef2c is evolutionarily conserved and plays a prominent role in the regulation of muscle genes (Black and Olson, 1998), it is a core cardiac- and muscle-specific transcription factor in regulatory network of lineage reprogramming (Materna et al. 2019; Potthoff and Olson, 2007). Mef2c shares homology within a basic-helix-loop-helix (bHLH) motif intermediated myogenic regulators (Anderson et al., 2017). The E-box DNA consensus sequence in the control region of Mef2c gene could bind to myogenic bHLH proteins, which activated some muscle-specific transcriptions through recognizing a conserved A/T-rich element (Anderson et al., 2017; Edmondson et al., 1994; Gossett et al., 1989).

Knockout of Mef2c gene would result in embryonic lethality, defective cardiac looping morphogenesis, and vascular malformations in mouse (Materna et al., 2019). Mutation (b10086) of Mef2c delayed the expression of differentiated makers (such as Actc1, Myh6, and Myl2) in zebrafish (Danio rerio) CMs, but final cardiac development was normal (Hinits et al., 2012).

However, a novel heterozygous Mef2c mutation, p.Y157X, was detected in an index patient with adult-onset dilated cardiomyopathy (DCM), and this mutation abolished the synergistic transactivation between Mef2c and Gata4, as well as Hand1, which indicated that Mef2c acted as a new gene contributed to human DCM. (Yuan et al., 2018). Mef2c expressed in the cardiac Purkinje fibers of Gallus made the specialized CMs with electrical conductivity (Takebayashi-Suzuki et al., 2001). Mef2c is also expressed in the sinus venosus, which contributes to the generation of CMs in atria and postnatal development in mouse and rat (Pereira et al., 2009).

Mef2c plays a critical role in regulating cardiac morphogenesis during cardiac reprogramming. γ-secretase inhibitor IX N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a classical Notch inhibitor, could enhance the binding of Mef2c to the promoter regions of structural genes in heart (Abad et al., 2017). DAPT cooperated with protein kinase B (PKB) kinase could enhance the conversion of mouse fibroblasts into iCMs through increasing the transcriptional activity of Mef2c (Abad et al., 2017; Song et al., 2012).

Mef2c and Nkx2.5 share the similar genetic pathways for the specification and differentiation of ventricular myocytes. Double mutants of Mef2c and Nkx2.5 in mouse, resulted in developing a heart with a single cardiac chamber, expressed atrial markers, but second heart field lacked ventricular markers (Materna et al., 2019; Vincentz et al., 2008). These results indicated that physical, functional, and genetic interactions between Nkx2.5 and Mef2c were necessary for the formation of a ventricle.

Nkx2.5

Nkx2.5, also called CSX/VSD3/HLHS2, belongs to NK-2 class homeodomain transcription factor family, which plays a crucial part in the specification and differentiation of myocardium in vertebrates and invertebrates (Jia et al., 2017; Zakariyah et al., 2018). Expression of Nkx2.5 gene will be gradually increased during the differentiation of cardiac progenitor cells (CPCs) into CMs (Stennard et al., 2005; Yue et al., 2018).

Nkx2.5 contributes to regulating the expression of myocardin required for cardiomyogenesis and can be used to mark the angioblasts, which contribute to the hemogenic endothelium of endocardium and dorsal aorta (Ruan et al., 2016; Zamir et al., 2017). Nkx2.5 can directly bond to a cis-regulatory element of Etv2 gene and then induce the expression of it, which is essential for the formation of embryonic and extraembryonic endothelium, endocardium, and blood lineages (Tanaka et al., 1999; Yue et al., 2018). Nkx2.5 is expressed in early embryo, which can initiate the cardiogenic differentiation and persist in heart development (Armiñán et al., 2009; Stennard et al., 2005). So Nkx2.5 is usually considered as the marker of cardiac development.

Specific mutation of Nkx2.5 gene could dramatically suppress the development of looping morphogenesis, trabeculation and integral heart, affect the formation of endocardial cushion, and inhibit the expression of heart and neural crest derivatives expressed 2 (Hand2) in CMs (Anderson et al., 2016; Yamagishi et al., 2001). Knockout of Nkx2.5 would develop hemodynamic insufficiency in mouse, which resulted in abnormal heart morphogenesis and early embryonic death, but cardiac commitment was not compromised (Lyons et al., 1995). Thus, Nkx2.5 is essential for myogenic and morphogenetic differentiation of the mammalian heart. Tinman, an equivalent of Nkx2.5, regulated the expression of NetrinB in the cardioblasts of the Drosophila dorsal vessel and participated in the formation of patterning in mesoderm derived from CPCs (Asadzadeh et al., 2016; Bodmer, 1993).

Nkx2.5 plays an important role in the lineage reprogramming of somatic cells and differentiation of pluripotent cells toward cardiac cells. Ruan et al. (2016) found that overexpression of Nkx2.5 could enhance the differentiation of mesenchymal stem cells derived from umbilical cord in human into CM-like cells. Change of microenvironment in the process of diversification and differentiation induced sequentially, the expression of Mesp1/2, Isl1, and Nkx2.5, in cardiovascular progenitors (Laugwitz et al., 2008; Zamir et al., 2017). Interaction of Nkx2.5 with Mesp1, Tbx5, Gata4, and Baf60c had the ability to convert fibroblasts into iCPCs, which could further differentiate into CMs, SMCs, and ECs for cardiac repair (Lalit et al., 2016).

Hand2

Hand2 belongs to the bHLH transcription factor family, and it can encode protein Hand1 and Hand2, which work in a complementary way (McFadden et al., 2005; Li and Liu, 2017). Hand2 contains a bHLH DNA-binding domain and a dimerization motif, which acts as a transcription-activating domain (Dai and Cserjesi, 2002; Li and Liu, 2017), and it is an ancestral regulator of heart development, which also regulates the development of limb bud and branchial arch in embryo as well as mediates the antiproliferative effects of progesterone on uterine epithelia (Anderson et al., 2016; Li et al., 2011).

In the cardiac development, Hand2 expressed in the epicardium and valve progenitor of the outflow tract (OFT) and the atrioventricular canal (AVC) contributes to the generation of the myocardial compartment of right ventricle and aortic arteries (VanDusen and Firulli, 2012; VanDusen et al., 2014).

Deficiency of Hand2 inhibited the formation of cardiac cushion in AVC of mouse embryo during epithelial/mesenchymal transition (EMT) (Laurent et al., 2017). p.L47P, a novel Hand2 loss-of-function mutation, significantly decreased the synergistic activation between Hand2 and Gata4 or Nkx2.5, which resulted in tetralogy of Fallot (Lu et al., 2016). Targeted deletion of Hand2 in cardiac neural crest cells would impair the expression of cardiac genes, interaction between cells and cells, regulation of cell cycle, proliferative control, and development of OFT (Holler et al., 2010; Liu et al., 2016). Connexin 43 was associated with the control of cell cycle and migration of cardiac neural crest, its expression could be increased by Hand2 in the developing heart (Simon et al., 2004).

Hand2 is a multifunctional DNA-binding factor, which has the ability to collaborate with Gata4, Nkx2.5, or Mef2c to induce lineage reprogramming (Thattaliyath et al., 2002; Zang et al., 2004). Tian et al. (2018) found that GMT and Hand2 (GMTH) could cooperatively in vitro reprogram mouse fibroblasts into CMs expressing cardiac-specific markers, such as α-myosin heavy chain (α-MHC), β-MHC, ANF, NK2 homeobox 5, and cTnT, the conversion efficiency of this process being higher than that of GMT. Laurent et al. (2017) proposed that the physical interaction between bHLH domain of Hand2 and the C-terminal zinc finger domain of Gata4 had the capability to convert fibroblasts into iCMs.

Collaboration of Hand2, Gata4, and transcriptional coactivator p300 could form a higher enhancer element, which synergistically activated the expression of cardiac-specific genes, including Cx43, MYH6, and so on (Dai et al., 2002; Laurent et al., 2017).

Therefore, heart-specific heterogeneous transcription factors, such as Gata4, Tbx5, Mef2c, Nkx2.5, and Hand2 play the pivotal roles in cardiac reprogramming. Enforced expression of them is intricately arranged to activate certain lineage-specific master genes, which are sufficient to induce lineage conversion in heart, but the efficiency of lineage reprogramming and genetic safety need to be further improved. Therefore, there are great interests to identify other novel factors involved in lineage reprogramming, including miRNAs, epigenetic regulators, and small molecules.

MicroRNAs

miRNAs, small noncoding silencing RNAs, can promote the degradation of target messenger RNAs (mRNAs) and inhibit their translation, and they are critically important in the posttranscriptional regulation of genes (Sandmaier and Telugu, 2015; Schraivogel and Meister, 2014). Target mRNAs are regulated by miRNAs prevailing through binding to their 3′-untranslated region (Muraoka et al., 2014). A single miRNA can act on the expression of a number of genes simultaneously through multiple pathways, it mediated gene regulation from common downregulation to mRNA-specific upregulation, the mRNA expression could be activated by the direct action of microribonucleoproteins (miRNPs), and/or could be indirectly relieved from miRNA-mediated repression by abrogating the action of repressive miRNPs. (Friedman et al., 2008; Valinezhad-Orang et al., 2014).

miRNA is small which makes it to easily access into cells through lipofection (Elmen et al., 2008), and allows the packing of multiple transcripts in the same delivery vector, increasing reprogramming efficiency, and functional homogeneity of reprogrammed cells (Bader et al., 2011;Sandmaier and Telugu, 2015). Therefore, miRNAs will be acted as a novel and potential strategy to efficiently achieve the regeneration of iCMs in vitro and in vivo.

Important roles of miRNAs in cardiac development

Most miRNAs are evolutionarily conservative, they have been recognized as a part of the regulatory networks on cardiac development (Fromm et al., 2015; Bartel, 2018). miRNA-1 and miRNA-133 have identical primary sequences and similar expression patterns of gene clusters specifically expressed in cardiac and skeletal muscle, they are collaboratively transcribed by a way of compensatory effects and share the commonality of functions during heart development (Chen et al., 2006; Rao et al., 2006; Zhao et al., 2005).

Overexpression of miRNA-1 reduced the proliferation of CMs and caused the atrioventricular block through directionally participating in the circulation of calcium channel, and Hand2, as a direct target of miRNA-1 may mediate these effects (Zhang et al., 2013; Zhao et al., 2005). Overexpression of miRNA-133 inhibited hypertrophic myocardial growth (Li et al., 2010), and knockdown of this miRNA-133 promoted hypertrophy in the adult heart (Carè et al., 2007).

miRNA-1 is distinguished from miRNA-1-1 and miRNA-1-2; deletion of miRNA-1-2 would cause incompletely penetrant developmental and electrophysiological phenotypes during cardiac development in mouse (Zhao et al., 2007). Cooperation of miRNA-1/miRNA-133a clusters could specify the cardiomyogenic lineage during the development of embryonic heart through adjusting the levels of myocardin, which plays an important role in the survival of CMs and maintenance of cardiac functions after birth (Hoofnagle et al., 2011; Wystub et al., 2013).

Other miRNAs, such as miRNA-208 and miRNA-499, are relevant to the contraction and proliferation of CMs. miRNA-208 regulated the expression of slow MHC in rat heart with a decrease of β-MHC and an increase of α-MHC in gene expression and protein content (Soci et al., 2013). Deletion of miRNA-208 presented a progressive decrease in cardiac contractility in mice at the age of 2 months (Rooij et al., 2007), and overexpression of miRNA-208 induced arrhythmia in the electrical conduction system of the heart (Callis et al., 2009). Deletion of miRNA-499 had no obvious effects on cardiac development, but its overexpression would result in hypertrophy of the heart (Shieh et al., 2011).

miRNA combo

Jayawardena et al. (2012) first proved that miRNA combo, including miRNA-1, miRNA-133, miRNA-208, and miRNA-499, could mediate the direct reprogramming of CFs into CM-like cells in vitro and in vivo. Those generated cells not only expressed CM markers, but also exhibited sarcomeric organization and spontaneous calcium flux in vitro (Jayawardena et al., 2012).

This process of reprogramming did not need continual upregulation of miRNAs, it may occur on the first days after introduction of miRNAs. Actually, only miRNA-1 was sufficient to induce cardiac reprogramming, but the efficiency of reprogramming and the maturation of converted cells were dramatically enhanced in combination with miRNA-133, miRNA-208, and miRNA-499 (Tilanthi et al., 2012). Janus kinases (JAK) inhibitor I could enhance 10-fold efficiency of this reprogramming by suppressing the pluripotency-promoting pathways of embryonic fibroblasts and promoting the transdifferentiation of them into iCMs (Efe et al., 2011).

Transfection of different miRNA combos (including miRNA 1, miRNA 133a, and miRNA 206; miRNA 1, miRNA 133a, and miRNA 208; miRNA 1, miRNA 206, and miRNA 208; miRNA 1, miRNA 133a, miRNA 208, and miRNA 499-5p; miRNA 1, miRNA 133a, miRNA 206, and miRNA 499-5p) into ischemic myocardia caused by MI could respectively reprogram fibrotic tissue into cardiomyocytic tissue, which presented the increased expression of the CM marker proteins, such as cardiac troponin, sarcomeric actinin, L-type calcium channel, and Troponin T2 (Dzau et al., 2018). Therefore, directly reprogramming of somatic cells into iCMs with miRNAs supplies an efficient and original approach to regenerate CMs for recovering the functions of the impaired heart.

miRNAs cooperated with transcription factors

miRNAs have the ability to enhance cardiac reprogramming through combining with transcription factors. miRNA-1 and miRNA-133 coupled with Gata4, Hand2, Tbx5 (GHT), and myocardin could convert human fibroblasts into iCMs, which expressed cardiac markers, sarcomeric-like structures, calcium transients, and spontaneous contractility (Nam et al., 2013). Although GHT and myocardin were enough to achieve this lineage conversion, the number of cTnT-positive cells would be increased through the promoting effects of miRNA-1 and miRNA-133 (Nam et al., 2013); those two miRNA and myocardin could form a negative feedback loop, which acted as a rheostat to regulate the activation of them, which was essential for the development of early heart (Wystub et al., 2013).

Combination of miRNA-133 with GMT could promote cardiac reprogramming by silencing signatures of fibroblast through suppression of EMT regulator Snai1 (Muraoka et al., 2014). Compared with the only use of GMT, overexpression of miRNA-133 could enhance reprogramming mediated by them and result in generating sevenfolds of beating iCMs from murine embryonic fibroblasts and shortening the duration of reprogramming (Muraoka et al., 2014).

Molecular mechanisms of cardiac reprogramming mediated by miRNAs

miRNAs can regulate and control the reprogramming of fibroblasts into iCMs, however, the molecular mechanisms on this strategy need to be further discovered. Overexpression of miRNAs that existed in cardiac lineage may downregulate master regulators expressed in fibroblasts or the transitional cells, which may result in disrupting the balance among master regulators of different lineages (Xu et al., 2015; Wang et al., 2017), for example, miRNA-133, may suppress the expression of master genes in fibroblasts through directly repressing Snai1 and upregulate the expression of master genes in CMs, promoting cardiac reprogramming (Muraoka et al., 2014).

Another possible mechanism is that miRNAs may downregulate the expression of some epigenetic regulators in CMs and promote the globally epigenetic changes to a certain context in vitro or in vivo (Morales et al., 2017; Xu et al., 2014), for example, miRNA combo, including miRNA-1, miRNA-133, miRNA-208, and miRNA-409 downregulated the expression of histone lysine N-methyltransferase Setdb2, which could alleviate the suppression of cardiac genes in fibroblasts (Vierbuchen and Werng, 2012). These possibilities still need further verification through more experimental evidences to deepen the broader understanding of lineage reprogramming.

Epigenetic Regulators

Lineage reprogramming refers to extensive epigenetic remodeling in different types of cells, exogenous factors such as transcription factors, small molecules could cooperate with or independent of epigenetic regulators to properly reactivate the epigenetic expression of the targeted cellular master genes.

Chromatin remodeling

Takeuchi and Bruneau (2009) showed that combination of enforced expression of Gata4 and Tbx5 with ATP-dependent BRG1/BRM-associated factor (BAF) chromatin remodeling complexes, such as Baf60c, could transdifferentiate noncardiac mesoderm cells of mouse embryos into iCMs. Baf60c may permit the binding of Gata4 to cardiac genes, which is essential for the conversion of different epigenetic states.

The nucleosome remodeling deacetylase (NuRD) complex, a chromatin regulatory complex that functions as a transcriptional corepressor in metazoans, was associated with the zinc finger transcription factor 281 (ZNF281) to potently stimulate the cardiac reprogramming by genome-wide association with Gata4 on cardiac enhancers (Ee et al., 2017; Zhou et al., 2017a). Therefore, chromatin modifiers can facilitate the lineage conversion through the coordination with cardiac transcription factors.

However, epigenetic barriers mediated by chromatin modifiers will hinder lineage reprogramming. The polycomb ring finger oncogene Bmi1 is a key epigenetic barrier during the early phase of iCM generation (Lessard and Sauvageau, 2003; Zhou et al., 2016).

Zhou et al. (2016) showed that reducing the level of the Bmi1 could significantly enhance the induction of beating iCMs from neonatal and adult mouse fibroblasts, and deletion of Bmi1 could eliminate the repression to endogenous Gata4 and substitute for exogenous Gata4 during this process. Knockdown of Bmi1 would increase the level of H3K4me3, reduce the level of repressive H2AK199 at cardiogenic loci, and derepress the expression of cardiogenic genes during reprogramming (Zhou et al., 2016). H3K4me3 was involved in the transcriptive activation of nearby genes, and monoubiquitination of H2AK119 was associated with gene repression (Sims et al., 2003; Wang et al., 2004).

Therefore, removing certain epigenetic barriers is adequate to efficiently generate functional iCMs when it cooperates with fewer transcription factors. Further studies should explore whether epigenetic regulators can substitute for transcription factors to induce cardiac lineage conversion in vitro and in vivo and the underlying mechanisms of activating the core gene regulatory network.

Histone-modifying enzymes

Histone-modifying enzymes could affect cardiac lineage conversion, which further emphasizes the significance of epigenetic regulators in this process. The H3K4 methyltransferase SETD7 controlled cardiac differentiation through reading H3K36 marks independently of its enzymatic activity, which associated with different cofactors, such as Nkx2.5, at stage-specific expression during the differentiation of CMs (Lee et al., 2018). Liu et al. (2016) showed that targeting Mll1-dependent H3K4 methyltransferase activity could guide cardiac lineage-specific reprogramming of fibroblasts. Inhibition of H3K4 methyltransferase with MM408 and MI503 significantly decreased the formation of adipocytes during iCM induction, which likely increased efficiency of iCMs by suppressing alternative lineage gene expression.

Histone deacetylase 1 (HDAC1) is a mediator for the commitment of cardiac mesenchymal cell (CMC) into cardiomyogenic lineage and paracrine signaling potency in vitro. Compared with naive CMCs, the benzamide class 1 isoform selective HDAC inhibitor entinostat (MS-275)-treated CMCs exhibited heightened capacity for myocyte-like differentiation in vitro and more efficient inhibition of myocardial fibrosis and greater increase in myocyte size in vivo, which suggested that HDAC inhibition enhanced the cardiac reparative capacity of CMCs likely through a paracrine mechanism that improved ventricular compliance and systolic function and augmented the growth and functions of myocytes (Moore et al., 2019).

Therefore, the histone-modifying enzymes may have an important effect on the process of lineage conversion because histone modifications are related to transcriptively active or silent chromatin states (Xu et al., 2015).

Small Molecules

Small molecules are focused on the induction of lineage reprogramming in recent years. Compared with transcription factors and miRNAs, small molecules have many advantages in lineage conversion, they are soluble, which results in being permeated into cells, more easily synthesized and preserved, less costly but effective, and more accessible to be adjusted in temporality and spatiality (Zhu et al., 2015); they will become a promising strategy avoiding the potential safety concerns of genetic manipulation (Cao et al., 2016). Both signal pathways and epigenetic modifications can be regulated by small molecules when they are involved in the conversion of different cellular types (Xu et al., 2015).

Functions of small molecules in reprogramming of cardiac lineage

Small molecules are widely used in the lineage reprogramming; they can promote the transition of somatic cells toward cardiogenic cells. Lalit et al. (2016) converted mouse cardiac, lung, and tail tip fibroblasts into iCPCs by overexpression of Mesp1, Tbx5, Gata4, Nkx2.5, and Baf60c, and generated cells could further differentiate into cardiac cells. Supplement of LIF (leukemia inhibitory factor, an activator for Wnt signaling pathway) and BIO (an activator for JAK/STAT signaling pathway) enabled iCPCs to robustly expand.

Meanwhile, inhibition of some signaling pathways also takes part in the reprogramming of cardiac lineage. Abad et al. (2017) found that inhibition of Notch signaling pathway by DAPT could promote the generation of iCMs and their cardiac-specific functions through increasing the transcriptional activity of Mef2c and strengthening the effective combination of Mef2c with cardiogenic promoter. The acceleration of signaling pathway also occurs on the direct cardiac reprogramming mediated by miRNAs, the induced efficiency could be increased 10-fold through supplement of JAK inhibitor I (Tilanthi et al., 2012).

The chemically defined reprogramming medium comprised a base tissue culture medium and insulin/transferrin/selenium (ITS) or ascorbic acid could increase the expression of cardiac genes and proteins in CFs and other fibroblasts, such as dermal fibroblasts and enhance the miRNA combo-mediated cardiac reprogramming of fibroblasts into iCMs (Wang et al., 2018).

Substitution of small molecules for exogenous transcription factors

The significance of small molecules in cardiac lineage reprogramming is further verified that they can replace certain transcription factors, for example, Wang et al. (2014) and Zhu et al. (2015) both reported that interaction between small-molecule cocktail SCPF (SB431542, CHIR99021, parnate, and forskolin) and only overexpression of Oct4 followed by treatment of bone morphogenetic protein 4 (BMP4) successfully converted mouse fibroblasts into functional iCMs, those molecules could substitute for exogenous transcription factors Sox2, Klf4, and c-Myc for the generation of intermediate cells.

It is encouraging that the small-molecule combination CRFVPTZ (CHIR99021, RepSox, forskolin, VPA, Parnate, TTNPB, and DZnep) and cardiac reprogramming medium (CRM) could generate automatically beating CM-like cells from mouse fibroblasts without actions of exogenous transcription factors (Cao et al., 2016; Fu et al., 2015).

Cao et al. (2016) identified the combination of small molecules 9C (CHIR99021, A83-01, BIX01294, AS8351, SC1, Y27632, OAC2, SU16F, and JNJ10198409) and growth factors enabled to reprogram human fibroblasts into iCMs that uniformly displayed contractile properties, which could be effectively adjusted through changing the concentration and composition of small molecules. The combination of 9C and sodium butyrate and SMER28 (NS) would enhance the duration of spontaneous beating of iCMs and the expression of myocardial markers, including cTNT, Cav3.2, and Kir 2.1 (Nan and Sheng, 2018).

In surprise, the pluripotent Yamanaka's factors without c-Myc (Oct4, Sox2, and Klf4) could be used to generate iCMs in a CM-favorable culture medium containing cardioinductive growth factor BMP4 and small-molecule JAK inhibitor, which could induce spontaneous contraction of iCMs (Efe et al., 2011). It was speculated that the beneficial culture condition may be a key factor to acquire specific cell lineage. Therefore, these researches offer a possibility that small molecules will reduce or abolish the requirement of exogenous transcription factors during cardiac lineage reprogramming, but complete replacement of them with small molecules needs to be further explored.

Mechanisms of Reprogramming in Cardiac Lineage

To investigate the mechanisms on the reprogramming of cardiac lineage induced by exogenous factors, we need to know how the regulatory network of the target cell is reactivated in reprogramming cells during conversion. Wapinski et al. (2013) proposed a hierarchical mechanism, which was responsible for the neuron conversion mediated with Brn2, Ascl1, and Myt1l (BAM). Effects of Gata4 on cardiac transition were similar to that of Ascl1, which acted as an “on-target pioneer factor” of neuron conversion. Gata4 could open chromatin structure of cardiac loci and integrate other transcription factors into their specific target sites, which activated the cellular conversion (Bono et al., 2018; Cirillo et al., 2002).

Meanwhile, the efficiency and functionality of lineage reprogramming may be influenced by stoichiometry of factors, higher levels of exogenous Mef2c, and lower levels of Gata4 and Tbx5 could significantly enhance the efficiency of conversion (Wang et al., 2015).

There is another speculation of “seesaw model” that exogenous factors are used to counteract lineage specifiers and break the epigenetic balances in the initial cells, a deviation from the balanced equilibrium for pluripotency directs cells to flow into divergent differentiated states (Shu et al., 2015). It may be verified that miRNA-133 directly repressed Snail, which resulted in the silence of fibroblastic signatures and the promotion of cardiac reprogramming (Muraoka et al., 2014), so the balance between different lineage specifiers plays a very important role in the converted direction of cell fate. It still needs more evidences to further verify the clear mechanisms on the molecular level.

Strategies for Improving the Functional Maturation of Converted Cells

Requirement of maturation for iCMs in regenerative medicine

With the development of reprogramming in the cardiac lineage, iCMs have been concerned to be applied in many fields of regenerative medicine, such as disease modeling, drug discovery, and particularly cell therapy. But these iCMs more closely resemble fetal cells than adult CMs, they exhibit mostly immature characteristics in structure, contractile performance, electrophysiology, and metabolism (Williams et al., 2015).

Chanda et al. (2014) found that the converted cells generated through lineage reprogramming failed to silence the expressive programs of the initial population, and they possessed an immature phenotype. When iCMs are used to build disease models or make drug testing, effects on iCMs will not be accurately predicted for their immature characteristics, because cardiovascular diseases usually occur in adults (Otsuji et al., 2010). Furthermore, in recent years, many studies attested that mature CMs were more suitable for strict researches and applications of regenerative medicine for their special features such as higher generation of contractile force, appropriate calcium-handling properties, and stable signal of electrophysiology (Dhahri et al., 2018; Yang et al., 2014a). Therefore, it is necessary to adopt strategies to promote the maturation of iCMs.

Extracellular matrix

Due to the important roles of mature cardiac cells in clinical applications, promising approaches have been explored to improve the maturation of iCMs (Gao et al., 2014). Kong et al. (2018) and Przybyt et al. (2015) indicated that extracellular matrix (ECM) influenced cellular behavior, such as spontaneous contraction and maturation, even modulated cardiac reprogramming efficiency. The development and maturation of CMs in rat (French et al., 2012) and human (Otsuji et al., 2010) would be promoted, being cultured with porcine ECM in two-dimensional (2D) culture system. Damaged areas of MI could be effectively repaired after mature iCMs were injected into those areas (Abadi et al., 2018; Singelyn et al., 2009).

Compared with 2D culture system, 3D microenvironment and architecture provided by cardiac ECM would likely support site-appropriate cell differentiation and spatial organization (Fong et al., 2016). Combination of cardiac ECM and a cardiac inductive cocktail consisted of oxytocin, ascorbic acid, α-tocopherol (Vitamin E), and β-mercaptoethanol could improve the differentiation of human adipose tissue-derived stem cells into CMs, which highly expressed connexin43 (Baghalishahi et al., 2018).

Effects of different ECM derived from fetal heart, neonatal heart, and adult heart on CMs were compared, and results indicated that CMs had better adhesion and expansion on fetal ECM (Williams et al., 2014). But ECM generated from adult heart in 3D culture system could promote the expression of maturation genes in CMs, regulation of calcium signaling, and response of iCMs derived from human pluripotent stem cells (hPSC-iCMs) to drugs (Fong et al., 2016).

High ECM-contenting 3D hydrogels (75% ECM, 25% collagen, no supplemental soluble growth factors) promoted maturation of iCMs derived from human ESCs (hESC-iCMs), which was attested by the striation patterns in cardiac troponin I and the upregulation of Cx43 gene (Duan et al., 2011). ECM-rigid substrates facilitated cardiac differentiation and collagen-soft substrates promoted the beating behavior of iCMs (Williams et al., 2015). Therefore, tunable property of cardiac ECM in 3D hybrid scaffolds is crucial for cardiovascular tissue engineering.

Long-term culture

One basic approach for improving the structural and functional maturation of iCMs is long-term culture. Compared with early stage counterparts (CMs which are derived from hPSC-iCMs after being cultured and differentiated in vitro for 20–40 days), late-stage counterparts (being cultured for 80–120 days) showed increased cell size and anisotropy, greater myofibril density and alignment, a 10-fold increase in the fraction of multinucleated CMs, better contractile performance, calcium handling properties and electrophysiology, and a robust induction of the key markers for cardiac structure (Lundy et al., 2013). These findings suggest that iCMs are capable of slowly maturing into cells, which have the phenotype more closely resembling adult CMs, but this process is time consuming.

Electric stimulation

Compared with long-term culture, electric stimulation for the maturation of iCMs is faster. Electrical signals could constantly stimulate CMs and promote synchronous contractions of CMs in vivo. Increase of sodium/calcium exchanger and action potential duration, enhanced conduction velocity, mitochondrial content, and activity were shown in neonatal rat ventricular myocyte (NRVM) monolayers after being electrically stimulated (Sathaye et al., 2006). Martherus et al. (2010) found that transcriptome of rat CMs was impacted following electrical stimulation, which displayed the upregulation of cardiac-specific genes, such as Myh6 and Cx43, through a genome-wide assessment. But electrical stimulation is not suitable for large-scale studies, as excessive electrical impulse will cause cell death.

Biochemical cues

Biochemical cues may be a strategy with less damage for maturation of iCMs. Tri-iodo-l-thyronine (T3) could promote the maturation of hPSC-iCMs, those matured cells had a larger cell size, longer sarcomere length, lower proliferative activity, higher contractile force generation, enhanced calcium-handling properties, and increased maximal mitochondrial respiration capacity (Yang et al., 2014b). Similarly, norepinephrine had the ability to enhance the mature level of NRVM and hESC-iCMs (Deng et al., 2000). It is interesting that hESC-iCMs displayed a decrease of action potential duration and hyperpolarization of both resting membrane potential and maximum diastolic potential after overexpression of miRNA-1 (Fu et al., 2012). Therefore, miRNA-1 may be regarded as a potential modulator for the maturation of iCMs.

Therefore, with the rapid development of lineage reprogramming, reprogrammed somatic cells has been one of the major ways to acquire desired cells. The feasibility of utilizing the combination of transcription factors to induce cardiac reprogramming had been testified by a number of researches (Table 1). Understanding the structure and functions of transcription factors in cardiac development is needful to analyze the mechanisms on the process of lineage reprogramming.

Table 1.

Summary of Factors for Reprogramming Fibroblasts into Induced Cardiomyocytes or Induced Cardiac Progenitor Cells Since 2010

Cardiac-specific lineage factors	Reprogramming factors	Target cell types	In vitro or in vivo	Species	References
Cardiac-specific lineage transcription factors	Gata4, Mef2c, and Tbx5	iCMs	In vitro	Mouse	Chen et al. (2012); Ieda et al. (2010); Inagawa et al. (2012); Vaseghi et al. (2016); Wang et al. (2015)
	Gata4, Mef2c, and Tbx5	iCMs	in vivo	Mouse	Chang et al. (2019); Qian et al. (2012)
	Gata4, Mef2c, and Tbx5	iCMs	Both	Mouse, human	Miyamoto et al. (2018)
	Tbx5, Mef2c, and Myocd	iCMs	In vitro	Mouse	Protze et al. (2012)
	Gata4, Mef2c, Tbx5, and Hand2	iCMs	Both	Mouse	Song et al. (2012)
	Gata4, Mef2c, Tbx5, and Hand2	iCMs	In vitro	Mouse	Hirai et al. (2013); Tian et al. (2018)
	Gata4, Mef2c, Tbx5, Nkx2.5, and Hand2	iCMs	In vitro	Mouse	Addis et al. (2013); Ifkovits et al. (2014)
	Gata4, Mef2c, Tbx5, Myocd, SRF, Mesp1, and SMARCD3	iCMs	In vitro	Mouse	Christoforou et al. (2013)
	Gata4, Mef2c, Tbx5, ESRRG, Mesp1, Myocardin, and ZFPM2	iCMs	Both	Mouse	Fu et al. (2013)
	ETS2 and Mesp1	iCPCs	In vitro	Human	Islas et al. (2012)
	Gata4, Tbx5, Nkx2.5, Mesp1, and Baf60c	iCPCs	Both	Mouse	Lalit et al. (2016)
miRNAs and/or no transcription factors	miRNA-1, miRNA-133, miRNA-208, and miRNA-499	iCMs	Both	Mouse	Jayawardena et al. (2012)
	miRNA-1, miRNA-133, Gata4, Hand2, Myocd, and Tbx5	iCMs	In vitro	Human	Nam et al. (2013)
	miRNA-133, Gata4, Mef2c, and Tbx5	iCMs	In vitro	Mouse, human	Muraoka et al. (2014)
	miRNA-1, miRNA-133a, and miRNA-208	iCMs	In vitro	Mouse	Dzau et al. (2018)
	miRNA-1, miRNA-206, and miRNA-208
	miRNA-1, miRNA-133a, miRNA-208, and miRNA-499-5p
	miRNA-1, miRNA-133a, miRNA-206, and miRNA-499-5p
Small molecules and/or no transcription factors	Small-molecule combination SCPF and Oct4	iCMs	In vitro	Mouse	Zhu et al. (2015)
	Small-molecule combination CRFVPTZ	iCMs	In vitro	Mouse	Fu et al. (2015)
	Small-molecule combination 9C	iCMs	In vitro	Human	Cao et al. (2016)
	Small-molecule combination 9C and NS	iCMs	In vitro	Human	Nan and Sheng (2018)

9C: CHIR99021, A83-01, BIX01294, AS8351, SC1, Y27632, OAC2, SU16F, and JNJ10198409; CRFVPTZ: CHIR99021 (C), RepSox (R), forskolin (F), valproic acid (V, VPA), Parnate (P), TTNPB (T), and DZnep (Z); NS: sodium butyrate and SMER28; SCPF: SB431542 (S), CHIR99021 (C), parnate (P), and forskolin (F).

iCMs, induced cardiomyocytes; iCPCs, induced cardiac progenitor cells; miRNAs, microRNAs.

Now, there is great interest in the identification of alternative factors to induce cardiac lineage conversion, including miRNAs, epigenetic regulators, and small molecules. Modified nanoparticles and miRNAs may be acted as novel, safer, and efficient delivery systems to facilitate lineage reprogramming (Zhou et al., 2018). Small molecules may be a compatible selection for generating iCMs. The maturation of converted cells is a slow and inefficient process to acquire functional heterogeneity, which is required to optimize protocols through more explorations.

Finally, understanding of the molecular mechanisms on lineage reprogramming is a challenge, which can be tractable with continuous investigations. We are anticipating that safe and matured functional cardiac cells can be ultimately generated for regenerative medicine, drug screening, and so on.

Footnotes

Acknowledgments

The authors thank the National Natural Science Foundation of China (Grant No. 31572488), the Based and Advanced Research Projects of Chongqing (Grant No. cstc2017jcyjAX0477) for the support to this work.

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

References

Abad

, Hashimoto

, Zhou

, Morales

M.G.

, Chen

, Bassel-Duby

, and Olson

E.N.

(2017). Notch inhibition enhances cardiac reprogramming by increasing MEF2C transcriptional activity. Stem Cell Reports, 8, 548–560.

Abadi

P.S.

, Garbern

J.C.

, Behzadi

, Hill

M.J.

, Tresback

J.S.

, Heydari

, Ejtehadi

M.R.

, Ahmed

, Copley

, Aghaverdi

, Lee

R.T.

, Farokhzad

O.C.

, and Mahmoudi

(2018). Engineering of mature human induced pluripotent stem cell-derived cardiomyocytes using substrates with multiscale topography. Adv. Funct. Mater. 28, 1707378.

Addis

R.C.

, Ifkovits

J.L.

, Pinto

, Kellam

L.D.

, Esteso

, Rentschler

, Christoforou

, Epstein

J.A.

, and Gearhart

J.D.

(2013). Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success. Mol. Cell. Cardiol. 60, 97–106.

Anderson

C.M.

, Hu

, Thomas

, Gainous

T.B.

, Celona

, Sinha

, Dickel

D.E.

, Heidt

A.B.

, Xu

S.M.

, Bruneau

B.G.

, Pollard

K.S.

, Pennacchio

L.A.

, and Black

B.L.

(2017). Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites. Development, 144, 1235.

Anderson

K.M.

, Anderson

D.M.

, McAnally

J.R.

, Shelton

J.M.

, Bassel-Duby

, and Olson

E.N.

(2016). Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development. Nature, 539, 433.

Ang

Y.S.

, Rivas

R.N.

, Ribeiro

A.J.S.

, Srivas

, Rivera

, Stone

N.R.

, Pratt

, Mohamed

T.M.A.

, Fu

J.D.

, Tippens

N.D.

, Li

, Narasimha

, Radzinsky

, Moon-Grady

A.J.

, Yu

, Pruitt

B.L.

, Snyder

M.P.

, and Srivastava

(2016). Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis. Cell, 167, 1734–1749.

Armiñán

, Gandía

, Bartual

, García-Verdugo

J.M.

, Lledó

, Mirabet

, Llop

, Barea

, Montero

J.A.

, and Sepúlveda

(2009). Cardiac differentiation is driven by NKX2.5 and GATA4 nuclear translocation in tissue-specific mesenchymal stem cells. Stem Cells Dev. 18, 907–908.

Asadzadeh

, Neligan

, Kramer

S.G.

, and Labrador

J.P.

(2016). Tinman regulates NetrinB in the cardioblasts of the drosophila dorsal vessel. PloS One 11, e0148526.

Bader

A.G.

, Brown

, Stoudemire

, and Lammers

(2011). Developing therapeutic microRNAs for cancer. Gene Ther. 18, 1121–1126.

10.

Bartel

D.P.

(2018). Metazoan microRNAs. Cell, 173, 20–51.

11.

Baghalishahi

, Efthekhar-Vaghefi

S.H.

, Piryaei

, Nematolahi-Mahanim

S.N.

, Mollaei

H.R.

, and Sadeghi

(2018). Cardiac extracellular matrix hydrogel together with or without inducer cocktail improves human adipose tissue-derived stem cells differentiation into cardiomyocyte-like cell. Biochem. Biophys. Res. Commun. 502, 215–225.

12.

Black

B.L.

, and Olson

E.N.

(1998). Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14, 167–196.

13.

Bodmer

(1993). The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development, 116, 719–729.

14.

Bono

C.D.

, Magli

T.R.

, and Robert

G.K.

(2018). Cardiac fields and myocardial cell lineages. In The ESC Textbook of Cardiovascular Development. J.M. Pérez-Pomares and R. Kelly, eds. (Oxford University Press, Oxford, UK) pp. 23–27.

15.

Boogerd

C.J.

, and Evans

(2016). TBX5 and NuRD divide the heart. Dev. Cell, 36, 242–244.

16.

Callis

T.E.

, Pandya

, Seok

H.Y.

, Tang

R.H.

, Tatsuguchi

, Huang

Z.P.

, Chen

J.F.

, Deng

, Gunn

, Shumate

, Willis

M.S.

, Selzman

C.H.

, and Wang

D.Z.

(2009). MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice. J. Clin. Invest. 119, 2772–2786.

17.

Cao

, Huang

, Zheng

, Spencer

C.I.

, Zhang

, Fu

J.D.

, Nie

, Xie

, Zhang

, Wang

, Ma

, Xu

, Shi

, Srivastava

, and Ding

(2016). Conversion of human fibroblasts into functional cardiomyocytes by small molecules. Science, 352, 1216–1220.

18.

Carè

, Catalucci

, Felicetti

, Bonci

, Addario

, Gallo

, Bang

M.L.

, Segnalini

, Gu

, Dalton

N.D.

, Elia

, Latronico

M.V.

, Hoydal

, Autore

, Russo

M.A.

, Dorn

G.W.

, Ellingsen

, Ruiz-Lozano

, Peterson

K.L.

, Croce

C.M.

, Peschle

, and Condorelli

(2007). MicroRNA-133 controls cardiac hypertrophy. Nat. Med. 13, 613–618.

19.

Chanda

, Ang

C.E.

, Davila

, Pak

, Mall

, Lee

Q.Y.

, Ahlenius

, Jung

S.W.

, Südhof

T.C.

, and Marius

(2014). Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem Cell Reports, 3, 282–296.

20.

Chang

Y.J.

, Lee

, Kim

, Kwon

Y.W.

, Kwon

, and Kim

(2019). Efficient in vivo direct conversion of fibroblasts into cardiomyocytes using a nanoparticle-based gene carrier. Biomaterials, 192, 500–509.

21.

Chen

J.F.

, Mandel

E.M.

, Thomson

J.M.

, Wu

, Callis

T.E.

, Hammond

S.M.

, Conlon

F.L.

, and Wang

D.Z.

(2006). The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat. Genet. 38, 228–233.

22.

Chen

J.X.

, Krane

, Deutsch

M.A.

, Wang

, Rav-Acha

, Gregoire

, Engels

M.C.

, Rajarajan

, Karra

, Abel

E.D.

, Wu

J.C.

, Milan

, and Wu

S.M.

(2012). Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5. Circ. Res. 111, 50–55.

23.

Christoforou

, Chellappan

, Adler

A.F.

, Kirkton

R.D.

, Wu

, Addis

R.C.

, Bursac

, and Leong

K.W.

(2013). Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming. PloS One 8, e63577.

24.

Cirillo

L.A.

, Lin

F.R.

, Cuesta

, Friedman

, Jarnik

, and Zaret

K.S.

(2002). Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol. Cell, 9, 279–289.

25.

Dai

Y.S.

, and Cserjesi

(2002). The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer. J. Biol. Chem. 277, 12604–12612.

26.

Dai

Y.S.

, Cserjes

, Markham

B.E.

, and Molkentin

J.D.

(2002). The transcription factors GATA4 and dHAND physically interact to synergistically activate cardiac gene expression through a p300-dependent mechanism. J. Biol. Chem. 277, 24390–24398.

27.

Deng

X.F.

, Rokosh

D.G.

, and Simpson

P.C.

(2000). Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes. Circ. Res. 87, 781–788.

28.

Dhahri

, Romagnuolo

, and Laflamme

M.A.

(2018). Training heart tissue to mature. Nat. Biomed. Eng. 2, 351–352.

29.

Duan

, Liu

, O'Neill

, Wan

L.Q.

, Freytes

D.O.

, and Vunjak-Novakovic

(2011). Hybrid gel composed of native heart matrix and collagen induces cardiac differentiation of human embryonic stem cells without supplemental growth factors. J. Cardiovasc. Transl. Res. 4, 605–615.

30.

Dzau

V.J.

, Maria

, and Tilanthi

(2018). Direct reprogramming of cells to cardiacmyocyte fate. UC Ptents. US 2018/0344776. A1.

31.

Edmondson

D.G.

, Lyons

G.E.

, Martin

J.F.

, and Olson

E.N.

(1994). Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development, 120, 1251–1263.

32.

L.S.

, McCannell

K.N.

, Tang

, Fernandes

, Hardy

R.W.

, Green

M.R.

, Chu

, and Fazzio

T.G.

(2017). An embryonic stem cell-specific NuRD complex functions through interaction with WDR5. Cell, 8, 1488–1496.

33.

Efe

J.A.

, Hilcove

, Kim

, Zhou

, Ouyang

, Wang

, Chen

, and Ding

(2011). Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat. Cell Biol. 13, 215–222.

34.

Elmen

, Lindow

, Schutz

, Lawrence

, Petri

, Obad

, Lindholm

, Hedtjarn

, Hansen

H.F.

, Berger

, Gullans

, Kearney

, Sarnow

, Straarup

E.M.

, and Kauppinen

(2008). Lna-mediated microRNA silencing in non-human primates. Nature, 452, 896–899.

35.

Evans

, and Felsenfeld

(1989). The erythroid-specific transcription factor Eryf1: a new finger protein. Cell, 58, 877–885.

36.

Fong

A.H.

, Romero-López

, Heylman

C.M.

, Keating

, Tran

, Sobrino

, Tran

A.Q.

, Pham

H.H.

, Fimbres

, Gershon

P.D.

, Botvinick

E.L.

, George

S.C.

, and Hughes

C.C.

(2016). Three-dimensional adult cardiac extracellular matrix promotes maturation of human induced pluripotent stem cell-derived cardiomyocytes. Tissue Eng. Part A, 22, 1016–1025.

37.

French

K.M.

, Boopathy

A.V.

, DeQuach

J.A.

, Chingozha

, Lu

, Christman

K.L.

, and Davis

M.E.

(2012). A naturally derived cardiac extracellular matrix enhances cardiac progenitor cell behavior in vitro. Acta Biomater. 8, 4357–4364.

38.

Friedman

R.C.

, Farh

K.K.

, Burge

C.B.

, and Bartel

D.P.

(2008). Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105.

39.

Fromm

, Billipp

, Peck

L.E.

, Johansen

, Tarver

J.E.

, King

B.L.

, Newcomb

J.M.

, Sempere

L.F.

, Flatmark

, Hovig

, and Peterson

K.J.

(2015). A uniform system for the annotation of vertebrate microRNA genes and the evolution of the human microRNAome. Annu. Rev. Genet. 49, 213–242.

40.

J.D.

, Rushing

S.N.

, Lieu

D.K.

, Chan

C.W.

, Kong

C.W.

, Geng

, Wilson

K.D.

, Chiamvimonvat

, Boheler

K.R.

, and Wu

J.C.

(2012). Circular specification and functional maturation of human embryonic stem cell-derived cardiomyocytes. PloS One 6, e27417.

41.

J.D.

, Stone

N.R.

, Liu

, Spencer

C.I.

, Qian

, Hayashi

, DelgadoOlguin

, Ding

, Bruneau

B.G.

, and Srivastava

(2013). Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state. Stem Cell Reports, 1, 235–247.

42.

, Huang

, Xu

, Gu

, Ye

, Jiang

, Qiu

, and Xie

(2015). Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails. Cell Res. 25, 1013–1024.

43.

Gao

, Liu

, Huang

, Guo

, Chen

, Zhang

, Zhao

, Peng

, Wang

, Xu

, Lu

, Yuan

, and Guo

(2014). The ECM-cell interaction of cartilage extracellular matrix on chondrocytes. Biomed. Res. Int. 2014, 648459.

44.

Garg

, Kathiriya

I.S.

, Barnes

, Marie

, Schluterman

M.K.

, King

I.N.

, Cheryl

, Butle

C.A.

, Rothrock

, Eapen

R.S.

, Hirayama-Yamadak

, Joo

, Matsuokak

, Jonathan

, Cohen

, and Srivastav

(2003). GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424, 443.

45.

Gentsch

G.E.

, Monteiro

R.S.

, and Smith

J.C.

(2017). Chapter five-cooperation between T-box factors regulates the continuous segregation of germ layers during vertebrate embryogenesis. Dev. Genes Evol. 122, 117–159.

46.

Ghosh

T.K.

, Song

F.F.

, Packham

E.A.

, Buxton

, Robinson

T.E.

, Ronksley

, Self

, Bonser

A.J.

, and Brook

J.D.

(2009). Physical interaction between TBX5 and MEF2C is required for early heart development. Mol. Cell Biol. 29, 2205–2218.

47.

Gossett

L.A.

, Kelvin

, Sternberg

, and Olson

E.N.

(1989). A new myocyte specific enhancer binding factor that recognizes a conserved element associated with multiple muscle specific genes. Mol. Cell. Biol. 9, 5022–5033.

48.

Hinits

, Pan

, Walker

, Dowd

, Moens

C.B.

, and Hughes

S.M.

(2012). Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation. Dev. Biol. 369, 199–210.

49.

Hirai

, Katoku-Kikyo

, Keirstead

S.A.

, and Kikyo

(2013). Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain. Cardiovasc. Res. 100, 105–113.

50.

Holler

K.L.

, Hendershot

T.J.

, Troy

S.E.

, Vincentz

J.W.

, Firulli

A.B.

, and Howard

M.J.

(2010). Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development. Dev. Biol. 341, 291–304.

51.

Hoofnagle

M.H.

, Neppl

R.L.

, Berzin

E.L.

, Teg Pipes

G.C.

, Olson

E.N.

, Wamhoff

B.W.

, Somlyo

A.V.

, and Owens

G.K.

(2011). Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. 300, H1707–H1721.

52.

Ieda

, Fu

J.D.

, Delgado-Olguin

, Vedantham

, Hayashi

, Bruneau

B.G.

, and Srivastava

(2010). Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 142, 375–386.

53.

Ifkovits

J.L.

, Addis

R.C.

, Epstein

J.A.

, and Gearhart

J.D.

(2014). Inhibition of TGFβ signaling increases direct conversion of fibroblasts to induced cardiomyocytes. PloS One 9, e89678.

54.

Inagawa

, Miyamoto

, Yamakawa

, Muraoka

, Sadahiro

, Umei

, Wada

, Katsumata

, Kaneda

, Nakade

, Kurihara

, Obata

, Miyake

, Fukuda

, and Ieda

(2012). Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5. Circ. Res. 111, 1147–1156.

55.

Iseoka

, Miyagawa

, Fukushima

, Saito

, Masuda

, Yajima

, Ito

, Sougawa

, Takeda

, Harada

, Lee

J.K.

, and Sawa

(2018). Pivotal role of non-cardiomyocytes in electromechanical and therapeutic potential of induced pluripotent stem cell-derived engineered cardiac tissue. Tissue Eng. Part A, 24, 287–300.

56.

Islas

J.F.

, Liu

, Weng

K.C.

, Robertson

M.J.

, Zhang

, Prejusa

, Harger

, Tikhomirova

, Chopra

, Iyer

, Mercola

, Oshima

R.G.

, Willerson

J.T.

, Potaman

V.N.

, and Schwartz

R.J.

(2012). Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors. Proc. Natl. Acad. Sci. U. S. A. 109, 13016–13021.

57.

Jayawardena

T.M.

, Egemnazarov

, Finch

E.A.

, Zhang

, Payne

J.A.

, Pandya

, Zhang

Z.P.

, Rosenberg

, Mirotsou

, and Dzau

V.J.

(2012). MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes. Circ. Res. 110, 1465.

58.

Jia

, Jensr

, Stefan

, Yuan

, Michail

, Carsten

, Mario

, Zhou

, and Thomas

(2017). Single cell RNA-seq and ATAC-seq indicate critical roles of Isl1 and Nkx2–Nkx5 for cardiac progenitor cell transition states and lineage settlement. BioRxiv 2, 210930.

59.

Kong

Y.P.

, Rioja

A.Y.

, Xufeng

, Yubing

, Jianping

, and Putnam

A.B.

(2018). A systems mechanobiology model to predict cardiac reprogramming outcomes on different biomaterials. Biomaterials, 181, 280–292.

60.

Laforest

, Dai

, Tyan

, Broman

, Weber

, and Ivan

(2018). Gata4 modulates a Tbx5-SERCA Ca²⁺-dependent pathway to maintain normal atrial rhythm. Circulation 138, A14876.

61.

Lalit

P.A.

, Salick

, Nelson

D.O.

, Squirrell

J.M.

, Shafer

C.M.

, Patel

N.G.

, Saeed

, Schmuck

E.G.

, Markandeya

Y.S.

, Wong

, Lea

M.R.

, Eliceiri

K.W.

, Hacker

T.A.

, Crone

W.C.

, Kyba

, Garry

D.J.

, Stewart

, Thomson

J.A.

, Downs

K.M.

, Lyons

G.E.

, and Kamp

T.J.

(2016). Lineage reprogramming of fibroblasts into proliferative induced cardiac progenitor cells by defined factors. Cell Stem Cell, 18, 354–367.

62.

Laugwitz

K.L.

, Moretti

, Caron

, Nakano

, and Chien

K.R.

(2008). Islet1 cardiovascular progenitors: a single source for heart lineages. Development, 135, 193–205.

63.

Laurent

, Girdziusaite

, Gamart

, Barozzi

, Osterwalder

, Akiyama

J.A.

, Lincoln

, Lopez-Rios

, Visel

, Zuniga

, and Zeller

(2017). Hand2 target gene regulatory networks control atrioventricular canal and cardiac valve development. Cell Rep. 19, 1602.

64.

Lee

, Shao

N.Y.

, Paik

D.T.

, Wu

, Guo

, Termglinchan

, Churko

J.M.

, Kim

, Kitani

, Zhao

M.T.

, Zhang

, Wilson

K.D.

, Karakikes

, Snyder

M.P.

, and Wu

J.C.

(2018). SETD7 drives cardiac lineage commitment through stage-specific transcriptional activation. Cell Stem Cell, 22, 428–444.

65.

Leifer

, Krainc

, Yu

Y.T.

, McDermott

, Breitbart

R.E.

, Heng

, Neve

R.L.

, Kosofsky

, Nadal-Ginard

, and Lipton

S.A.

(1993). MEF2C, a MADS/MEF2-family transcription factor expressed in a laminar distribution in cerebral cortex. Proc. Natl. Acad. Sci. U. S. A. 90, 1546–1550.

66.

Lessard

, and Sauvageau

(2003). Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature, 423, 255–260.

67.

F.M.

, and Liu

W.Y.

(2017). Genome-wide identification, classification, and functional analysis of the basic helix-loop-helix transcription factors in the cattle, Bos Taurus. Mamm. Genome, 28, 176–197.

68.

, Cao

, Ma

X.J.

, Wang

H.J.

, Zhang

, Luo

, Chen

, Wu

, Meng

, Zhang

, Yuan

, Ma

, and Huang

G.Y.

(2013). Roles of miR-1-1 and miR-181c in ventricular septal defects. Int. J. Cardiol. 168, 1441–1446.

69.

, Kannan

, DeMayo

F.J.

, Lydon

J.P.

, Cooke

P.S.

, Yamagishi

, Srivastava

, Bagchi

M.K.

, and Bagchi

I.C.

(2011). The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2. Science, 331, 912–916.

70.

, Lin

, Yang

X.S.

, and Chang

(2010). NFATc4 is negatively regulated in miR-133a-mediated cardiomyocyte hypertrophic repression. Am. J. Physiol. Heart Circ. Physiol. 298, H1340–H1347.

71.

Liu

, Lei

, Karatas

, Li

, Wang

, Gnatovskiy

L. D

, ou

, Wang

, Qian

, and Wang

(2016). Targeting Mll1 H3K4 methyltransferase activity to guide cardiac lineage specific reprogramming of fibroblasts. Cell Discov. 2, 16036.

72.

C.X.

, Gong

H.R.

, Liu

X.Y.

, Wang

, Zhao

C.M.

, Huang

R.T.

, Xue

, and Yang

Y.Q.

(2016). A novel Hand2 loss-of-function mutation responsible for tetralogy of Fallot. Int. J. Mol. Med. 37, 445–451.

73.

Lundy

S.D.

, Zhu

W.Z.

, Regnier

, and Laflamme

M.A.

(2013). Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells Dev. 22, 1991–2002.

74.

Lyons

, Parsons

L.M.

, Hartley

, Andrews

J.E.

, Robb

, and Harvey

R.P.

(1995). Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2–Nkx5. Genes Dev. 9, 1654–1666.

75.

Martherus

R.S.

, Vanherle

S.J.

, Timmer

E.D.

, Zeijlemaker

V.A.

, Broers

J.L.

, Smeets

H.J.

, Geraedts

J.P.

, and AJyoubi

T.A.

(2010). Electrical signals affect the cardiomyocyte transcriptome independently of contraction. Physiol. Genomics 42A, 283–289.

76.

Masumoto

, Ikuno

, Takeda

, Fukushima

, Marui

, Katayama

, Shimizu

, Ikeda

, Okano

, Sakata

, and Yamashita

J.K.

(2014). Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration. Sci. Rep. 4, 6716.

77.

Materna

S.C.

, Sinha

, Barnes

R.M.

, Kelly

L.B.

, and Black

B.L.

(2019). Cardiovascular development and survival require Mef2c function in the myocardial but not the endothelial lineage. Dev. Biol. 445, 170–177.

78.

McFadden

D.G.

, Barbosa

A.C.

, Richardson

J.A.

, Schneider

M.D.

, Srivastava

, and Olson

E.N.

(2005). The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner. Development, 132, 189–201.

79.

Miyamoto

, Akiyama

, Tamura

, Isomi

, Yamakawa

, Sadahiro

, Muraoka

, Kojima

, Haginiwa

, Kurotsu

, Tani

, Wang

, Qian

, Inoue

, Ide

, Kurokawa

, Yamamoto

, Seki

, Aeba

, Yamagishi

, Fukuda

, and Ieda

(2018). Direct in vivo reprogramming with sendai virus vectors improves cardiac function after myocardial infarction. Cell Stem Cell, 22, 91–103.

80.

Mohammadi

M.M.

, Shirvani

, Gigina

, Cordero

, Dobreva

, Engelhardt

, Bauersachs

, and Heineke

(2018). Neonatal mice adapt to pressure overload by inducing cardiomyocyte proliferation and angiogenesis. Circulation 138, A10882.

81.

Moore

J.B.

, Tang

X.L.

, Zhao

, Fischer

A.G.

, Wu

W.J.

, Uchida

, Gumpert

A. M.

, Stowers

, Wysoczynski

, and Bolli

(2019). Epigenetically modified cardiac mesenchymal stromal cells limit myocardial fibrosis and promote functional recovery in a model of chronic ischemic cardiomyopathy. Basic Res. Cardiol. 114, 3.

82.

Morales

, Monzo

, and Navarro

(2017). Epigenetic regulation mechanisms of microRNA expression. Biomol. Concepts, 8, 203–212.

83.

Müller

C.W.

, and Herrmann

B.G.

(1997). Crystallographic structure of the T domain-DNA complex of the Brachyury transcription factor. Nature, 389, 884–888.

84.

Muraoka

, Yamakawa

, Miyamoto

, Sadahiro

, Umei

, Isomi

, Nakashima

, Akiyama

, Wada

, Inagawa

, Nishiyama

, Kaneda

, Fukuda

, Takeda

S. T

, ohyama

, Hashimoto

, Kawamura

, Goshima

, Aeba

, Yamagishi

, Fukuda

, and Ieda

(2014). MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures. EMBO J. 33, 1565–1581.

85.

Nam

Y.J.

, Song

, Luo

, Daniel

, Lambeth

, West

, Hill

J.A.

, DiMaio

J.M.

, Baker

L.A.

, Bassel-Duby

, and Olson

E.N.

(2013). Reprogramming of human fibroblasts toward a cardiac fate. Proc. Natl. Acad. Sci. U. S. A. 110, 5588–5593.

86.

Nan

, and Sheng

(2018). Methods for reprogramming differentiated non-cardiac cells into cardiomycytes. US Patent, 9, 982, 231.

87.

Otsuji

T.G.

, Minami

, Kurose

, Yamauchi

, Tada

, and Nakatsuji

(2010). Progressive maturation in contracting cardiomyocytes derived from human embryonic stem cells: qualitative effects on electrophysiological responses to drugs. Stem Cell Res. 4, 201–213.

88.

Papaioannou

V.E.

(2014). The T-box gene family: emerging roles in development, stem cells and cancer. Development, 141, 3819–3833.

89.

Pereira

A.H.M.

, Clemente

C.F.

, Cardoso

A.C.

, Theizen

T.H.

, Rocco

S.A.

, Judice

C.C.

, Guido

M.C.

, Pascoal

V.D.

, Lopes-Cendes

, Souza

J.R.

, and Franchini

K.G.

(2009). MEF2C silencing attenuates load-induced left ventricular hypertrophy by modulating mTOR/S6K pathway in mice. PloS One 4, e8472.

90.

Potthoff

M.J.

, and Olson

E.N.

(2007). MEF2: a central regulator of diverse developmental programs. Development, 134, 4131–4140.

91.

Protze

, Khattak

, Poulet

, Lindemann

, Tanaka

E.M.

, and Ravens

(2012). A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells. J. Mol. Cell Cardiol. 53, 323–332.

92.

Przybyt

, Luyn

M.J.

, and Harmsen

M.C.

(2015). Extracellular matrix components of adipose derived stromal cells promote alignment, organization, and maturation of cardiomyocytes in vitro. J. Biomed. Mater. Res. A, 103, 1840–1848.

93.

Qian

, Huang

, Spencer

C.I.

, Foley

, Vedantham

, Liu

, Conway

S.J.

, Fu

J.D.

, and Srivastava

(2012). In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature, 485, 593–598.

94.

Rao

P.K.

, Kumar

R.M.

, Farkhondeh

, Baskerville

, and Lodish

H.F.

(2006). Myogenic factors that regulate expression of muscle-specific microRNAs. Proc. Natl. Acad. Sci. U. S. A. 103, 8721–8726.

95.

Rooij

E.V.

, Sutherland

L.B.

, Qi

, Richardson

J.A.

, Hill

, and Olson

E.N.

(2007). Control of stress-dependent cardiac growth and gene expression by a microRNA. Science, 316, 575–579.

96.

Ruan

, Zhu

, Yin

, and Chen

(2016). Overexpressing NKx2.5 increases the differentiation of human umbilical cord drived mesenchymal stem cells into cardiomyocyte-like cells. Biomed. Pharmacother. 78, 110–115.

97.

Sadahiro

, Isomi

, Muraoka

, Kojima

, Haginiwa

, Kurotsu

, Tamura

, Tani

, Tohyama

, Fujita

, Miyoshi

, Kawamura

, Goshima

, Iwasaki

Y.W.

, Murano

, Saito

, Oda

, Andersen

, Kwon

, Uosaki

, Nishizono

, Fukuda

, and Ieda

(2018). Tbx6 induces nascent mesoderm from pluripotent stem cells and temporally controls cardiac versus somite lineage diversification. Cell Stem Cell, 23, 382–395.

98.

Sandmaier

S.E.

, and Telugu

B.P.

(2015). MicroRNA-mediated reprogramming of somatic cells into induced pluripotent stem cells. Methods Mol. Biol. 1330, 29–36.

99.

Sathaye

, Bursac

, Sheehy

, and Tung

(2006). Electrical pacing counteracts in trinsic shortening of action potential duration of neonatal rat ventricular cells in culture. J. Mol. Cell Cardiol. 41, 633–641.

100.

Schraivogel

, and Meister

(2014). Import routes and nuclear functions of Argonaute and other small RNA silencing proteins. Trends Biochem. Sci. 39, 420–431.

101.

Shieh

J.T.

, Huang

, Gilmore

, and Srivastava

(2011). Elevated miR-499 levels blunt the cardiac stress response. PloS One 6, e19481.

102.

Shu

, Wu

, Li

, Shao

, Zhao

, Tang

, Yang

, Shen

, Zuo

, Yang

, Shi

, Chi

, Zhang

, Gao

, Shu

, Yuan

, He

, Tan

, Zhao

, and Deng

(2015). Induction of pluripotency in mouse somatic cells with lineage specifiers. Cell 161, 1229.

103.

Simon

A.M.

, McWhorter

A.R.

, Dones

J.A.

, Jackson

C.L.

, and Chen

(2004). Heart and head defects in mice lacking pairs of connexins. Dev. Biol. 265, 369–383.

104.

Sims

R.J.

, Nishioka

, and Reinberg

(2003). Histone lysine methylation: a signature for chromatin function. Trends Genet. 19, 629–639.

105.

Singelyn

J.M.

, DeQuach

J.A.

, Seif-Naraghi

S.B.

, Littlefield

R.B.

, Schup-Magoffin

P.J.

, and Christman

K.L.

(2009). Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering. Biomaterials, 30, 5409–5416.

106.

Soci

U.P.R.

, Tiago

, Rosa

K.T.

, Maria

C.I.

, Phillips

M.I.

, and Edilamar

M.O.

(2013). The role of microRNA-208a in cardiac hypertrophy induced by aerobic physical training. FASEB J. 27, 975.4.

107.

Song

, Nam

Y.J.

, Luo

, Qi

, Tan

, Guo

N.H.

, Acharya

, Smith

C.L.

, Tallquist

M.D.

, Neilson

E.G.

, Hill

J.A.

, Bassel-Duby

, and Olson

E.N.

(2012). Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature, 485, 599–604.

108.

Spiridon

M.R.

, Petris

A.O.

, Gorduza

E.V.

, Petras

A.S.

, Popescu

, and Caba

(2018). Holt-Oram syndrome with multiple cardiac abnormalities. Cardiol. Res. 9, 324–329.

109.

Srivastava

(2006). Making or breaking the heart: from lineage determination to morphogenesis. Cell, 126, 1037–1048.

110.

Stennard

F.A.

, Costa

M.W.

, Elliott

D.A.

, Rankin

, Haast

S.J.

, Lai

, McDonald

L.P.

, Niederreither

, Dolle

, Bruneau

B.G.

, Zorn

A.M.

, and Harvey

R.P.

(2005). Cardiac T-box factor Tbx20 directly interacts with Nkx2–Nkx5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev. Biol. 262, 206–224.

111.

Tabei

, Kawaguchib

, Kanazawa

, Tohyama

, Hirano

, Handa

, Hishikawa

, Teratani

, Kunita

, Fukuda

, Mugishima

, Suzuki

, Nakajima

, Seki

, Kishino

, Okada

, Yamazaki

, Okamoto

, Shimizu

, Kobayashi

, Tabata

, Fujita

, and Fukuda

(2019). Development of a transplant injection device for optimal distribution and retention of human iPSC-derived cardiomyocytes. J. Heart Lung Transpl. 38, 203–214.

112.

Takahashi

, Tanabe

, Ohnuki

, Narita

, Ichisaka

, Tomoda

, and Yamanaka

(2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.

113.

Takebayashi-Suzuki

, Pauliks

L.B.

, Eltsefon

, and Mikawa

(2001). Purkinje fibers of the avian heart express a myogenic transcription factor program distinct from cardiac and skeletal muscle. Dev. Biol. 234, 390–401.

114.

Takeuchi

J.K.

, and Bruneau

B.G.

(2009). Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors. Nature, 459, 708–711.

115.

Tanaka

, Wechsler

S.B.

, Lee

I.W.

, Yamasaki

, and Lawitts

(1999). Complex modular cis-acting elements regulate expression of the cardiac specifying homeobox gene Csx/Nkx2.5. Development, 126, 1439–1450.

116.

Thattaliyath

B.D.

, Firulli

B.A.

, and Firulli

A.B.

(2002). The basic-helix-loop-helix transcription factor HAND2 directly regulates transcription of the atrial naturetic peptide gene. J. Mol. Cell Cardiol. 34, 1335–1344.

117.

Tian

, Wang

, Hou

, Li

, Chen

, Deng

, Zhu

, Zhao

, and He

W.F.

(2018). Optimization and enrichment of induced cardiomyocytes derived from mouse fibroblasts by reprogramming with cardiac transcription factors. Mol. Med. Rep. 17, 3912–3920.

118.

Tsai

S.F.

, Martin

D.I.

, Zon

, D'Andrea

A.D.

, and Wong

(1989). Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Cell, 339, 446–451.

119.

Valinezhad-Orang

A.V.

, Safaralizadeh

, and Kazemzadeh-Bavili

(2014). Mechanisms of miRNA-mediated gene regulation from common downregulation to mRNA-specific upregulation, Int. J. Genomics 2014, 970607.

120.

Vandusen

N.J.

, and Firulli

A.B.

(2012). Twist factor regulation of non-cardiomyocyte cell lineages in the developing heart. Differentiation, 277, 79–88.

121.

VanDusen

N.J.

, Vincentz

J.W.

, Firulli

B.A.

, Howard

M.J.

, Rubart

, and Firulli

A.B.

(2014). Loss of Hand2 in a population of Periostin lineage cells results in pronounced bradycardia and neonatal death. Dev. Biol. 388, 149–158.

122.

Vanlerberghe

, Jourdain

A.S.

, Ghoumid

, Frenois

, Mezel

, Vaksmann

, Lenne

, Delobel

, Porchet

, Cormier-Daire

, Smol

, Escande

, Manouvrier-Hanu

, and Petit

(2019). Holt-Oram syndrome: clinical and molecular description of 78 patients with TBX5 variants. Eur. J. Hum. Genet. 27, 360–368.

123.

Vaseghi

H.R.

, Yin

, Zhou

, Wang

, Liu

, and Qian

(2016). Generation of an inducible fibroblast cell line for studying direct cardiac reprogramming. Genesis, 54, 398–406.

124.

Vierbuchen

, and Werng

(2012). Molecular roadblocks for cellular reprogramming. Mol. Cell. 47, 827–838.

125.

Vincentz

J.W.

, Barnes

R.M.

, Firulli

B.A.

, Conway

S.J.

, and Firulli

A.B.

(2008). Cooperative interaction of Nkx2.5 and Mef2c transcription factors during heart development. Dev. Dyn. 237, 3809–3819.

126.

Wang

, Wang

, Erdjament-Bromage

, Vidal

, Tempst

, Jones

R.S.

, and Zhang

(2004). Role of histone HzA ubiquitination in polycomb silencing. Nature, 431, 873–878.

127.

Wang

, Cao

, Spencer

C.I.

, Nie

, Ma

, Xu

, Zhang

, Wang

, Srivastava

, and Ding

(2014). Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4. Cell Rep. 6, 951–960.

128.

Wang

, Liu

J.D.

, and Qian

(2017). In vivo lineage reprogramming of fibroblasts to cardiomyocytes for heart regeneration. In Stem Cell Biology and Regenerative Medicine. (Humana Press, New York, NY) pp. 45–63.

129.

Wang

, Liu

, Yin

, Asfour

, Chen

, Li

, Bursac

, Liu

, and Qian

(2015). Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming. Circ. Res. 116, 237–244.

130.

Wang

X.W.

, Conrad

, and Dzau

(2018). Composition and methods for the prepositions and methods for the reprogramming of cells into cardiomyocytes. US Patent 2018/0042969. A1.

131.

Wang

, Shi

, Liu

, and Meng

(2016). Hypoxia enhances direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells. Cell Reprogram. 18, 1–7.

132.

Wapinski

O.L.

, Vierbuchen

, Qu

, Lee

Q.Y.

, Chanda

, Fuentes

D.R.

, Giresi

P.G.

, Ng

Y.H.

, Marro

, Neff

N.F.

, Drechsel

, Martynoga

, Castro

D.S.

, Webb

A.E.

, Südhof

T.C.

, Brunet

, Guillemot

, Chang

H.Y.

, and Wernig

(2013). Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell, 155, 621–635.

133.

Williams

, Budina

, Stoppe

W.L.

, Sullivan

K.E.

, Emani

S.M.

, and Black

L.D.

(2015). Cardiac extracellular matrix-fibrin hybrid scaffolds with tunable properties for cardiovascular tissue engineering. Acta Biomater. 14, 84–95.

134.

Williams

, Quinn

K.P.

, Georgakoudi

, and Black

L.D.

(2014). Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro. Acta Biomater. 10, 194–204.

135.

Wystub

, Besser

, Bachmann

, Boettger

, and Braun

(2013). MiR-1/133a clusters cooperatively specify the cardiomyogenic lineage by adjustment of myocardin levels during embryonic heart development. PloS Genet. 9, e1003793.

136.

, Du

, and Deng

(2015). Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell, 16, 119–c134.

137.

Yamagishi

, Yamagishi

, Nakagawa

, Harvey

R.P.

, Olson

E.N.

, and Srivastava

(2001). The combinatorial activities of Nkx2.5 and dHAND are essential for cardiac ventricle formation. Dev. Biol. 2, 190–203.

138.

Yang

, Pabon

, and Murry

C.E.

(2014a). Engineering adolescence: maturation of human pluripotent stem cell derived cardiomyocytes. Circ. Res. 114, 511–523.

139.

Yang

, Rodriguez

, Pabon

, Fischer

K.A.

, Reinecke

, Regnier

, Sniadecki

N.J.

, Ruohola-Bakr

, and Murry

C.E.

(2014b). Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells. J. Mol. Cell Cardiol. 72, 296–304.

140.

Yuan

, Qiu

Z.H.

, Wang

X.H.

, Sun

Y. M.

, Wang

, Li

R.G.

, Liu

, Zhang

, Shi

H.Y.

, Zhao

, Jiang

W.F.

, Liu

, Qiu

X.B.

, Qu

X.K.

, and Yang

Y.Q.

(2018). Mef2c loss-of-function mutation associated with familial dilated cardiomyopathy. Clin. Chem. Lab Med. 56, 502–511.

141.

Yue

, Jiang

M.L.

, He

, Zhang

, Gu

, Liu

, Li

, and Zhao

(2018). The transcription factor Foxc1a in zebrafish directly regulates expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. J. Biol. Chem. 293, 638–650.

142.

Zakariyah

A.F.

, Rajgara

R.F.

, Horner

, Cattin

M.E.

, Blais

, Skerjanc

I.S.

, and Brugon

P.G.

(2018). In vitro modeling of congenital heart defects associated with an Nkx2.5 mutation revealed a dysregulation in BMP/Notch-mediated signaling. Stem Cell, 36, 514–526.

143.

Zamir

, Singh

, Nathan

, Patrick

, Yifa

, Yahalom-Ronen

, Arraf

A.A.

, Schultheiss

T.M.

, Suo

, J.-D.J., Han

, Peng

, Jing

, Wang

, Palpant

P.P.

, Tam

R.P.

, and Harvey

E.T.

(2017). Nkx2.5 marks angioblasts that contribute to hemogenic endothelium of the endocardium and dorsal aorta. eLife 6, e20994.

144.

Zang

M.X.

, Li

, Wang

J.B.

, and Jia

H.T.

(2004). Cooperative interaction between the basic helix-loop-helix transcription factor dHAND and myocyte enhancer factor 2C regulates myocardial gene expression. J. Biol. Chem. 279, 54258–54263.

145.

Zhang

, Ai

, Zheng

, and Peng

(2017). Associations of Gata4 genetic mutations with the risk of congenital heart disease: a meta-analysis. Medicine 96, e6857.

146.

Zhao

, Ransom

J.F.

, Li

, Vedantham

, von Drehle

, Muth

A.N.

, Tsuchihashi

, McManus

M.T.

, Schwartz

R.J.

, and Srivastava

(2007). Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell, 129, 303–317.

147.

Zhao

, Samal

, and Srivastava

(2005). Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature, 436, 214–220.

148.

Zhou

H.Y.

, Morales

M.G.

, Hashimoto

, Dickson

M.E.

, Song

K.H.

, Ye

W.D.

, Kim

M.S.

, Niederstrasser

, Wang

Z.N.

, Chen

, Posner

B.A.

, Rhonda

B.D.

, and Olson

E.N.

(2017a). ZNF281 enhances cardiac reprogramming by modulating cardiac and inflammatory gene expression. Gene Dev. 31, 1770–1783.

149.

Zhou

, Liu

, Xiang

, Olson

, Guzzetta

, Zhang

, Ivan

, Moskowitz

I.P.

, and Xie

(2017b). Gata4 potentiates second heart field proliferation and Hedgehog signaling for cardiac septation. Proc. Natl. Acad. Sci. U. S. A. 114, E1422–E1431.

150.

Zhou

, Alimohamadi

, Wang

, Liu

, Wall

J.B.

, Yin

, Liu

, and Qian

(2018). A loss of function screen of epigenetic modifiers and splicing factors during early stage of cardiac reprogramming. Stem Cells Int. 2018, 1–14.

151.

Zhou

, Wang

, Vaseghi

H.R.

, Liu

, Lu

, Alimohamadi

, Yin

, Fum

J.D.

, Wang

G.G.

, Liu

, and Qian

(2016). Bmi1 is a key epigenetic barrier to direct cardiac reprogramming. Cell Stem Cell, 18, 382–395.

152.

Zhu

, Wang

, and Ding

(2015). Reprogramming fibroblasts toward cardiomyocytes, neural stem cells and hepatocytes by cell activation and signaling-directed lineage conversion. Nat. Protoc. 10, 959–973.