Direct Reprogramming Improves Cardiac Function and Reverses Fibrosis in Chronic Myocardial Infarction.

BACKGROUND: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. METHODS: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. RESULTS: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. CONCLUSIONS: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.

Direct reprogramming with Sendai virus vectors repaired infarct hearts at the chronic stage.

Adult hearts have limited regenerative capacity. Hence, after acute myocardial infarction (MI), dead myocardial tissues are digested by immune cells and replaced by fibrosis, leading to ventricular remodeling and heart failure at the chronic stage. Direct reprogramming of the cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) with cardiac transcription factors, including Gata4, Mef2c, and Tbx5 (GMT), may have significant potential for cardiac repair. Sendai virus (SeV) vectors expressing GMT have been reported to reprogram the mouse cardiac fibroblasts into iCMs without any risk of insertional mutagenesis. In vivo reprogramming improved the cardiac function after acute MI in immunodeficient mice. However, it is unknown whether the newly generated iCMs could exist in infarct hearts for a prolonged period and SeV-GMT can improve cardiac function after MI at the chronic stage in immunocompetent mice. Here, we show that SeV vectors efficiently infect CFs in vivo and reprogram them into iCMs, which existed for at least four weeks after MI, in fibroblast-linage tracing mice. Moreover, SeV-GMT improved cardiac function and reduced fibrosis and collagen I expression at 12 weeks after MI in immunocompetent mice. Thus, direct cardiac reprogramming with SeV vectors could be a promising therapy for MI.

Tbx6 induces cardiomyocyte proliferation in postnatal and adult mouse hearts.

Cardiovascular disease is a leading cause of death worldwide. Mammalian cardiomyocytes (CMs) proliferate during embryonic development, whereas they largely lose their regenerative capacity after birth. Defined factors expressed in cardiac progenitors or embryonic CMs may activate the cell cycle and induce CM proliferation in postnatal and adult hearts. Here, we report that the overexpression of Tbx6, enriched in the cardiac mesoderm (progenitor cells), induces CM proliferation in postnatal and adult mouse hearts. By screening 24 factors enriched in cardiac progenitors or embryonic CMs, we found that only Tbx6 could induce CM proliferation in primary cultured postnatal rat CMs. Intriguingly, it did not induce the proliferation of cardiac fibroblasts. We next generated a recombinant adeno-associated virus serotype 9 vector encoding Tbx6 (AAV9-Tbx6) for transduction into mouse CMs in vivo. The subcutaneous injection of AAV9-Tbx6 into neonatal mice induced CM proliferation in postnatal and adult mouse hearts. Mechanistically, Tbx6 overexpression upregulated multiple cell cycle activators including Aurkb, Mki67, Ccna1, and Ccnb2 and suppressed the tumor suppressor Rb1. Thus, Tbx6 promotes CM proliferation in postnatal and adult mouse hearts by modifying the expression of cell cycle regulators.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.

Distinct expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after myocardial infarction.

The coronary vascular system is critical for myocardial growth and cardiomyocyte survival. However, the molecular mechanism regulating coronary angiogenesis remains elusive. Vascular endothelial growth factor (VEGF) regulates angiogenesis by binding to the specific receptors Flk1 and Flt1, which results in different functions. Despite the importance of Flk1 and Flt1, their expression in the coronary vasculature remains largely unknown due to the lack of appropriate antibodies for immunostaining. Here, we analyzed multiple reporter mice including Flk1-GFP BAC transgenic (Tg), Flk1-LacZ knock-in, Flt1-DsRed BAC Tg, and Flk1-GFP/Flt1-DsRed double Tg animals to determine expression patterns in mouse hearts during cardiac growth and after myocardial infarction (MI). We found that Flk1 was expressed in endothelial cells (ECs) with a pattern of epicardial-to-endocardial transmural gradients in the neonatal mouse ventricle, which was downregulated in adult coronary vessels with development. In contrast, Flt1 was homogeneously expressed in the ECs of neonatal mouse hearts and expression was maintained until adulthood. After MI, expression of both Flk1 and Flt1 was induced in the regenerating coronary vessels at day 7. Intriguingly, Flk1 expression was downregulated thereafter, whereas Flt1 expression was maintained in the newly formed coronary vessels until 30 days post-MI, recapitulating their expression kinetics during development. This is the first report demonstrating the spatiotemporal expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after MI; thus, this study suggests that these factors have distinct and important functions in coronary angiogenesis.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression.

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Soft Matrix Promotes Cardiac Reprogramming via Inhibition of YAP/TAZ and Suppression of Fibroblast Signatures.

Direct cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to ∼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming.

Progress and Challenge of Cardiac Regeneration to Treat Heart Failure.

Cardiac muscle has limited proliferative capacity, and regenerative therapies are highly in demand as a new treatment strategy. Pharmacological and non-pharmacological therapies have been developed, but these medical therapies have limited effects to cure patients with severe heart failure. Moreover, heart transplantation is limited due to the low number of donor organs. Thus, heart regeneration holds great potential to offer innovative therapy to treat heart failure patients. Currently, there are several strategies for heart regeneration. Transplantation of somatic stem cells was safe and modestly improved cardiac function after myocardial infarction mainly through paracrine mechanisms. Alternatively, new cardiomyocytes could be generated from induced pluripotent stem cells (iPSCs) to transplant into injured hearts. However, several issues remain to be resolved prior to using iPSC-derived cardiomyocytes, such as a potential risk of tumorigenesis and poor survival of transplanted cells in the injured heart. More recently, direct cardiac reprogramming has emerged as a novel technology to regenerate damaged myocardium by directly converting endogenous cardiac fibroblasts into induced cardiomyocyte-like cells to restore cardiac function. Following our first report of cardiac reprogramming, an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming, and cardiac reprogramming in human cells were reported by many investigators. While these previous studies have advanced regenerative research, many challenges remain. Here, we review the current status of cardiac regenerative technology, a great hope to treat cardiovascular diseases.

Role of cyclooxygenase-2-mediated prostaglandin E2-prostaglandin E receptor 4 signaling in cardiac reprogramming.

Direct cardiac reprogramming from fibroblasts can be a promising approach for disease modeling, drug screening, and cardiac regeneration in pediatric and adult patients. However, postnatal and adult fibroblasts are less efficient for reprogramming compared with embryonic fibroblasts, and barriers to cardiac reprogramming associated with aging remain undetermined. In this study, we screened 8400 chemical compounds and found that diclofenac sodium (diclofenac), a non-steroidal anti-inflammatory drug, greatly enhanced cardiac reprogramming in combination with Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2. Intriguingly, diclofenac promoted cardiac reprogramming in mouse postnatal and adult tail-tip fibroblasts (TTFs), but not in mouse embryonic fibroblasts (MEFs). Mechanistically, diclofenac enhanced cardiac reprogramming by inhibiting cyclooxygenase-2, prostaglandin E2/prostaglandin E receptor 4, cyclic AMP/protein kinase A, and interleukin 1ß signaling and by silencing inflammatory and fibroblast programs, which were activated in postnatal and adult TTFs. Thus, anti-inflammation represents a new target for cardiac reprogramming associated with aging.

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction.

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration.

Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification.

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming.