MEF2C/p300-mediated epigenetic remodeling promotes the maturation of induced cardiomyocytes.

Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where MEF2C acts as a pioneer factor with GATA4 and TBX5 (GT). However, the generation of functional and mature iCMs is inefficient, and the molecular mechanisms underlying this process remain largely unknown. Here, we found that the overexpression of transcriptionally activated MEF2C via fusion of the powerful MYOD transactivation domain combined with GT increased the generation of beating iCMs by 30-fold. Activated MEF2C with GT generated iCMs that were transcriptionally, structurally, and functionally more mature than those generated by native MEF2C with GT. Mechanistically, activated MEF2C recruited p300 and multiple cardiogenic TFs to cardiac loci to induce chromatin remodeling. In contrast, p300 inhibition suppressed cardiac gene expression, inhibited iCM maturation, and decreased the beating iCM numbers. Splicing isoforms of MEF2C with similar transcriptional activities did not promote functional iCM generation. Thus, MEF2C/p300-mediated epigenetic remodeling promotes iCM maturation.

Dermal fibroblast-like cells reprogrammed directly from adipocytes in mouse.

In deep burns, early wound closure is important for healing, and skin grafting is mainly used for wound closure. However, it is difficult to achieve early wound closure in extensive total body surface area deep burns due to the lack of donor sites. Dermal fibroblasts, responsible for dermis formation, may be lost in deep burns. However, fat layers composed of adipocytes, lying underneath the dermis, are retained even in such cases. Direct reprogramming is a novel method for directly reprograming some cells into other types by introducing specific master regulators; it has exhibited appreciable success in various fields. In this study, we aimed to assess whether the transfection of master regulators (ELF4, FOXC2, FOXO1, IRF1, PRRX1, and ZEB1) could reprogram mouse adipocytes into dermal fibroblast-like cells. Our results indicated the shrinkage of fat droplets in reprogrammed mouse adipocytes and their transformation into spindle-shaped dermal fibroblasts. Reduced expression of PPAR-2, c/EBP, aP2, and leptin, the known markers of adipocytes, in RT-PCR, and enhanced expression of anti-ER-TR7, the known anti-fibroblast marker, in immunocytochemistry, were confirmed in the reprogrammed mouse adipocytes. The dermal fibroblast-like cells, reported here, may open up a new treatment mode for enabling early closure of deep burn wounds.

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.

Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.

Our knowledge of pluripotent stem cell (PSC) biology has advanced to the point where we now can generate most cells of the human body in the laboratory. PSC-derived cardiomyocytes can be generated routinely with high yield and purity for disease research and drug development, and these cells are now gradually entering the clinical research phase for the testing of heart regeneration therapies. However, a major hurdle for their applications is the immature state of these cardiomyocytes. In this Review, we describe the structural and functional properties of cardiomyocytes and present the current approaches to mature PSC-derived cardiomyocytes. To date, the greatest success in maturation of PSC-derived cardiomyocytes has been with transplantation into the heart in animal models and the engineering of 3D heart tissues with electromechanical conditioning. In conventional 2D cell culture, biophysical stimuli such as mechanical loading, electrical stimulation and nanotopology cues all induce substantial maturation, particularly of the contractile cytoskeleton. Metabolism has emerged as a potent means to control maturation with unexpected effects on electrical and mechanical function. Different interventions induce distinct facets of maturation, suggesting that activating multiple signalling networks might lead to increased maturation. Despite considerable progress, we are still far from being able to generate PSC-derived cardiomyocytes with adult-like phenotypes in vitro. Future progress will come from identifying the developmental drivers of maturation and leveraging them to create more mature cardiomyocytes for research and regenerative medicine.

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.

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.

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.

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.

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.

Positional desaturation due to persistent left superior vena cava draining into the left atrium.

Persistent left superior vena cava (PLSVC) is a rare congenital anomaly whose prevalence is 0.3 % of general population. The majority of PLSVC drain into right atrium (RA) through the coronary sinus without clinical harm. However, in about 10 % of patients with PLSVC, it drains into left atrium (LA) causing right-to-left shunt. Here, we present a 60-year-old male patient with a PLSVC draining into LA, who developed dyspnea and desaturation depending on the body position after trans-catheter coil embolization of coronary to pulmonary artery fistulas. PLSVC draining into LA should be included in the differential diagnosis of positional desaturation.

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.

Analysis of cardiomyocyte movement in the developing murine heart.

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle.

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.

Time-lapse imaging of cell cycle dynamics during development in living cardiomyocyte.

Mammalian cardiomyocytes withdraw from the cell cycle shortly after birth, although it remains unclear how cardiomyocyte cell cycles behave during development. Compared to conventional immunohistochemistry in static observation, time-lapse imaging can reveal comprehensive data in hard-to-understand biological phenomenon. However, there are no reports of an established protocol of successful time-lapse imaging in mammalian heart. Thus, it is valuable to establish a time-lapse imaging system to enable the observation of cell cycle dynamics in living murine cardiomyocytes. This study sought to establish time-lapse imaging of murine heart to study cardiomyocyte cell cycle behavior. The Fucci (fluorescent ubiquitination-based cell cycle indicator) system can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei red, green and yellow, respectively, in living mammalian cells, and could therefore be useful to visualize the real-time cell cycle transitions in living murine heart. To establish a similar system for time-lapse imaging of murine heart, we first developed an ex vivo culture system, with the culture conditions determined in terms of sample state, serum concentration, and oxygen concentration. The optimal condition (slice culture, oxygen concentration 20%, serum concentration 10%) successfully mimicked physiological cardiomyocyte proliferation in vivo. Time-lapse imaging of cardiac slices from E11.5, E14.5, E18.5, and P1 Fucci-expressing transgenic mice revealed an elongated S/G2/M phase in cardiomyocytes during development. Our time-lapse imaging of murine heart revealed a gradual elongation of the S/G2/M phase during development in living cardiomyocytes.

Direct reprogramming of fibroblasts into myocytes to reverse fibrosis.

Heart disease is a major cause of morbidity and mortality worldwide. The low regenerative capacity of adult human hearts has thus far limited the available therapeutic approaches for heart failure. Therefore, new therapies that can regenerate damaged myocardium and improve heart function are urgently needed. Although cell transplantation-based therapies may hold great potential, direct reprogramming of endogenous cardiac fibroblasts, which represent more than half of the cells in the heart, into functional cardiomyocytes in situ may be an alternative strategy by which to regenerate the heart. We and others demonstrated that functional cardiomyocytes can be directly generated from fibroblasts by using several combinations of cardiac-enriched factors in mouse and human. In vivo gene delivery of cardiac reprogramming factors generates new cardiac muscle and improved heart function after myocardial infarction in mouse. This article reviews recent progress in cardiac reprogramming research and discusses the perspectives and challenges of this new technology for future regenerative therapy.

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.

Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.