Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation [mean ± SD]: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Metabolism-based cardiomyocytes production for regenerative therapy.

Human pluripotent stem cells (hPSCs) are currently used in clinical applications such as cardiac regenerative therapy, studying disease models, and drug screening for heart failure. Transplantation of hPSC-derived cardiomyocytes (hPSC-CMs) can be used as an alternative therapy for heart transplantation. In contrast to differentiated somatic cells, hPSCs possess unique metabolic programs to maintain pluripotency, and understanding their metabolic features can contribute to the development of technologies that can be useful for their clinical applications. The production of hPSC-CMs requires stepwise specification during embryonic development and metabolic regulation is crucial for proper embryonic development. These metabolic features have been applied to hPSC-CM production methods, such as mesoderm induction, specifications for cardiac progenitors, and their maturation. This review describes the metabolic programs in hPSCs and the metabolic regulation in hPSC-CM production for cardiac regenerative therapy.

Associations between plasma nesfatin-1 levels and the presence and severity of coronary artery disease.

Nesfatin-1 is a recently identified anorexigenic peptide mainly secreted from the brain and adipose tissue. Although nesfatin-1 may have pro-inflammatory and apoptotic properties, the association between plasma nesfatin-1 levels and coronary artery disease (CAD) has not been clarified yet. We investigated plasma nesfatin-1 levels in 302 patients undergoing elective coronary angiography. Of the 302 study patients, CAD was present in 172 (57%), of whom 67 had 1-vessel, 49 had 2-vessel, and 56 had 3-vessel disease. Compared with 130 patients without CAD, 172 with CAD had higher plasma nesfatin-1 levels (median 0.21 vs. 0.17 ng/mL, P < 0.01). A stepwise increase in nesfatin-1 levels was found depending on the number of > 50% stenotic coronary vessels: 0.17 in CAD(-), 0.20 in 1-vessel, 0.21 in 2-vessel, and 0.22 ng/mL in 3-vessel disease (P < 0.05). A high nesfatin-1 level (> 0.19 ng/mL) was found in 43% of patients with CAD(-), 55% of those with 1-vessel, 55% of those with 2-vessel, and 68% of those with 3-vessel disease (P < 0.05). Nesfatin-1 levels significantly correlated with the number of > 50% stenotic coronary segments (r = 0.14, P < 0.02). In multivariate analysis, plasma nesfatin-1 levels were a significant factor for CAD independent of atherosclerotic risk factors. The odds ratio for CAD was 1.71 (95% CI 1.01-2.91) for high nesfatin-1 level of > 0.19 ng/mL (P < 0.05). Thus, plasma nesfatin-1 levels were found to be high in patients with CAD and were associated with CAD independent of atherosclerotic risk factors, suggesting that high nesfatin-1 levels in patients with CAD may play a role in the development of coronary atherosclerosis.

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.

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.

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.

Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.

RATIONALE: After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. OBJECTIVE: To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. METHODS AND RESULTS: Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. CONCLUSIONS: GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

Seamless and non-destructive monitoring of extracellular microRNAs during cardiac differentiation from human pluripotent stem cells.

Monitoring cardiac differentiation and maturation from human pluripotent stem cells (hPSCs) and detecting residual undifferentiated hPSCs are indispensable for the development of cardiac regenerative therapy. MicroRNA (miRNA) is secreted from cells into the extracellular space, and its role as a biomarker is attracting attention. Here, we performed an miRNA array analysis of supernatants during the process of cardiac differentiation and maturation from hPSCs. We demonstrated that the quantification of extracellular miR-489-3p and miR-1/133a-3p levels enabled the monitoring of mesoderm and cardiac differentiation, respectively, even in clinical-grade mass culture systems. Moreover, extracellular let-7c-5p levels showed the greatest increase with cardiac maturation during long-term culture. We also verified that residual undifferentiated hPSCs in hPSC-derived cardiomyocytes (hPSC-CMs) were detectable by measuring miR-302b-3p expression, with a detection sensitivity of 0.01%. Collectively, we demonstrate that our method of seamlessly monitoring specific miRNAs secreted into the supernatant is non-destructive and effective for the quality evaluation of hPSC-CMs.

Scalable production of homogeneous cardiac organoids derived from human pluripotent stem cells.

Three-dimensional (3D) cultures are known to more closely mimic in vivo conditions compared with 2D cultures. Cardiac spheroids (CSs) and organoids (COs) are useful for 3D tissue engineering and are advantageous for their simplicity and mass production for regenerative therapy and drug discovery. Herein, we describe a large-scale method for producing homogeneous human induced pluripotent stem cell (hiPSC)-derived CSs (hiPSC-CSs) and COs without scaffolds using a porous 3D microwell substratum with a suction system. Our method has many advantages, such as increased efficiency and improved functionality, homogeneity, and sphericity of hiPSC-CSs. Moreover, we have developed a substratum on a clinically relevant large scale for regenerative therapy and have succeeded in producing approximately 40,000 hiPSC-CSs with high sphericity at once. Furthermore, we efficiently produced a fused CO model consisting of hiPSC-derived atrial and ventricular cardiomyocytes localized on opposite sides of one organoid. This method will facilitate progress toward hiPSC-based clinical applications.

Heart-derived collagen promotes maturation of engineered heart tissue.

Although the extracellular matrix (ECM) plays essential roles in heart tissue engineering, the optimal ECM components for heart tissue organization have not previously been elucidated. Here, we focused on the main ECM component, fibrillar collagen, and analyzed the effects of collagens on heart tissue engineering, by comparing the use of porcine heart-derived collagen and other organ-derived collagens in generating engineered heart tissue (EHT). We demonstrate that heart-derived collagen induces better contraction and relaxation of human induced pluripotent stem cell-derived EHT (hiPSC-EHT) and that hiPSC-EHT with heart-derived collagen exhibit more mature profiles than those with collagens from other organs. Further, we found that collagen fibril formation and gel stiffness influence the contraction, relaxation, and maturation of hiPSC-EHT, suggesting the importance of collagen types III and type V, which are relatively abundant in the heart. Thus, we demonstrate the effectiveness of organ-specific collagens in tissue engineering and drug discovery.

Metabolism in Human Pluripotent Stem Cells and Cardiomyocytes for Regenerative Therapy.

Pluripotent stem cells (PSCs), which include embryonic stem cells and induced pluripotent stem cells, have the potential for unlimited self-renewal and proliferation and the ability to differentiate into all three embryonic germ layers. Human PSCs (hPSCs) are used in drug discovery screening, disease models, and regenerative medicine. These cells maintain a transcriptional regulatory network based on a set of unique transcription factors to maintain their stem cell properties. Downstream of such transcriptional regulatory networks, various stem cell-specific metabolic programs are used to produce energy and metabolites as necessary. hPSCs and differentiated cells utilize different metabolic programs for self-renewal ability and maintenance of quiescence. Understanding the different metabolic features of hPSCs and differentiated cells can contribute to the development of technologies that are useful for regenerative medicine, such as the purification of differentiated cells. This review describes the unique metabolic programs active in hPSCs and their differences from somatic cells, with a focus on cardiomyocytes.

Recurrence of Myopericarditis Following mRNA COVID-19 Vaccination in a Male Adolescent.

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease has spread worldwide, resulting in health and economic crises. Vaccination against SARS-CoV-2 infection is considered a valid prevention measure to control this pandemic. There have been reports of cases of myopericarditis following mRNA COVID-19 vaccination. We present a case of a 20-year-old man with recurrent myopericarditis following an initial episode of influenza virus-induced myopericarditis and after a second dose of the mRNA-1273 Moderna COVID-19 vaccine. Careful attention should be paid to patients with a history of myocarditis following COVID-19 vaccination.

COVID-19 est causée par le coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2). La maladie qui s'est répandue dans le monde a entraîné des crises sanitaire et économique. La vaccination contre l'infection à SRAS-CoV-2 est considérée comme une mesure de prévention valide pour juguler la pandémie. Des cas de myopéricardite ont été déclarés après le vaccin à ARNm contre la COVID-19. Nous présentons le cas d'un homme de 20 ans qui a eu une myopéricardite récurrente après un épisode de myopéricardite induite par le virus de l'influenza et après une deuxième dose du vaccin à ARNm-1273 contre la COVID-19 de Moderna. Il faudrait porter une attention particulière aux patients qui ont des antécédents de myocardite après la vaccination contre la COVID-19.

Tryptophan Metabolism Regulates Proliferative Capacity of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) have a unique metabolic signature for maintenance of pluripotency, self-renewal, and survival. Although hPSCs could be potentially used in regenerative medicine, the prohibitive cost associated with large-scale cell culture presents a major barrier to the clinical application of hPSC. Moreover, without a fully characterized metabolic signature, hPSC culture conditions are not optimized. Here, we performed detailed amino acid profiling and found that tryptophan (TRP) plays a key role in the proliferation with maintenance of pluripotency. In addition, metabolome analyses revealed that intra- and extracellular kynurenine (KYN) is decreased under TRP-supplemented conditions, whereas N-formylkynurenine (NFK), the upstream metabolite of KYN, is increased thereby contributing to proliferation promotion. Taken together, we demonstrate that TRP is indispensable for survival and proliferation of hPSCs. A deeper understanding of TRP metabolism will enable cost-effective large-scale production of hPSCs, leading to advances in regenerative medicine.

Sudden cardiac death due to ventricular fibrillation in a case of giant cell myocarditis.

A 70-year-old woman was admitted to our hospital complaining of shortness of breath. She was diagnosed with acute decompensated heart failure due to left ventricular dysfunction. Her symptoms began to improve with standard therapy for heart failure with diuretics, noninvasive pressure ventilation, and inotropes, but paroxysmal atrial fibrillation and premature ventricular contractions (PVCs) occurred. After treatment with amiodarone, the number of PVCs decreased, and the left ventricular wall motion gradually improved. However, on day 28, ventricular fibrillation and cardiopulmonary arrest occurred suddenly, and she could not be resuscitated. She was diagnosed with giant cell myocarditis via an autopsy. The autopsy revealed diffuse inflammatory cells that comprised giant cells and eosinophils as well as cellular degeneration and necrosis. .

High Plasma Levels of Legumain in Patients with Complex Coronary Lesions.

AIM: The degradation of the vascular extracellular matrix is important for atherosclerosis. The cysteine protease legumain was shown to be upregulated in atherosclerotic plaques, especially unstable plaques. However, no study has reported blood legumain levels in patients with coronary artery disease (CAD). METHODS: We investigated plasma legumain and C-reactive protein (CRP) levels in 372 patients undergoing elective coronary angiography. RESULTS: CAD was found in 225 patients. Compared with patients without CAD, those with CAD had higher CRP levels (median 0.60 [0.32, 1.53] vs. 0.46 [0.22, 0.89] mg/L, P＜0.001), but no difference was found in legumain levels between patients with and without CAD (median 5.08 [3.87, 6.82] vs. 4.99 [3.84, 6.88] ng/mL). A stepwise increase in CRP was found depending on the number of ＞50% stenotic vessels: 0.55 mg/L in 1-vessel, 0.71 mg/L in 2-vessel, and 0.86 mg/L in 3-vessel diseases (P＜0.001). However, legumain did not differ among 1-, 2-, and 3-vessel diseases (5.20, 4.93, and 5.01 ng/mL, respectively). Of 225 patients with CAD, 40 (18%) had complex lesions. No difference was found in CRP levels between patients with CAD with and without complex lesions (0.60 [0.34, 1.53] vs. 0.60 [0.32, 1.51] mg/L). Notably, legumain levels were higher in patients with CAD with complex lesions than without such lesions (6.05 [4.64, 8.64] vs. 4.93 [3.76, 6.52] ng/mL, P＜0.01). In multivariate analysis, legumain levels were not a factor for CAD, but were a factor for complex lesions. The odds ratio for complex lesions was 2.45 (95% CI=1.26-4.79) for legumain ＞5.5 ng/mL. CONCLUSION: Plasma legumain levels were associated with the presence of complex coronary lesions.

Blood levels of heme oxygenase-1 versus bilirubin in patients with coronary artery disease.

OBJECTIVE: Heme oxygenase-1 (HO-1) degrades heme to CO, iron, and biliverdin/bilirubin. Although serum bilirubin levels were often reported in patients with coronary artery disease (CAD), HO-1 levels in patients with CAD and the association between HO-1 and bilirubin levels have not been clarified. METHODS: We measured plasma HO-1 and serum total bilirubin levels in 262 patients undergoing coronary angiography. RESULTS: HO-1 levels were higher in patients with CAD than without CAD (median 0.46 vs. 0.35 ng/mL, P < 0.01), but bilirubin were lower in patients with CAD than without CAD (0.69 vs. 0.75 mg/dL, P < 0.02). Notably, HO-1 levels in CAD(-), 1-vessel, 2-vessel, and 3-vessel disease were 0.35, 0.51, 0.45, and 0.44 ng/mL, and were highest in 1-vessel disease (P < 0.05). Bilirubin levels in CAD(-), 1-vessel, 2-vessel, and 3-vessel disease were 0.75, 0.70, 0.68, and 0.66 mg/dL (P = NS). No correlation was found between HO-1 and bilirubin levels. In multivariate analysis, HO-1 levels were a significant factor for CAD independent of atherosclerotic risk factors and bilirulin levels. Odds ratio for CAD was 2.32 (95%CI = 1.29-4.17) for high HO-1 (>0.35 ng/mL). CONCLUSIONS: Patients with CAD were found to have high HO-1 and low bilirubin levels in blood, but no correlation was found between HO-1 and bilirubin levels.

Pacemaker malfunction after acute myocardial infarction in a patient with wrap-around left anterior descending artery supplying the right ventricular apex.

A 50-year-old man with a dual-chamber pacemaker was admitted to our hospital complaining of chest pain. Anterior ST segment elevation myocardial infarction (STEMI) was diagnosed. Emergency coronary angiography revealed total occlusion of the proximal left anterior descending artery (LAD), and primary percutaneous coronary intervention was performed. Angiograms showed that the LAD was wrapped around the apex of both ventricles. On day 8, ventricular fibrillation and cardiopulmonary arrest occurred due to elevation of the pacing threshold because of pacemaker malfunction. The pacemaker was upgraded to an implantable cardioverter-defibrillator and the lead was inserted into the right ventricular septum. Myocardial scintigraphy with thallium-201 and technetium-99m pyrophosphate located the infarct zone around the apex of both ventricles. We conclude that pacing failure of the right ventricular lead occurred in this case of LAD occlusion due to a LAD supplying the right ventricular apex. Clinicians should be aware of the possibility of pacemaker failure in patients presenting with anterior STEMI due to a wrap-around LAD. .

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.