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 Tcf21^iCre/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 Tcf21^iCre/Tomato/MGTH2A and Tcf21^iCre/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.

中文翻译：

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直接重编程可改善慢性心肌梗死的心脏功能并逆转纤维化

背景：由于成年心肌细胞再生能力较差，心肌梗死（MI）后常驻的心脏成纤维细胞（CF）合成细胞外基质形成纤维化，导致心功能障碍和心力衰竭。目前尚缺乏能够使慢性心肌梗死的心肌再生和逆转纤维化的疗法。心脏转录因子的过度表达，包括Mef2c/Gata4/Tbx5/Hand2 (MGTH)，可以直接将 CF 重编程为诱导心肌细胞 (iCM)，并改善急性 MI 下的心脏功能。然而，体内心脏重编程修复已形成疤痕的慢性心肌梗死的能力尚未确定。方法：我们生成了一种新型 Tcf21 ^iCre/reporter/MGTH2A 转基因小鼠系统，其中他莫昔芬治疗可以诱导常驻 CF 中的 MGTH 和报告基因表达，用于心脏重编程和成纤维细胞谱系追踪。我们首先在体外和体内测试了该转基因系统对急性心肌梗死的功效。^{接下来，我们使用 Tcf21 iCre} /Tomato/MGTH2A 和 Tcf21 ^iCre分析了慢性 MI 下的体内心脏重编程和融合事件分别为/mTmG/MGTH2A小鼠。通过微阵列和单细胞RNA测序来确定体内重编程的心脏修复机制。结果：我们证实了转基因体外和体内心脏重编程对急性心肌梗死的疗效。在慢性 MI 中，体内心脏重编程将约 2% 的常驻 CF 转化为 iCM，其中大多数 iCM 是通过真正的心脏重编程而不是通过与心肌细胞融合产生的。心脏重编程显着改善了慢性心肌梗死的心肌收缩并减少了纤维化。微阵列分析显示，MGTH 的过度表达激活了心脏程序，同时抑制了慢性 MI 中的成纤维细胞和炎症特征。单细胞 RNA 测序表明常驻 CF 由 7 个亚簇组成，其中，慢性心肌梗死时促纤维化 CF 数量增加。心脏重编程通过将促纤维化 CF 转化为静止抗纤维化状态来抑制慢性 MI 中的成纤维细胞基因表达。MGTH 过表达部分通过抑制 Meox1（成纤维细胞活化的核心调节因子）诱导抗纤维化作用。结论：这些结果表明，心脏重编程可以通过心肌再生和减少纤维化来修复慢性 MI。这些发现为开发慢性心肌梗死和心力衰竭的新疗法提供了机会。MGTH 过表达部分通过抑制 Meox1（成纤维细胞活化的核心调节因子）诱导抗纤维化作用。结论：这些结果表明，心脏重编程可以通过心肌再生和减少纤维化来修复慢性 MI。这些发现为开发慢性心肌梗死和心力衰竭的新疗法提供了机会。MGTH 过表达部分通过抑制 Meox1（成纤维细胞活化的核心调节因子）诱导抗纤维化作用。结论：这些结果表明，心脏重编程可以通过心肌再生和减少纤维化来修复慢性 MI。这些发现为开发慢性心肌梗死和心力衰竭的新疗法提供了机会。