Chemical Enhancement of Direct Cardiac Reprogramming In Vitro and In Vivo

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 (GMT), can convert fibroblasts into iCMs, albeit with low efficiency in vitro. Here, we screened 5,500 compounds in primary cardiac fibroblasts and found that a combination of the transforming growth factor (TGF)-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency eight-fold when added to GMT-overexpressing cardiac fibroblasts. The small-molecules also enhanced the speed and the quality of cell conversion, as we observed beating cells as early as 1 week after reprogramming compared to 6–8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared to those exposed to only GMT. Human cardiac reprogramming was similarly enhanced upon TGF-b and WNT inhibition and was achieved most efficiently with GMT plus Myocardin. Thus, TGF-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.

将原位心脏成纤维细胞重编程为诱导式心肌样细胞（induced cardiomyocyte-like cells，下称iCMs），是心脏再生领域极具潜力的治疗策略。三种心脏转录因子——Gata4、Mef2c与Tbx5的联合应用（下称GMT），可将成纤维细胞转化为iCMs，但该方法在体外的转化效率偏低。本研究在原代心脏成纤维细胞中对5500种化合物进行筛选，发现当向过表达GMT的心脏成纤维细胞中联合加入转化生长因子β抑制剂（transforming growth factor β inhibitor，下称TGF-β抑制剂）SB431542与WNT抑制剂（WNT inhibitor）XAV939后，重编程效率可提升8倍。此类小分子同时还优化了细胞转化的速度与质量：相较于仅使用GMT的对照组需等待6~8周才能观测到搏动的心肌细胞，联用小分子的实验组最早可在重编程启动仅1周后便观察到自主搏动的心肌细胞。体内实验层面，心肌梗死（myocardial infarction）模型小鼠于梗死后连续接受GMT、SB431542与XAV939联合处理2周后，相较于仅接受GMT治疗的对照组小鼠，其体内重编程效率与心脏功能均得到了显著改善。人类心脏重编程经TGF-β与WNT联合抑制处理后同样实现了效率提升，其中以GMT联合心肌素（Myocardin）的组合方案转化效率最高。综上表明，TGF-β与WNT抑制剂可联合增强GMT介导的体外及体内定向心肌重编程效率，为心脏再生领域提供一套更具鲁棒性的技术平台。