Transcription Factors MYOCD, SRF, Mesp1 and SMARCD3 Enhance the Cardio-Inducing Effect of GATA4, TBX5, and MEF2C during Direct Cellular Reprogramming

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca2+ transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.对特定组合的主调控基因（master regulator genes）进行瞬时过表达，可有效诱导细胞重编程（cellular reprogramming）：即从分化细胞谱系中获得替代性的预期表型。这在心脏再生医学中尤为重要——心脏自身缺乏修复能力，但同时拥有大量成纤维细胞。本研究评估了十种转录因子（transcription factors）将小鼠胚胎成纤维细胞（mouse embryonic fibroblasts）重编程为心肌样细胞（cardiomyocyte-like cells）的心脏诱导能力。单独过表达转录因子MYOCD与SRF，或联合Mesp1与SMARCD3，可增强此前报道的GATA4、TBX5及MEF2C所具备的基础且必要的心脏诱导效应。具体而言，包含五个或七个转录因子的组合可增强心脏报告载体的激活，并诱导心脏特异性基因的上调。全基因表达分析同样证实，当使用MYOCD与SRF时，心脏诱导效果显著更强。横纹交叉细胞的检测高度依赖细胞培养条件，添加丙戊酸与JAK抑制剂可提升该检测效果。尽管我们在重编程细胞中检测到了钙瞬态振荡，但并未观察到静息膜电位的显著变化，也未发现自发收缩的细胞。本研究进一步阐明了参与心脏细胞重编程的转录网络的心脏诱导效应，为心脏再生治疗所需的稳健实验方案的合理设计提供了助力。