Michael E. De Bakey Department of Surgery, Baylor College of Medicine, 1 Moursund St, Houston, TX-77030, USA.
Department of Surgery, Mount Sinai Hospital, New York, NY, 10029, USA.
Sci Rep. 2022 Jul 6;12(1):11416. doi: 10.1038/s41598-022-15559-y.
Direct cell reprogramming represents a promising new myocardial regeneration strategy involving in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocytes. Adult human cells are relatively resistant to reprogramming, however, likely because of epigenetic restraints on reprogramming gene activation. We hypothesized that modulation of the epigenetic regulator gene p63 could improve the efficiency of human cell cardio-differentiation. qRT-PCR analysis demonstrated significantly increased expression of a panel of cardiomyocyte marker genes in neonatal rat and adult rat and human cardiac fibroblasts treated with p63 shRNA (shp63) and the cardio-differentiation factors Hand2/Myocardin (H/M) versus treatment with Gata4, Mef2c and Tbx5 (GMT) with or without shp63 (p < 0.001). FACS analysis demonstrated that shp63+ H/M treatment of human cardiac fibroblasts significantly increased the percentage of cells expressing the cardiomyocyte marker cTnT compared to GMT treatment with or without shp63 (14.8% ± 1.4% versus 4.3% ± 1.1% and 3.1% ± 0.98%, respectively; p < 0.001). We further demonstrated that overexpression of the p63-transactivation inhibitory domain (TID) interferes with the physical interaction of p63 with the epigenetic regulator HDAC1 and that human cardiac fibroblasts treated with p63-TID+ H/M demonstrate increased cardiomyocyte marker gene expression compared to cells treated with shp63+ H/M (p < 0.05). Whereas human cardiac fibroblasts treated with GMT alone failed to contract in co-culture experiments, human cardiac fibroblasts treated with shp63+ HM or p63-TID+ H/M demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes. These findings demonstrate that p63 silencing provides enhanced rat and human cardiac fibroblast transdifferentiation into induced cardiomyocytes compared to a standard reprogramming strategy. p63-TID overexpression may be a useful reprogramming strategy for overcoming epigenetic barriers to human fibroblast cardio-differentiation.
直接细胞重编程代表了一种很有前途的心肌再生新策略，涉及到将心脏成纤维细胞原位转分化为诱导性心肌细胞。然而，成年人类细胞相对难以进行重编程，这可能是由于重编程基因激活受到表观遗传限制。我们假设，调节表观遗传调节因子基因 p63 可以提高人类细胞心脏分化的效率。qRT-PCR 分析表明，与用 Gata4、Mef2c 和 Tbx5（GMT）处理相比，用 p63 shRNA（shp63）和心脏分化因子 Hand2/Myocardin（H/M）处理新生大鼠和成年大鼠及人类心脏成纤维细胞可显著增加一组心肌细胞标记基因的表达（p<0.001）。FACS 分析表明，与用 GMT 处理相比，shp63+ H/M 处理人类心脏成纤维细胞可显著增加表达心肌细胞标记物 cTnT 的细胞百分比（分别为 14.8%±1.4%、4.3%±1.1%和 3.1%±0.98%；p<0.001）。我们进一步表明，p63 转录激活抑制结构域（TID）的过表达会干扰 p63 与表观遗传调节剂 HDAC1 的物理相互作用，而用 p63-TID+ H/M 处理的人类心脏成纤维细胞与用 shp63+ H/M 处理的细胞相比，心肌细胞标记基因的表达增加（p<0.05）。虽然单独用 GMT 处理的人类心脏成纤维细胞在共培养实验中不能收缩，但用 shp63+ H/M 或 p63-TID+ H/M 处理的人类心脏成纤维细胞在电刺激时表现出钙瞬变，并与周围的新生心肌细胞同步收缩。这些发现表明，与标准重编程策略相比，p63 沉默可增强大鼠和人类心脏成纤维细胞向诱导性心肌细胞的转分化。p63-TID 的过表达可能是克服人类成纤维细胞心脏分化的表观遗传障碍的一种有用的重编程策略。
