Department of Cardiology & National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital & Medical School of Chinese PLA, Beijing, China.
Department of Cardiology, The Affiliated Hospital of Northwest University, Xi'an No.3 Hospital, Xi'an, China.
Stem Cells Dev. 2020 Sep 15;29(18):1229-1239. doi: 10.1089/scd.2020.0025. Epub 2020 Aug 18.
Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising source for cardiac regenerative therapy, and ideal for in vitro cell modeling of cardiovascular diseases and drug screening. Recent studies have shown that rapamycin can promote cardiomyocyte differentiation in various stem cells. However, how rapamycin affects cardiomyocyte differentiation of iPSCs is still not fully understood. This study aimed to investigate the effect of rapamycin on cardiomyocyte differentiation based on embryoid body (EB) method. First, to determine the autophagy induction protocol, different concentrations of rapamycin were applied in hEBs on day 6. The autophagy was most significant when applying rapamycin at 1 μM for 48 h, demonstrating by the LC3II/LC3I ratio and p62 expression. Then, 1 μM rapamycin was applied for 48 h at different time points of cardiomyocyte differentiation to investigate the role of rapamycin in this process. Compared with control, rapamycin applied on days 0-4 of differentiation significantly decreased the proportion of beating EBs and expression of cardiomyocyte-specific genes, while rapamycin applied on days 4-14 significantly increased them. Among all groups, rapamycin applied on days 4-6 achieved highest cardiomyocyte differentiation efficiency. Furthermore, using autophagy inhibitor NHCl and GSK-3β inhibitor CHIR-99021, we found rapamycin-induced autophagy promoted cardiomyocyte differentiation at middle stage by negatively regulating the Wnt/β-catenin signaling pathway. These results suggest that rapamycin regulates EB-based cardiomyocyte differentiation in a stage-dependent manner, and the negative regulation of Wnt/β-catenin signaling pathway by autophagy was involved in the prodifferentiation effect of rapamycin at middle stage.
诱导多能干细胞衍生的心肌细胞（iPSC-CMs）是心脏再生治疗的有前途的来源，非常适合心血管疾病的体外细胞建模和药物筛选。最近的研究表明，雷帕霉素可以促进各种干细胞中的心肌细胞分化。然而，雷帕霉素如何影响 iPSC 的心肌细胞分化尚不完全清楚。本研究旨在基于胚状体（EB）方法研究雷帕霉素对心肌细胞分化的影响。首先，为了确定自噬诱导方案，在第 6 天的 hEB 中应用不同浓度的雷帕霉素。当在 1μM 下应用 48h 时，自噬最明显，表现为 LC3II/LC3I 比值和 p62 表达。然后，在不同的心肌细胞分化时间点应用 1μM 雷帕霉素 48h，以研究雷帕霉素在该过程中的作用。与对照组相比，分化第 0-4 天应用雷帕霉素显著降低了搏动 EB 的比例和心肌细胞特异性基因的表达，而分化第 4-14 天应用雷帕霉素则显著增加了它们的表达。在所有组中，分化第 4-6 天应用雷帕霉素达到了最高的心肌细胞分化效率。此外，使用自噬抑制剂 NHCl 和 GSK-3β 抑制剂 CHIR-99021，我们发现雷帕霉素诱导的自噬通过负向调节 Wnt/β-catenin 信号通路促进中期的心肌细胞分化。这些结果表明，雷帕霉素以阶段依赖的方式调节基于 EB 的心肌细胞分化，自噬对 Wnt/β-catenin 信号通路的负向调节参与了雷帕霉素在中期的促分化作用。
