The regulation of cardiomyocyte differentiation is a fundamental aspect of cardiac development and regenerative medicine. PTEN plays important roles during embryonic development. However, its role in cardiomyocyte differentiation remains unknown. In this study, a low-cost protocol for cardiomyocyte differentiation from mouse embryonic stem cells (ESCs) is presented and it is shown that Pten deletion potently suppresses cardiomyocyte differentiation. Transcriptome analysis shows that the expression of a series of cardiomyocyte marker genes is downregulated in Pten^−/− cardiomyocytes. Pten ablation induces Dnmt3b expression via the AKT/FoxO3a pathway and regulates the expression of a series of imprinted genes, including Igf2. Double knockout of Dnmt3l and Dnmt3b rescues the deficiency of cardiomyocyte differentiation of Pten^−/− ESCs. The DNA methylomes from wild-type and Pten^−/− embryoid bodies and cardiomyocytes are analyzed by whole-genome bisulfite sequencing. Pten deletion significantly promotes the non-CG (CHG and CHH) methylation levels of genomic DNA during cardiomyocyte differentiation, and the non-CG methylation levels of cardiomyocyte genes and Igf2 are increased in Pten^−/− cardiomyocytes. Igf2 or Igf1r deletion also suppresses cardiomyocyte differentiation through the MAPK/ERK signaling pathway, and IGF2 supplementation partially rescues the cardiomyocyte differentiation. Finally, Pten conditional knockout mice are generated and the role of PTEN in cardiomyocyte differentiation is verified in vivo.

中文翻译：

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Pten 通过 Dnmt3 调节非 CG 甲基化来调节心肌细胞分化

心肌细胞分化的调节是心脏发育和再生医学的一个基本方面。PTEN 在胚胎发育过程中发挥着重要作用。然而，它在心肌细胞分化中的作用仍然未知。在这项研究中，提出了一种用于从小鼠胚胎干细胞 (ESC) 分化心肌细胞的低成本方案，并表明Pten缺失有效地抑制了心肌细胞分化。转录组分析显示一系列心肌细胞标记基因的表达在Pten ^-/-心肌细胞中下调。Pten消融通过 AKT/FoxO3a 途径诱导Dnmt3b表达并调节一系列印迹基因的表达，包括免疫球蛋白2。Dnmt3l和Dnmt3b的双重敲除挽救了Pten ^-/- ESC的心肌细胞分化缺陷。通过全基因组亚硫酸氢盐测序分析来自野生型和Pten ^{-/-胚状体和心肌细胞的 DNA 甲基化组。}Pten缺失显着促进了心肌细胞分化过程中基因组 DNA 的非 CG（CHG 和 CHH）甲基化水平，并且Pten ^-/-心肌细胞中心肌细胞基因和Igf2的非 CG 甲基化水平增加。Igf2或Igf1r缺失还通过 MAPK/ERK 信号通路抑制心肌细胞分化，补充 IGF2 部分挽救了心肌细胞分化。最后，产生了Pten条件性敲除小鼠，并在体内验证了 PTEN 在心肌细胞分化中的作用。