Department of Cardiology, Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu 225001, P.R. China.
Department of Cardiology, Northern Jiangsu People's Hospital, Yangzhou, Jiangsu 225001, P.R. China.
Mol Med Rep. 2021 Aug;24(2). doi: 10.3892/mmr.2021.12255. Epub 2021 Jun 29.
Studies have shown that histone H3 at lysine 9 (H3K9me2) is an important epigenetic modifier of embryonic development, cell reprogramming and cell differentiation, but its specific role in cardiomyocyte formation remains to be elucidated. The present study established a model of 5‑Azacytidine‑induced differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes and, on this basis, investigated the dimethylation of H3K9me2 and its effect on cardiomyocyte formation by knockdown of H3K9me2 methylase, euchromatic histone‑lysine N‑methyltransferase 2 (G9a) and H3K9me2 lysine demethylase 3A (KDM3A). The results demonstrated that, in comparison with the normal induction process, the knockdown of G9a could significantly reduce the H3K9me2 level of the MSCs in the induced model. Reverse transcription‑quantitative (RT‑q) PCR demonstrated that the expression of cardiac troponin T(cTnT) was significantly increased. In addition, flow cytometry demonstrated that the proportion of cTnT‑positive cells was significantly increased on day 21. With the knockdown of KDM3A, the opposite occurred. In order to explore the specific way of H3K9me2 regulating cardiomyocyte formation, western blotting and RT‑qPCR were used to detect the expression of key transcription factors including GATA binding protein 4 (GATA‑4), NK2 Homeobox 5 (Nkx2.5) and myocyte enhancer factor 2c (MEF2c) during cardiomyocyte formation. The decrease of H3K9me2 increased the expression of transcription factors GATA‑4, Nkx2.5 and MEF2c in the early stage of myocardial development while the increase of H3K9me2 inhibited the expression of those transcription factors. Accordingly, it was concluded that H3K9me2 is a negative regulator of cardiomyocyte formation and can participate in cardiomyocyte formation by activating or inhibiting key transcription factors of cardiomyocytes, which will lay the foundation for the optimized induction efficiency of cardiomyocytes in and clinical applications.
研究表明，组蛋白 H3 赖氨酸 9 （H3K9me2）是胚胎发育、细胞重编程和细胞分化的重要表观遗传修饰物，但它在心肌细胞形成中的具体作用仍有待阐明。本研究建立了 5-氮杂胞苷诱导大鼠骨髓间充质干细胞（MSCs）分化为心肌细胞的模型，并在此基础上通过敲低 H3K9me2 甲基转移酶 euchromatic histone-lysine N-methyltransferase 2（G9a）和 H3K9me2 赖氨酸去甲基化酶 3A（KDM3A）研究 H3K9me2 的二甲基化及其对心肌细胞形成的影响。结果表明，与正常诱导过程相比，敲低 G9a 可显著降低诱导模型中 MSCs 的 H3K9me2 水平。逆转录-定量（RT-q）PCR 表明心肌肌钙蛋白 T（cTnT）的表达显著增加。此外，流式细胞术表明 cTnT 阳性细胞的比例在第 21 天显著增加。敲低 KDM3A 则相反。为了探索 H3K9me2 调节心肌细胞形成的具体方式，使用 Western blot 和 RT-qPCR 检测了包括 GATA 结合蛋白 4（GATA-4）、NK2 同源盒 5（Nkx2.5）和肌细胞增强因子 2c（MEF2c）在内的关键转录因子在心肌细胞形成过程中的表达。H3K9me2 的减少增加了心肌发育早期转录因子 GATA-4、Nkx2.5 和 MEF2c 的表达，而 H3K9me2 的增加抑制了这些转录因子的表达。因此，H3K9me2 是心肌细胞形成的负调节剂，可通过激活或抑制心肌细胞的关键转录因子参与心肌细胞形成，为心肌细胞的优化诱导效率和临床应用奠定基础。
