Department of Haematology, Taizhou People's Hospital, Taizhou, Jiangsu 225300, P.R. China.
Department of Cardiology, Taizhou People's Hospital, Taizhou, Jiangsu 225300, P.R. China.
Mol Med Rep. 2023 Feb;27(2). doi: 10.3892/mmr.2022.12908. Epub 2022 Dec 9.
Biological pacemakers, made of pacemaker-like cells, are promising in the treatment of bradyarrhythmia; however, the inefficiency of stem cell differentiation into pacemaker-like cells has limited their clinical application. Previous studies have reported that histone H3 at lysine 9 (H3K9) methylation is widely involved in the proliferation and differentiation of cardiomyocytes, but the specific role of H3K9 dimethylation (H3K9me2) in the formation of pacemaker cells remains unclear. The present study evaluated the functional role of H3K9me2 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) into pacemaker-like cells. Rat BMSCs pretreated with the euchromatic histone lysine methyltransferase 2 (G9a) inhibitor BIX01294 were transfected with a T-box 18 overexpression plasmid to induce BMSCs to form pacemaker-like cells. The induced pacemaker-like cells were analyzed using reverse transcription-quantitative PCR (RT-qPCR) and immunofluorescence to assess the efficiency of differentiation. The enrichment of H3K9me2 in the hyperpolarized-activated cyclic nucleotide-gated cation channel (HCN)4 promoter region was assessed by chromatin immunoprecipitation (ChIP). In addition, BIX01294 was injected into rats, and the protein and mRNA expression levels of HCN4 were assessed using western blotting and RT-qPCR. After interference with G9a using BIX01294, ChIP results demonstrated that H3K9me2 levels in the promoter region of HCN4 were markedly decreased. Immunofluorescence and RT-qPCR demonstrated that the protein expression levels of certain cardio-specific proteins in the treated group were significantly higher compared with those in the untreated group. experiments demonstrated that interference with G9a could cause pathological hypertrophy. Furthermore, and inhibition of G9a could increase the differentiation and proliferation of pacemaker-like cells by decreasing the levels of H3K9me2 in the promoter region of HCN4 gene.
生物起搏器由类似起搏器的细胞制成，在治疗心动过缓方面具有广阔的应用前景；然而，干细胞向类似起搏器的细胞分化效率低下限制了其临床应用。先前的研究表明，组蛋白 H3 在赖氨酸 9 位的甲基化（H3K9）广泛参与心肌细胞的增殖和分化，但 H3K9 二甲基化（H3K9me2）在起搏细胞形成中的具体作用尚不清楚。本研究评估了 H3K9me2 在骨髓间充质干细胞（BMSC）向起搏样细胞分化中的功能作用。用 euchromatic histone lysine methyltransferase 2（G9a）抑制剂 BIX01294 预处理大鼠 BMSC 后，转染 T-box 18 过表达质粒诱导 BMSC 形成起搏样细胞。通过逆转录定量 PCR（RT-qPCR）和免疫荧光分析评估分化效率。通过染色质免疫沉淀（ChIP）评估 H3K9me2 在超极化激活环核苷酸门控阳离子通道（HCN）4 启动子区域的富集情况。此外，将 BIX01294 注射到大鼠体内，通过 Western blot 和 RT-qPCR 评估 HCN4 的蛋白和 mRNA 表达水平。用 BIX01294 干扰 G9a 后，ChIP 结果表明 HCN4 启动子区域的 H3K9me2 水平明显降低。免疫荧光和 RT-qPCR 表明，与未处理组相比，处理组中某些心型特异性蛋白的蛋白表达水平显著升高。实验表明，干扰 G9a 可导致病理性肥大。此外，抑制 G9a 可通过降低 HCN4 基因启动子区域的 H3K9me2 水平，增加起搏样细胞的分化和增殖。
