基于干细胞技术进行TRPC1调节心肌肥厚发生的分子机制研究

Cardiac hypertrophy is an adaptive response to the myocardial work overload, leading to heart failure, severe arrhythmias and sudden cardiac death, which is a serious threat to human health. Mechanical stress is the most important factor to initiate the cardiac hypertrophy. Recent studies have shown that TRPC1, a mechanical stress-sensitive ion-channel gene, is up-regulated in cardiac hypertrophic mice, indicating that TRPC1 may play an important role in regulating the development of cardiac hypertrophy. However, the underlying mechanism is illusive. Using CRISPR/Cas genomic editing technology, we have successfully established TRPC1 knock-out (KO) H9 human embryonic stem cell line. In this study, we will firstly generate H9-derived cardiomyocytes (H9-CMs) using the cardiac differentiation protocol, reveal the phenotype of the TRPC1 KO H9-CMs to elucidate the role of TRPC1 in regulating cardiac function under physiological condition; we will then expose H9-CMs with phenylephrine (PE) to establish the cellular hypertrophic model, confirm and validate the regulatory role of TRPC1 in cardiac hypertrophy, screen cardiac hypertrophy-related gene targets that are associated with TRPC1, and deeply analyze down-stream signaling pathways. We believe this project will fully elucidate the role of TRPC1 in the development of cardiac hypertrophy and its underlying mechanism, which will provide new clues for developing new diagnosis and/or treatment approaches for cardiac hypertrophy.

心肌肥厚是心肌工作超负荷的一种适应性反应，最终导致心力衰竭、恶性心律失常及心源性猝死，严重威胁人类健康。机械牵拉是心肌肥厚最重要的始动因素。TRPC1是一种机械牵拉敏感性离子通道，应用小鼠模型的研究显示了TRPC1表达与心肌肥厚之间存在正相关性，提示TRPC1对心肌肥厚发生起着关键作用。然而，TRPC1如何调节心肌肥厚的分子机制并不清楚。前期研究中，本项目组应用CRISPR技术成功建立了TRPC1敲除的H9人胚胎干细胞株。本项目将应用心肌分化技术获取H9来源的心肌细胞（H9-CMs），揭示生理状态下TRPC1对于心肌细胞功能的调节作用；采用苯肾上腺素诱导H9-CMs建立“人源性”心肌肥厚模型，明确TRPC1对于心肌肥厚的调节作用，筛选心肌肥厚发生中与TRPC1相关的靶基因和信号通路。通过以上研究，本项目将阐明TRPC1调节心肌肥厚发生的分子机制，为发展心肌肥厚诊断和治疗新方法打下基础。

心肌肥厚是一种对负荷增加的适应性反应，其典型特征是左心室重量增加和左心室壁增厚。我们的结果表明，肥厚型心肌病和心力衰竭患者的心脏组织中瞬时受体电位通道1（TRPC1）的表达显著高于正常人。为了更好地解析TRPC1调控人心肌细胞肥大的机制，我们用CRISPR/Cas9基因组编辑技术进行了TRPC1基因敲除，成功建立了人多能干细胞系。我们证明，TRPC1基因敲除显著减弱佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）诱导的心肌细胞肥大表型，这与NF-κB信号通路的异常激活相关。相反，TRPC1基因的过度表达可诱导心肌细胞肥大，而抑制NF-κB可逆转心肌细胞肥大。我们建立了一个稳定的人源性心肌细胞肥大模型，阐明了TRPC1介导的心肌肥大的分子机制，通过靶向TRPC1有助于开发治疗HCM和HF的药物。