FSTL1促进心肌细胞增殖与心脏再生机理的研究

Mammalian heart cannot regeneration after myocardial infarction because mature cardiomyocytes cannot proliferate. An important goal of cardiac regenerative medicine is to find ways to promote cardiomyocyte proliferation. We found FSTL1 secreted from the epicardium can promote cardiomyocyte proliferation and cardiac regeneration, however it is not clear which protein is the FSTL1 receptor and which signaling pathway is downstream of FSTL1 in cardiomyocytes. Our preliminary data showed that FSTL1 secreted from the cardiomyocyte is highly N-linked glycosylated and not able to promote regeneration, and FSTL1 can active Jak2-dependent signaling pathway. Therefore, we hypothesize that N-linked glycosylation can regulate the function of FSTL1 to promote cardiomyocyte proliferation, which is transduced by JAK2-dependent inflammation response signaling pathways. To test our hypothesis, we propose to study the mechanism of FSTL1 promoting cardiomyocyte proliferation and cardiac regeneration. We will analyze the role of N-linked glycosylation in FSTL1’s ability to promote cardiomyocyte proliferation through mutating Asparagines (Ns) in FSTL1 and testing its function, and generating mutant mice carrying FSTL1 with reduced N-linked glycosylation, and examinating its role in cardiac development and regeneration. We will also identify the receptor(s) and downstream signaling pathway(s) of FSTL1 in cardiomyocyte through siRNA screen and protein mass spectrometry, and further optimize methods to promote cardiomyocyte proliferation through manipulating the identified pathway(s). This study will provide new drug target(s) and possible therapeutic method(s) to treat myocardial infarction.

哺乳动物心脏受损后因心肌细胞失去增殖能力难以再生，如何刺激心肌细胞增殖是心脏再生医学的重要课题。申请人发现心外膜分泌的FSTL1可促进心肌细胞增殖与心脏再生，但其在心肌细胞中的受体和下游信号通路还未被明确鉴定。前期研究发现心肌细胞分泌的高糖基化FSTL1无促增殖作用，而JAK2可被FSTL1激活，因此申请人推测N连接糖基化可调控FSTL1促心肌增殖的功能，而FSTL1通过激活依赖JAK2的炎症反应信号通路促进心肌细胞增殖。为验证此假设，本项目将针对FSTL1促进心肌增殖与心脏再生的机理进行研究。通过对天冬酰胺位点的突变，检验N连接糖基化对FSTL1功能的影响，并建立FSTL1重要天冬酰胺突变的小鼠模型，检验其对心脏发育与再生过程的作用；通过siRNA筛选、蛋白质谱等方法鉴定FSTL1在心肌细胞中的受体和下游信号通路，优化促进心肌增殖的方法，为治疗心肌梗死提供新的药物靶点和治疗手段。

哺乳动物心脏受损后因心肌细胞失去增殖能力难以再生，如何刺激心肌细胞增殖是心脏再生医学的重要课题。为寻找促进心肌细胞增殖的方法和研究心肌细胞增殖的机制，项目原计划鉴定N糖基化对Fstl1促进心肌细胞增殖功能的影响，但前合作者于项目执行初期（2018年）将此结果独立发表，导致项目原计划内容失去新颖性。因此项目组围绕本项目宏观目标，调整研究计划，进行了针对心肌细胞增殖的高内涵筛选，以发现新的可以促进心肌细胞增殖的小分子和信号通路。项目组通过针对人类iPSC来源的心肌细胞增殖的高内涵筛选，发现L型钙离子通道抑制剂可促进心肌细胞增殖，其中尼莫地平可在体外促进心肌细胞增殖，并在小鼠心梗模型中促进心脏再生；机制研究发现尼莫地平影响L型钙离子通道和Wnt信号受体Lrp5的相互作用，激活Wnt信号通路，导致心肌细胞的增殖。项目组通过高内涵筛选，发现miR-199a-3p促进心肌细胞增殖的下游功能性靶点基因Cd151和Ppp2r5e，并发现Cd151通过p38激酶抑制心肌细胞增殖，而Ppp2r5e通过黏着斑抑制心肌细胞增殖。项目组还通过单细胞测序技术揭示了心肌球细胞（一种心脏前体细胞）的内部异质性、鉴定出功能特异性亚群，并在体内验证Sca-1阳性细胞为促进心脏损伤修复的主要细胞类群。综上，项目组利用高内涵筛选和干细胞测序，发现了新的调控心肌细胞增殖的小分子和信号通路，和促进心脏再生和修复的小分子药物和心脏前体细胞类型，为理解心肌细胞的增殖调控提供了新的分子机制，也为治疗心脏疾病提供了新的药物靶点和可行性方案。