Rnd3/ROCK1信号轴在介导心肌细胞凋亡过程中的作用研究

We have previosly demonstrated that ROCK1 activation in turn promoted caspase-3 activation, which resulted in a positive feed-forward regulatory loop promoting.Rnd3 is an endogenous repressor of ROCK1, howerer the biological fuction of Rnd3 in the heart remains unexplored.Very recently, we found significant decreases in Rnd3 protein levels in human failing heart.Moreover, the mice with complete genetic deletion of the Rnd3 gene predispose to the embryonice lethality with heart failure.A significant amount of apoptotic cells were observed in the Rnd3-null mice heart along with the increase in ROCK1 activity. Our preliminary data suggest a potential mechanism in which the downregulation of Rnd3 results in apoptotic cardiomyopathy partially through ROCK1 activation.Thus, by taking advantage of our established Rnd3 haploinsufficient knockout mouse model(Rnd3+/-) as well as a double-knockout mouse line(Rnd3+/-/ROCK1-/-), this proposal is to investigate the pathological function of Rnd3 downregulation in heart failure, and to determine the molecular mechanism of Rnd3/ROCK1 signaling axis in mediating the apoptosis of cardiomyocytes.The outcome of this proposal could be a new therapeutic strategy in the future treatment of cardiac apoptosis, and provide a novel theory and experimental basis for prevention of clinical heart failure.

本课题组前期证实心肌细胞中ROCK1激活促进caspase-3活性增强的正反馈机制在心肌细胞凋亡过程中发挥重要作用。研究表明Rnd3是 ROCK1的内源性抑制剂，但Rnd3在心脏中的作用尚不明了。近期我们发现心衰患者的心脏中Rnd3蛋白表达下调，进一步研究发现Rnd3基因敲除纯合子小鼠极易发生心功能不全性胚胎致死，且心肌细胞凋亡明显并伴ROCK1活性显著增加。上述预实验结果提示Rnd3表达下调可能通过激活ROCK1来介导心肌细胞凋亡进而引起心衰。本项目将利用Rnd3基因敲除鼠（Rnd3+/-）和Rnd3、ROCK1双基因敲除鼠（Rnd3+/-/ROCK1-/-）模型，从整体水平和细胞水平研究Rnd3表达下调在心衰发生发展中的病理作用，并探讨Rnd3/ROCK1信号轴介导心肌细胞凋亡的分子调节机制，其成果将可能成为心肌细胞凋亡干预的新着眼点，为临床心力衰竭的防治提供新的理论和实验依据。

心肌细胞凋亡致心力衰竭是心血管领域亟待解决的难题。本项目假设Rnd3 表达下调通过激活ROCK1 来介导心肌细胞凋亡进而引起心衰，利用Rnd3 基因敲除鼠（Rnd3+/-）和Rnd3、ROCK1 双基因敲除鼠（Rnd3+/-/ROCK1-/-）为模型，结果发现：(1) 较WT小鼠相比，Rnd3+/-小鼠在压力超负荷条件下（建立小鼠主动脉弓缩窄术模型，TAC）出现明显的心力衰竭，且心脏中检测到显著的心肌细胞凋亡伴随caspase-3活性增加和Rho激酶活性升高。 (2) 采用Rho激酶抑制剂fasudil可部分恢复Rnd3 +/-基因敲除小鼠行TAC 手术后的小鼠心脏功能并减轻凋亡性心肌病。(3) Rnd3+/-/ROCK1-/-小鼠同样的可部分而不是完全恢复Rnd3基因缺陷介导的心肌细胞凋亡和心力衰竭表型。综上，本项目数据表明，Rnd3基因对维持小鼠的心脏功能有重要作用，Rnd3基因缺失的小鼠心脏容易发生心力衰竭，而Rho激酶信号的激活是导致这一过程的部分原因。