αB-晶体蛋白在mTOR活化引起的心肌肥厚中的作用

Hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in adolescence and athletes. Its underlying mechanisms remain poorly understood and there is no effective treatment for this disease. Mammalian target of rapamycin (mTOR) signaling is critical in maintaining the hemostasis of cardiomyocytes. Hyperactivation of mTOR led to cardiac hypertrophy in mice. Our recent studies have demonstrated that αB-crystallin is a downstream target protein of mTOR signaling and one of the central controllers of cell growth, proliferation and cancer development. Consequently, we hypothesize that activation of mTOR induces cardiac hypertrophy through upregulation of αB-crystallin and inhibition of αB-crystallin abolishes mTOR activation-induced cardiac hypertrophy. To test this hypothesis, we will generate an mTOR activation-induced cardiac hypertrophy mouse model through inducible TSC1 knockout in cardiomyocytes. These mice will then be crossmated to αB-crystallin knockout mice to check whether the hypertrophy phenotype of TSC1 knockout mice could be ameliorated or even disappears. Besides that, we will create a pressure overload-induced cardiac hypertrophy model as control through transverse aortic arch constriction, evaluate the relationship between the expression level of αB-crystallin, mTOR activity and cardiac hypertrophy, and examine the correlation between mTOR activity and the level of αB-crystallin in biopsyed myocardial tissues from HCM patients. In a word, we will elucidate the role of αB-crystallin in the mTOR activation-induced cardiac hypertrophy in this study. And the completion of this project may expand our understanding of the pathogenesis of cardiomyopathy, which may provide a better therapeutic strategy for the treatment of HCM.

肥厚型心肌病是青少年和运动员心源性猝死的首要原因，其发病机制不甚明了，缺乏有效的治疗方法。已知雷帕霉素靶蛋白（mTOR）信号通路过度活化可引起小鼠肥厚型心肌病。我们发现αB-晶体蛋白为mTOR通路下游的一种效应蛋白，对细胞增生起决定性作用。因此推断：mTOR活化通过上调αB-晶体蛋白的表达引起心肌肥厚，阻断该蛋白的表达可治疗mTOR活化引起的心肌病。本课题拟通过可诱导性敲除心肌mTOR抑制基因Tsc1，建立mTOR活化导致的心肌肥厚动物模型，在此基础上敲除aB-晶体蛋白基因，观察心肌肥厚能否减轻或消失。结合横向主动脉缩窄致心肌肥厚模型，研究αB-晶体蛋白与mTOR活性、心肌肥厚的关系。并在肥厚型心肌病患者心肌组织中验证mTOR活性与αB-晶体蛋白表达的相关性。总之，本研究旨在阐明αB-晶体蛋白在mTOR活化引起心肌肥厚发生中的作用，为临床防治肥厚型心肌病提供新的理论依据和治疗靶点。

背景：TSC1或TSC2基因突变或者缺失会导致mTOR信号通路活化。mTOR是一种丝氨酸/苏氨酸蛋白激酶，受外界生长因子和营养信号刺激激活后，调控细胞生长，增殖，代谢等多种生命过程。晶体蛋白（αB-crystallin）是一种小分子热休克蛋白，具有分子伴侣的功能，在维持心脏动态平衡中发挥重要作用。本实验室前期在mTOR活化的成纤维细胞模型和裸鼠成瘤模型中发现，mTOR可通过上调αB-crystallin促进肿瘤发生。.方法：本课题首先构建诱导型心脏敲除Tsc1小鼠(Tsc1-cKO)、αB-crystallin敲除小鼠(αB-KO)、诱导型心脏组织双敲Tsc1; αB-crystallin小鼠(dKO)及对照小鼠(Control)，从形态学、组织学、细胞超微结构、转录水平、蛋白质表达等几个层面分析mTOR下游分子αB-crystallin在心肌病中的作用。.结果：组织学染色分析发现Tsc1-cKO组小鼠发生心肌细胞损伤：Tsc1-cKO组小鼠心脏纤维化增生，dKO小鼠心脏纤维化明显缓解，细胞超微结构结果也观察到了相同的结果；Tsc1-cKO组小鼠心肌细胞能够检测到细胞凋亡，在其他三种基因型小鼠未能检测到；四种基因型小鼠心肌细胞PCNA染色差别不大，但是Tsc1-cKO和dKO组小鼠心脏成纤维细胞PCNA染色阳性增强，即成纤维细胞处于增殖旺盛阶段；细胞超微结构结果显示Tsc1-cKO组小鼠心肌细胞线粒体肿胀，嵴减少、肌纤维变性、细胞连接异常，dKO组小鼠线粒体、肌纤维、细胞连接结构恢复正常。Tsc1-cKO组小鼠发生心脏肥大，心功能减弱，50%小鼠发生死亡；dKO组小鼠心脏体积恢复正常，心功能改善，20%生死亡，即敲除αB-crystallin减少心脏Tsc1敲除小鼠心脏肥大，改善小鼠心功能，降低小鼠死亡率。进一步分析分子机制发现，αB-crystallinα敲除抑制mTOR信号通路，因此TSC1通过抑制αB-crystallinα调控小鼠心功能和心肌细胞存活。.结论：mTOR下游分子αB-crystallin在心肌病中发挥重要作用，TSC1通过抑制αB-crystallin调控小鼠心功能和心肌细胞存活。