CRP3巯基亚硝基化修饰在心肌肥大中的作用及机制研究

The mechanism of myocardial hypertrophy is unknown. Protein S-nitrosylation is regulated by NO synthase (NOS), thioredoxin (Trx) and S-nitrosogluthathione reductase (GSNOR) and so on. Our preliminary experiments showed that there was more S-nitrosylation at cysteine and glycine-rich protein 3 (CRP3) in hypertrophic myocardium. Bioinformatics analysis suggests that cysteines play a vital role in maintaining the function of CRP3 protein. And there was lower expression of GSNOR but higher expression of iNOS in hypertrophic cardiomyocytes, while there was no significant change of nNOS, eNOS and Trx expression. Ang Ⅱ increased binding activity between CRP3 and calcineurin, as well as enhanced NFAT dephosphorylation of CaN's downstream in myocardial cell. Based on these preliminary experiments, the research will use models of cardiomyocyte hypertrophy associated with gene silencing or adenovirus transfection, combined with iNOS knockout mice, cardiomyocyte-specific knockout and overexpression of GSNOR mice subjected to myocardial hypertrophy. The aim of the project is to validate the hypothesis that S-nitrosylation at special cysteine sites of CRP3 by iNOS and/or GSNOR is to regulate CaN-NFAT signal pathway and to promote myocardial hypertrophy. The significance of our research is to discover the novel mechanism of myocardial hypertrophy and provide more unambiguous therapeutic targets for prevention or treatment of cardiovascular diseases.

心肌肥大机制不明，蛋白质巯基亚硝基化修饰受NO合酶（NOS）、硫氧还原蛋白（Trx）和亚硝基谷胱甘肽还原酶（GSNOR）等调节。预实验发现：肥大心肌组织中半胱氨酸和甘氨酸富集蛋白3（CRP3）巯基亚硝基化修饰增加，生物信息学分析提示半胱氨酸在维持CRP3蛋白质功能中作用突出；肥大心肌细胞中GSNOR表达减少而iNOS增加，nNOS、eNOS和Trx均无明显变化；Ang Ⅱ明显增加心肌细胞中CRP3与CaN的结合，增强CaN下游NFAT的去磷酸化水平。因此，我们将联合基因沉默、半胱氨酸位点突变腺病毒转染等手段，运用心肌细胞和iNOS-/-小鼠、心肌特异性敲除或过表达GSNOR小鼠心肌肥大模型验证科学假说：iNOS和/或GSNOR调节CRP3蛋白质半胱氨酸发生巯基亚硝基化修饰，调控CaN-NFAT信号通路，促进心肌肥大。本项目可望揭示心肌肥大新机制，为心肌肥大的防治提供新的干预靶点和思路。

研究背景：心肌肥厚是多种心血管疾病的独立危险因素，是心肌重构的主要表现。蛋白质巯基亚硝基化修饰（S-nitrosylation，SNO）是一种由一氧化氮介导的蛋白质翻译后修饰，参与多种心血管疾病的调节。本研究利用心肌肥厚病人的临床样本以及不同的心肌肥厚在体和离体模型探讨心肌肥厚时肌肉Lim蛋白（CRP3）巯基亚硝基化修饰的水平并探讨CRP3巯基亚硝基化修饰（SNO-CRP3）在心肌肥厚中的作用与机制。.研究结果：我们首先发现SNO-CRP3在心肌肥厚病人、动物心肌肥厚组织和心肌肥厚细胞模型中显著增高。利用LC-MS/MS筛选主动脉缩窄术（TAC）组织中CRP3的修饰位点，结果发现：CRP3的巯基亚硝基化修饰发生在半胱氨酸Cys79位点。将79位点突变C79A，转染NRCMs，给予血管紧张素II（AngII）刺激，结果显示：将79位点突变后，可明显改善AngII诱导的心肌细胞肥厚水平。同时小鼠在体过表达CRP3半胱氨酸79位点突变的腺相关病毒和构建点突变的Knock in小鼠，TAC造模，结果表明79位点突变后可以明显降低外源性SNO-CRP3水平，同时79位点突变可明显逆转TAC诱导的小鼠心肌肥厚水平和心脏功能降低。机制上，我们利用质谱在Ang II 刺激的心肌细胞中筛选出一种可与CRP3结合的蛋白质TLR3，SNO-CRP3明显促进CRP3与TLR3的结合作用，并且79位点突变后明显抑制两者的结合效应。同时敲低TLR3或者在TLR3敲除鼠中，给予肥厚刺激，明显抑制心肌肥厚水平。此外SNO-CRP3增加了TLR3和受体相互作用蛋白3 （Receptor-interacting protein 3，RIP3）之间复合物的形成。同时我们检测到TLR3/RIP3诱导p65/nod样受体pyrin域包含3（NOD-like receptor pyrin domain containing 3，NLRP3）炎症小体激活，给予NLRP3抑制剂或者敲低NLRP3可以明显抑制心肌肥厚。.结论及科学意义：我们的研究结果揭示了SNO-CRP3在心肌肥厚中的重要作用，并证实SNO-CRP3通过激活TLR3介导的RIP3和NLRP3炎性小体激活促进心肌肥厚，为心肌肥厚的研究和治疗提供了新的潜在靶点。