阿诺碱受体和钙调神经磷酸激酶在压力超负荷早期心肌代偿性纤维化中的作用和机制研究

It has been known that adaptive increases in myocardial firosis is important for maintaining of cardiac function after pressure overload.Recently we have reported that in ryanodine receptor type 2 (RyR-2)deficient mice, heart function was early impaired and myocardial fibrosis and calcineurin (CaN) activity were decreased after pressure overload, suggesting that RyR-2-CaN regulates adaptive myocardial fibrosis. Growth factors BMP-2 and TGFbeta are important factors regulating myocardial fibrosis while cardiac fibroblasts are main cell types contributing myocardial fibrosis. We therefore suppose that RyR-2 and CaN in cardiomyocytes control production and secretion of BMP-2 and TGFbeta which act on cardiac fibroblasts by paracrine mechanism to maintain adaptive myocardial fibrosis and heart function. This project aims to examine whether RyR-2-CaN regulate expression and secretion of BMP-2 and TGFbeta in cardiomyocytes and whether the secreted BMP-2 and TGFbeta exert effect on cardiac fibroblasts and adaptive myocardial fibrosis through paracrine mechanism in pressure overload mice and culture cell models by using the methods of pharmocological drugs, gene overexpresion and gene knockdown to upregulate or downregulate RyR-2 and CaN both in vitro and in vivo.

心肌间质纤维化稳态是维持正常心功能的基础。我们近期发现，在阿诺碱受体（RyR-2）缺失小鼠，压力超负荷状态下较早发生心功能损害，并伴有心肌纤维化的缺少和钙调神经磷酸激酶（CaN）的下调，提示RyR-2-CaN与心肌代偿性纤维化有关。细胞因子BMP-2和TGFb是调节心肌纤维化的重要因子，而心肌成纤维细胞是产生心肌细胞外基质的主要细胞。据此推测，心肌细胞内RyR-2-CaN调控心肌细胞产生和分泌BMP-2和TGFb，通过旁分泌机制作用于心肌成纤维细胞而维持心肌间质纤维化的稳态。当RyR-2-CaN活性不足时，这种稳态发生紊乱而引起心肌纤维化减少。本实验拟通过药物、转基因、基因敲低等手段上调或下调心肌细胞内RyR-2-CaN，观察压力超负荷后心肌细胞产生和分泌BMP-2和TGFb的变化及对心肌成纤维细胞活性和功能的影响，阐明RyR-2-CaN在压力超负荷时心肌代偿性纤维化中的作用和机制。

阿诺碱受体（RyR-2）介导的钙离子释放是影响心肌肥厚的重要因素，然而RyR-2在心肌纤维化中的作用仍是有待阐明的重要问题。本研究中，我们对RyR-2进行敲减，构建心肌肥厚模型，，利用荧光定量PCR、蛋白免疫印迹和酶联免疫吸附等分子和细胞生物学对其分析，发现在心肌细胞中RyR-2正调控生长因子TGFβ的表达和分泌，TGFβ通过旁分泌作用抑制成纤维细胞基质金属蛋白酶的表达进而促进胶原的表达，从而导致心肌纤维化。本研究丰富对高血压心肌肥厚早期代偿机制的认识，为更加科学有效的干预高血压心脏损害提供新的线索和思路。