内质网应激UPR-ERAD级联对心肌梗死后缝隙连接重构的影响及稳心颗粒的干预研究

The abnormal electrophysiological substrate induced by gap junction remodeling is an important cause of sudden cardiac death after myocardial infarction. Our previous National Natural Science Fund program found that wenxin granule can reduce the inhibition of connexins synthesis induced by miR-1. But there is no research on connexins degradation aspect in this program. Endoplasmic reticulum stress the unfolded protein response (UPR) - endoplasmic reticulum associated degradation (ERAD) cascade is a necessary pathway in regulation of protein degradation. But how does it affect myocardial gap junction remodeling is the research blind spot. The hypothesis of current program is that endoplasmic reticulum stress UPR-ERAD cascade is one of the mechanisms to regulate gap junction remodeling after myocardial infarction, and wenxin granule can intervene in this cascade to protect the gap junction. Current program will establish a rat model of myocardial infarction, and will study the initiation, regulation and execution mechanism of UPR-ERAD cascade, further will study the relationship between UPR-ERAD cascade and gap junction remodeling; Cell experiment will verify the specific ways of connexins degradation and effects of wenxin granule under conditions of excessive activation or inhibition by use of UPR-ERAD specific agonists or antagonists. If the current hypothesis is true, a relatively complete molecular mechanism of the synthesis and degradation of connexins will be described through the current and the previous programs which complement each other. At the same time, the current program also provides a new target and a potential alternative and complementary traditional Chinese medicine for the prevention and treatment of arrhythmia after myocardial infarction.

缝隙连接重构导致的电生理基质异常是心梗后心源性猝死的重要原因。前一个基金发现稳心颗粒能减轻miR-1对缝隙连接蛋白的合成抑制，但对其降解的影响不明。内质网应激未折叠蛋白反应（UPR）-内质网相关降解（ERAD）级联是调节蛋白降解的必经通路，其如何影响心肌缝隙连接重构的病理机制目前是研究盲点。项目假说：内质网应激UPR-ERAD级联是调控心梗后缝隙连接重构的机制之一，稳心颗粒能干预这一级联改善缝隙连接重构。本项目拟建立心梗大鼠模型，研究UPR-ERAD级联的启动、调节、执行机制及其与缝隙连接重构的相关性；细胞实验借助UPR-ERAD特异性激动或阻滞剂，验证UPR-ERAD级联过度激活或抑制条件下引起缝隙连接降解的具体途径及稳心颗粒的作用。如假说成立，将与前一个基金相辅相成，较完整的阐述心梗后缝隙连接蛋白合成和降解稳态失衡的分子机制，同时也为心梗后心律失常的防治提供一个新靶点和可供选择的中药。

心肌梗死是威胁人类健康的严重疾病，绝大多数心肌梗死病人都会发生心律失常，缝隙连接重构导致的电生理基质异常是心梗后心源性猝死的重要原因。在心梗后心律失常病理过程中缝隙连接蛋白代谢异常的分子机制仍需深入研究。内质网应激未折叠蛋白反应（UPR）-内质网相关降解（ERAD）级联是调节蛋白降解的必经通路，其影响心肌缝隙连接的病理机制和中医药的干预作用值得关注。本项目首先firstly采用冠脉结扎法制作心肌梗死大鼠模型，研究心脏组织病理的变化与心律失常的关联，分析缝隙连接蛋白43（Cx43）与内质网应激UPR通路的变化及相关关系。接着nextly，从基因和蛋白水平分别观察稳心颗粒对缝隙连接相关基因、蛋白以及心脏胶原表达的干预作用。最后eventually，从动物和细胞不同层面，进一步研究了稳心颗粒干预内质网应激UPR-ERAD级联的启动、调节、执行过程保护缝隙连接蛋白的分子机制。研究结果显示，心脏组织病理变化是导致心梗后心律失常的重要因素，特别是Cx43与心律失常高度相关，并且内质网应激UPR通路与Cx43表达异常存在关联，是保护Cx43的重要靶点。进一步研究显示，稳心颗粒能够调节microRNA表达而改善缝隙连接及胶原重构，稳心颗粒保护Cx43的机制为：作用于蛋白激酶R样内质网激酶（PERK）、活化转录因子6（ATF6）和X盒结合蛋白-1（XBP1）等而抑制UPR的过度激活，作用于甘露糖苷酶-Ⅰ样蛋白（EDEM）和泛素酶而调节ERAD的泛素化降解过程，此外稳心颗粒对自噬-溶酶体途径的过度激活也有部分作用，但结合细胞水平验证结果发现，稳心颗粒保护Cx43的最主要机制是在UPR-ERAD级联上游启动环节抑制UPR的过度激活和在下游执行环节作用于凋亡信号分子从而抑制Cx43经胱天蛋白酶水解途径降解。本研究为揭示心肌梗死后心律失常与缝隙连接重构的关系，以及缝隙连接蛋白代谢失衡的分子机制提供了实验室依据，同时也为心梗后心律失常的防治提供了新的靶标途径和可供选择的中药。