miR-1促进EGFR配体非依赖性活化在急性心肌缺血再灌注性心律失常中的作用

Acute myocardial ischemia/reperfusion (I/R) arrhymias is an important cause for sudden death of coronary heart disease. Our recent study found that activation of epidermal growth factor receptor (EGFR) was involved in the genesis of reperfusion arrhythmias(RA) by enhancing tyrosine phosphorylation of cardiac Na+ and L-type Ca2+ channels. However, the relevant regulatory factors resulting in ligand-independent EGFR activation remains unclear. In tumor research field, there are many reports on the regulation of EGFR by miRNAs, but whether miR-1 may promote EGFR activation is not understood. Consequently, the aim of this study is to establish acute myocardial I/R model in anaesthetized rats, on one hand at whole level, miRNAs microarray analysis is used to observe the changes of miR-1 expression and EGFR phosphorylation in rat hearts during I/R in order to investigate the correlation between miR-1 and EGFR activation; on the other hand at organ and cellular levels, electrophysiology techniques, molecular biology methods and a cell model of miR-1 low expression are adopted to investigate the changes of ion channel current, the genes and proteins phosphorylation levels of ion channel and EGFR in order to explore the mechanisms of EGFR activation by miR-1. The present study is designed to elucidate the effects and mechanisms of miR-1 on EGFR activation during myocardial I/R, which provide a new train of thought to prevention and treatment reperfusion arrhymias.

急性心肌缺血再灌注性心律失常是冠心病猝死的重要原因。我们最近的研究发现，表皮生长因子受体（EGFR）活化通过激活心肌钠通道和L型钙通道可能是诱发再灌注性心律失常（RA）的重要机制，但EGFR配体非依赖性活化的上游调控因子尚未阐明。虽然miRNAs调控EGFR在肿瘤研究领域屡见报道，但miR-1能否促进EGFR的活化仍不得而知。本课题拟建立大鼠急性心肌缺血再灌注损伤模型，采用miRNAs芯片观察缺血再灌注过程中，心肌组织miR-1、磷酸化EGFR表达的改变，在整体水平探讨miR-1在RA过程中与EGFR活化的相关性；并进一步通过电生理、分子生物学、miR-1低表达模型等方法分析离子通道电流、离子通道和EGFR的基因、蛋白磷酸化水平的变化，从离体和细胞水平探讨miR-1活化EGFR的机制。旨在阐明miR-1在心肌缺血再灌注时对EGFR活化的影响及其分子机制，为RA的发病机制与干预提供新的思路。

我们以前的研究发现，表皮生长因子受体（EGFR）活化通过激活心肌钠通道和L型钙通道可能是诱发缺血再灌注性心律失常的重要机制，但EGFR活化的上游调控因子尚未阐明。最近研究揭示，miR-1可能靶向心脏的PP2A B56而具有致心律失常的潜能。本课题拟在整体、细胞和分子水平探讨在心肌缺血再灌注时miR-1对EGFR活化的影响及其机制。. 我们首先采用了大鼠急性心肌缺血再灌注损伤模型。结果表明，心肌缺血再灌注早期miR-1的水平上调，伴有PP2A B56 mRNA和蛋白表达下降，EGFR活化增加。其后，在缺氧复氧心肌细胞上也得到了相似结果。. 为进一步明确miR-1、PP2A和EGFR三者之间的关系，我们建立了miR-1低表达和高表达心肌细胞模型。研究发现，miR-1高表达可以抑制PP2A B56的表达并活化EGFR，此结果与缺氧复氧过程中的改变相似。反之，抑制miR-1能对抗缺氧复氧造成的PP2A B56的下调以及EGFR的活化。. 膜片钳的结果表明，EGFR特异性抑制剂Tyrphostin AG556显著抑制了成年大鼠原代心室肌细胞电压依赖性的INa和ICa, L，而EGFR的配体EGF能明显增加INa和ICa, L。与此同时，PP2A抑制剂冈田酸增加了电压依赖性的INa和ICa, L。反之，AG556预孵育能对抗冈田酸的上述作用。. 综上所述，在心肌缺血再灌注期间，由于miR-1上调，通过负向调控PP2A B56，促进EGFR激酶的活化以及钠通道和钙通道的开放，可能是诱发各种再灌注性心律失常的机制之一。