Caveolin-3的SUMO化对心肌钠离子通道新的调控机制的研究

The serious arrhythmia rate is high in ischemic heart diseases，and the mechanism is far from being clarified. The voltage-gated sodium channel Nav1.5 is the key structure for determining cardiac excitability and conduction. Our previous study found that Nav1.5 is significantly reduced in myocardial ischemia, but how Nav1.5 is regulated in myocardial ischemia, is largely unknown. In recent years, the relationship between Nav1.5 and Caveolin-3, one of Nav1.5 associated protein in cardiomyocytes,is compelling. It may link ischemia induced signaling pathways to abnormal Nav1.5. Based on the fact that Caveolin-3 is the target of a small ubiquitin-related modifier (SUMO) modification we hypothesize that in myocardial ischemia enhanced Caveolin-3 SUMOylation, is responsible for decreased Nav1.5 expression and functional abnormalities. By using site-directed mutagenesis to mutate SUMO sites on the plasmid of Caveolin-3, and direct importing recombinant adeno-associated virus (AAV9-ShRNA-PIASy) into in vivo rat heart, for the purpose of specific blocking PIASy, an E3 ligase of Caveolin-3 SUMOylation, we further explore such intervention strategies for prevention and treatment of ischemic arrhythmias. This study will be very important for us to understand pathogenisis of acute ischemic arrhythmia, and find new therapeutic targets.

缺血性心脏病人严重心律失常发生率高，发生机制远未阐明。电压门控性钠通道Nav1.5是决定心肌电兴奋和传导的关键结构。我们前期研究发现心肌缺血时Nav1.5明显下调，但缺血如何调控Nav1.5尚不清楚。近年，一种Nav1.5相关蛋白Caveolin-3和Nav1.5关系引人注目，它可能将缺血所激活的信号通路与异常的Nav1.5联系起来。本课题围绕心肌缺血时Caveolin-3 被小泛素相关修饰物SUMO 化事件，验证心肌缺血时上述事件是造成Nav1.5表达和功能异常的重要机制这一假设。采用对Caveolin-3上的SUMO位点定点突变和将重组腺相关病毒（AAV9-ShRNA-PIASy）直接导入活体大鼠心脏以特异性阻断SUMO E3连接酶-PIASy激活的方法，进一步探讨此种干预策略预防和治疗缺血性心律失常的可能性。本研究对探明急性缺血性心律失常发病机制、寻找新的治疗靶点有重要意义。

心肌细胞Nav1.5的表达和功能异常是心肌缺血再灌注（I/R) 期间严重心律失常发生的主要病理基础。Cav-3不仅是Nav1.5的重要调适蛋白，其本身亦可受转录后分子调控，影响其与结合分子间的相互作用。本项目研究PIASy介导的Cav-3SUMO 化在Cav-3-Nav1.5 相互作用和再灌注心律失常中的作用。结果显示，心肌I/R时严重心律失常的发生与Nav1.5的下调和功能异常密切相关，I/R时PIASy的上调促进Cav-3的SUMO化，使与Cav-3结合的Nav1.5减少，膜上Nav1.5的表达量下调，是导致严重心律失常的重要机制。而应用AVV9介导的心肌活体PIASy-RNA干扰可减少I/R时Cav-3 SUMO化修饰增加，使膜上Nav1.5量部分恢复，同时室性心律失常发生率明显降低。进一步的研究发现I/R 时因延迟性钠电流增加和胞内钠-钙离子的依次增加，经CaMKII激活及线粒体途径，使再灌注期间线粒体自噬明显增加，细胞凋亡亦增加；钠通道选择性抑制剂-Ranolazine 显著增加I/R时Nav1.5的表达，同时降低再灌注时的过度线粒体自噬和细胞凋亡，对I/R心肌有保护作用。本研究确定PIASy 介导的Cav-3 SUMO化可能是I/R导致严重心律失常的重要靶点，对临床应用针对性干扰进行预防治疗有重要意义。