内源性CaMKⅡ活性抑制因子在心梗修复期室性心律失常中的作用及机制研究

Ventricular arrhythmias are the most important causes for the continuously-rising mortality in patients with myocardial infarction (MI), but currently not only is the underlying mechanism still unclear but also is there short of effective treatment approach. Our cDNA microarray results showed that the expression of ALK4, CaMKⅡN1, PCP4, Smad2, Smad3, and Smad4 were decreased in the border zone of the myocardial infarction in mice and the results were confirmed by Western blotting. It is well-known that CaMKⅡ is deeply involved in the pathogenesis of ventricular arrhythmias post-MI and that CaMKⅡN1 and PCP4 are endogenous CaMKⅡ inhibitors. Therefore, we generated ALK4 and CaMKⅡN1-knockout mice. The ECG recording successfully captured spontaneous ventricular arrhythmias and Western blotting demonstrated an increase in the CaMKⅡ activity in the myocardial tissue of these mice. Most importantly, ALK4-Smad2/3/4 constitutes a famous signal pathway regulating gene transcription/expression. The expression of CaMKⅡN1 in neonatal mice myocardial cells was up-regulated by treatment of activin, a specific ligand to activate ALK4. However, the expression of PCP4 was unchanged. Next in this study, we plan ① to analyze the cardiac electrophysiological characteristics and the pattern of ventricular arrhythmias in mice knocked-out of the three genes by mimicking the post-MI down-regulation of CaMKⅡN1, ALK4, and PCP4; ② to determine the role of ALK4-Smad2/3/4 pathway in the mechanism responsible for the post-MI down-regulated CaMKⅡN1; ③ to explore the mechanisms responsible for the down-regulation of PCP4 by two methods. One is to test whether it is caused by a potentiated degradation resulted from an increased activity of calpain, a Ca2+-dependent proteinase. The activity of calpain is usually upregulated under hypoxia/ischemia. Another is to test whether PCP4’s expression is suppressed by a series of traditional promoter/transcriptional factor binding interactions; ④ at last, for eagerly looking for potential treatment approaches, to test whether over-expression of CaMKⅡN1 and/or PCP4 in mice could attenuate the occurrence of the ventricular arrhythmias. Our study will expand the understanding of the pathological mechanisms of the ventricular arrhythmias after MI and provide potential therapeutic targets.

心梗后室性心律失常发生凶险、机理不明、缺乏有效的治疗手段。预实验显示小鼠梗死边缘区ALK4、CaMKⅡN1、PCP4、Smads表达明显下调。CaMKⅡN1和PCP4是CaMKⅡ内源性抑制因子，CaMKⅡ在心梗后室性心律失常中起关键作用。我们在CaMKⅡN1-/-小鼠观察到频发室性心律失常及CaMKⅡ活性增强。预实验提示CaMKⅡN1表达是ALK4-Smads信号通路调节的，而PCP4下调可能与其降解增加和转录减少有关。故提出假设：CaMKⅡN1和PCP4表达下调使CaMKⅡ持续激活而导致心梗后室性心律失常。本项目拟研究：CaMKⅡN1和PCP4在心脏电生理特性和室性心律失常中的作用及机制；ALK4-Smads信号通路对CaMKⅡN1表达的调控作用；PCP4表达下调的机制；CaMKⅡN1和PCP4高表达对心梗后室性心律失常的抑制作用。通过以上研究为心梗后室性心律失常的防治提供新的治疗靶点。

心梗后室性心律失常发生凶险、机理不明、缺乏有效的治疗手段。心梗后CaMKⅡ激活是发生室性心律失常的重要机制，而CaMK2N1、PCP4是已知的重要的内源性CaMKⅡ抑制因子，极有可能影响心梗后室性心律失常发生。本项目研究了CaMK2N1、PCP4对心梗修复期心脏重构和室性心律失常的影响及机制，主要成果如下：（1）心梗后梗死边缘区CaMK2N1表达下调。CaMK2N1敲除引起心梗后心肌细胞钙调控异常；明显增加心梗炎症期巨噬细胞极化，M1促炎型巨噬细胞与心肌细胞偶联，激活KCa3.1影响心梗后心肌细胞复极异质性；同时，CaMK2N1敲除可通过炎症小体途径，增加心梗修复期炎症介导的心肌纤维化过程。以上心脏重构易化了心梗后室性心律失常的发生。机制研究发现心梗后CaMK2N1下调受ALK4/Smad3通路调节。（2）PCP4主要表达于心脏浦肯野细胞，PCP4敲除引起浦肯野细胞中INa-L明显增大，心梗修复期室性心律失常发生率明显增加。而浦肯野细胞INa-L较心室肌细胞有更明显的频率依赖性，且其电流衰减及通道恢复频率依赖性也更大，这揭示了浦肯野细胞作为心梗后室性心律失常发生重要起源的原因。机制研究发现PCP4转录减少及Calpain介导的PCP4蛋白降解增加是导致心梗后PCP4表达下调的机制。（3）取向碳纳米管薄膜构建仿生型心肌组织片可诱导心肌细胞取向生长、降低复极离散度、改善信号传导，实现心肌细胞同步收缩，这为心脏补片发挥心梗后心肌修复作用提供了重要基础。我们正利用一种负载可透膜性CaMK2N1和PCP4的复合补片，探索上调CaMK2N1和PCP4通过抑制CaMKII活性，发挥降低心梗后室性心律失常发生的作用。（4）心梗后交感神经激活在心梗后室性心律失常中发挥重要作用，我们通过拓展研究探索干预交感神经的新方法。首次将立体定向放射消融（SBRT）用于交感神经的干预，发现SBRT星状神经节可导致心梗后心脏去交感神经化，明显减少心梗后室性心律失常的发生。后续，我们将检测去交感神经化的心脏组织中CaMK2N1和PCP4的表达量变化，深入研究二者在这一过程中的作用。综上，本研究阐明了CaMK2N1和PCP4调控心梗后室性心律失常的分子机制，二者有望成为治疗心梗后室性心律失常的新靶点。本项目累计资助发表SCI论文26篇，累计影响因子169.1分，申请专利2项。