CaMEK基因靶向干预防治动脉硬化性心肌缺血再灌注损伤的机制研究

Atherosclerosis (AS) is the basic cause of coronary heart disease. Recently, we used ApoE knockout AS mouse model, we found that the pathological changes of AS increase the sensitivity to myocardial ischemia-reperfusion injury. The cardiac ERK1/2 pathway activity significantly decreased when compared with the healthy control group, but the mechanism is not clear, CaMEK gene can specifically activate ERK1/2 signaling pathway to protect against myocardial ischemic injury. Accordingly, this study intends to use the model of myocardial ischemia-reperfusion injury to explore the susceptibility mechanism of ERK1/2 pathway in AS related myocardial ischemia. Meanwhile, we construct the double-stranded recombinant AAV9 vector which can express CaMEK gene rapidly. Via in vivo and in vitro transfection experiments, we can clarify regulated role of CaMEK in critical target genes of ERK1/2 pathway at the cellular, tissue and whole animal level. Then molecular mechanisms of the ERK1/2 pathway activation against the myocardial ischemia injury could be revealed. Based on intervention experiments of exogenous CaMEK importation in AS mice, we can clarify the effects and molecular mechanisms of ERK1/2 signaling pathway targeted activation in the prevention of myocardial ischemia-reperfusion injury of AS. The utility of exogenous CaMEK as myocardial ischemia therapeutic target of AS can be identified, it can also provide new ideas and basis to alleviate and improve the prognosis of ischemia-reperfusion injury in patients with coronary heart disease.

动脉粥样硬化（AS）是冠心病的基本病因。我们最近利用ApoE基因敲除AS小鼠模型，发现AS病理变化加重心肌缺血再灌注损伤的敏感性，且心肌ERK1/2通路活性较健康对照组显著降低，但机制不清楚，CaMEK 基因能特异性激活ERK1/2信号通路，抵抗心肌缺血损伤。据此，本课题拟采用心肌缺血再灌注损伤模型，探讨ERK1/2通路在AS心肌缺血损伤易感性中的作用机制；同时，构建快速表达CaMEK基因的双链重组AAV9载体，通过体内外转染实验，在细胞、组织及动物整体水平上，明确CaMEK对ERK1/2通路关键靶基因的调控，揭示ERK1/2通路激活抵抗心肌缺血损伤的分子机制；基于AS鼠体内外源导入CaMEK干预实验，阐明靶向激活心肌ERK1/2通路防治AS心肌缺血再灌注损伤的效果及分子机制，确定外源CaMEK作为AS心肌缺血损伤治疗靶标的效用，为减轻冠心病患者缺血再灌注损伤、改善预后提供新的思路和依据。

本项目基于缺血后适应(IPost)对急性心肌梗死患者再灌注治疗并未呈现出明显的心脏保护作用以及动脉粥样硬化（AS）作为急性心肌梗死基本病因的临床现象，首次开展ERK1/2信号通路在AS心肌缺血再灌注损伤中的作用机制及其靶向干预效果研究。首先在活体动物整体水平发现IPost处理通过在再灌注早期激活ERK1/2信号通路（磷酸化ERK1/2，p70S6K，GSK3β）减轻健康小鼠的心肌缺血再灌注损伤，ERK1/2信号通路的激活是参与介导IPost心肌保护作用的重要分子机制。动脉硬化的病理改变取消了缺血后适应的心肌保护作用与ERK1/2信号通路未有效激活有关。其次，利用杆状病毒载体系统搭建规模化制备腺相关病毒载体的技术平台，研发理想的可用于心脏疾病基因靶向治疗的双链重组AAV9载体，可有效驱动靶基因在心肌内靶向的高效快速表达，据此构建携带CaMEK基因的双链重组AAV9载体（dsAAV9-CaMEK），尾静脉转染可高效激活ERK1/2信号转导通路。另外，在体外细胞水平建立乳鼠心肌细胞缺氧/复氧(H/R)及氧化应激损伤模型，发现外源性导入CaMEK基因，可以通过激活ERK1/2信号通路抑制心肌细胞的自噬效应，减少因H/R及H2O2造成的心肌细胞凋亡，且能够稳定线粒体功能，提高心肌细胞活力，从而减轻缺氧/复氧及氧化应激对心肌细胞造成的损伤。最后，在组织及活体动物整体水平，发现靶向激活心肌ERK1/2信号通路可以通过抑制自噬、促进心肌细胞存活、缩小梗死面积、抑制心肌纤维化并改善心脏功能等多种效应，最终有效防治AS心肌I/R损伤，CaMEK基因靶向转导有望成为AS病理性心肌对抗缺血再灌注损伤的有效手段。