HSPA12A对急性心肌梗死的保护作用及机制研究： 对exosome/miR145介导的巨噬细胞“Adam17/MerTK/胞葬功能”信号的正调节

Impaired efferocytosis caused accumulation of apoptotic cardiomyocytes and exaggerated inflammation，both of which are critical for myocardial injury of acute myocardial infarction (AMI)..HSPA12A is a novel heat shock protein which biological function is barely known. During the previous NSFC studies, we observed that HSPA12A was downregulated in cardiomyocytes after AMI, and cardiomyocyte-specific overexpression of HSPA12A attenuated AMI-induced cardiac injury. Our preliminary data also suggest that this action of HSPA12A could be mediated by increasing cardiomyocyte-derived exosomes-loaded miR145, which in turn activating macrophage “miR145/MerTK/efferocytosis” signaling system..To test this possibility, we will fully evaluate the protection of HSPA12A against AMI using cardiomyocyte-specific HSPA12A transgenic and knockout mice. We will also investigate whether the protection of cardiomyocyte HSPA12A against AMI is achieved by the activation of macrophage “miR145/MerTK/efferocytosis” signaling using both mice and cell culture models. The results will provide novel clues for identifying potential protective targets of AMI based on the inter-cellular interactions of “cardiomyocyte signaling-macrophage efferocytosis”.

胞葬(Efferocytosis)是指吞噬细胞清除凋亡细胞、产生抗炎因子的过程，而胞葬功能受损是急性心肌梗死(AMI)的关键病理机制。.HSPA12A是功能尚不甚明确的热休克蛋白。前期国家自然基金研究中，我们发现：HSPA12A在AMI心肌细胞中表达减少，而心肌细胞高表达HSPA12A减轻AMI损伤，且该作用可能是通过增加心肌细胞miR145表达、并经exosome到达巨噬细胞、进而激活其MerTK介导的胞葬功能而实现的。.本项目将在前期基础上，采用心肌细胞特异性HSPA12A转基因鼠、基因敲除鼠，综合评价HSPA12A对AMI的保护作用，并结合细胞模型，阐明该作用是否通过心肌细胞exosome负载的miR145来激活巨噬细胞“Adam17/MerTK/胞葬功能”信号活性，快速清除凋亡细胞、抑制炎症反应而实现的。研究结果将从“心肌细胞-巨噬细胞”相互影响的角度，为AMI防治提供基础数据。

背景: 心肌梗死是世界范围内的高发、高危险疾病，及时进行血流再灌注是其根本治疗途径，而再灌注可致心肌缺血再灌注 (Myocardial ischemia reperfusion, MI/R) 损伤。无氧酵解在心肌缺血期间的作用已经得到广泛关注和较为深入的研究，而再灌注期间的有氧糖酵解的作用、及其调控尚未完全阐明。组蛋白H3乳酸化是糖酵解转化的表观遗传学标记。本研究将探索热休克蛋白A12A(heat shock protein A12A, HSPA12A)在MI/R损伤中的作用、以及该作用是否与H3乳酸化有关。.方法: 采用WT和HSPA12A基因敲除鼠进行MI/R实验。采用原代乳大鼠心肌细胞缺氧/复氧(Hypoxia/reoxygenation, H/R)实验进行体外模拟MI/R。心功能测定采用二维心脏超声心电图。糖酵解流采用细胞外乳酸测定、细胞内糖酵解相关基因表达进行分析。心肌细胞损伤采用TUNEL 染色、live/dead 染色、LDH 渗漏、心肌细胞搏动频率和节律进行评估。分子信号分析采用免疫印迹、免疫荧光染色、和免疫共沉淀-免疫印迹进行分析。 .结果: 在心肌缺血后的再灌注期间, 心肌细胞中的HSPA12A表达下降、并伴有 糖酵解流抑制。值得关注的是，HSPA12A基因敲除加重小鼠MI/R诱导的糖酵解抑制、心肌细胞死亡、心功能不全。功能获得、功能缺失实验表明，HSPA12A是支持心肌细胞抵抗H/R应激所必须的，该保护作用是通过维持再灌注期间的有氧糖酵解、H3乳酸化而介导的。进一步研究发现，HSPA12A增加Smurf1-依赖性Hif1α 蛋白质稳定性，进而激活Hif1α下游糖酵解靶基因表达、维持糖酵解稳态和H3乳酸化，最终促进心肌细胞存活、进而减轻MI/R损伤。.结果: HSPA12A可能是治疗心肌缺血/在灌输损伤的潜在靶点，HSPA12A 的这种保护作用是通过维持心肌细胞中Smurf1介导的Hif1α 蛋白稳定性、从而维持心肌细胞在再灌注期间的有氧酵解活性和H3乳酸化稳态而实现的。