DRAM1通过调控自噬流改善急性心肌梗死心脏预后的机制研究

Acute myocardial infarction (AMI) is one of the most common heart diseases that cause morbidity and mortality worldwide. Our previous studies have shown that autophagy was activated quickly after AMI, and however, autophagy substrate P62 began to accumulate along with the prolonged ischemia. Our results indicated that autophagy flux was impaired in the latter phase of the AMI. The damage-regulated autophagy modulator 1 (DRAM1), a novel P53 target gene, is a lysosomal protein and has been reported to be essential in P53-dependent autophagy activation and apoptosis. The preliminary data showed that DRAM1 expression was upregulated in the infarct border zone of the AMI mice. Overexpression of DRAM1 in the cardiomycytes alleviated P62 accumulation and cell apoptosis caused by hypoxia significantly. On the basis of the literature and the preliminary data we proposed the hypothesis: DRAM1 will improve cardiomycyte autophagy flux and reduce cell apoptosis after ischemia by promoting autophagy-lysosome degradation pathway. To address this issue, we will use the mouse acute myocardial infarction model in vivo and H9C2 cells oxygen glucose deprivation model in vitro, first observing the effect of DRAM1 in the hearts of AMI and then exploring the mechanism by which DRAM1 improve the impaired autophagy flux after AMI. Our study will provide a strong rationale for developing the novel mechanism and approach to effectively improve the prognosis of AMI.

急性心肌梗死(AMI)是全球范围内严重危害人类健康的重要疾病之一。前期研究发现，自噬在AMI后早期被迅速激活，但自噬底物P62随缺血时间延长发生堆积，提示自噬流在AMI 中后期受损。损伤调节自噬调控基因1(DRAM1)是 p53下游的溶酶体膜蛋白。预实验表明，DRAM1在AMI小鼠心梗周边区表达明显增高；在心肌细胞中过表达DRAM1缓解长时缺氧引起的P62堆积并明显减轻心肌细胞凋亡。鉴于此，我们提出设想：DRAM1可改善缺血后心肌细胞自噬流，从而减少心肌细胞凋亡、逆转AMI心肌重构。为证明这一猜想，拟在实验性小鼠AMI模型中研究DRAM1对AMI后心脏功能及心肌重构的影响并探讨其作用是否依赖于自噬流；并从细胞和分子水平研究DRAM1与自噬相关蛋白及溶酶体功能蛋白是否存在相互作用，以期明确DRAM1改善自噬流、影响AMI心脏预后的分子机制。本研究将揭示改善心肌缺血后自噬流受损的新机制。

急性心肌梗死(Acute Myocardial Infarction，AMI)是全球范围内严重危害人类健康的重要疾病之一。AMI患者的预后与心肌坏死面积的大小及存活心肌的数量密切相关。然而，临床上仍缺少特异性的逆转心梗后心肌重构的药物，因此，寻找特异性逆转心肌重构并具有心肌细胞保护作用的药物靶点，有望为AMI的防治提供新的治疗策略。本项目组在前期研究的基础上，通过整体动物和细胞水平借助RNA-seq、免疫荧光、透射电镜及免疫印迹等多种手段，发现溶酶体膜蛋白DRAM1在AMI后表达下降，这一现象可能与其DNA甲基化变化有关。项目组进一步采用冠状动脉左前降支永久性结扎建立实验性AMI小鼠模型，小鼠心室室壁多点注射过表达DRAM1腺病毒载体，经超声心动图、组织病理检测等方法，发现过表达DRAM1可明显改善缺血引起的心肌细胞自噬流障碍，延缓心肌细胞凋亡逆转AMI心肌重构。同时，在细胞水平，通过Gain-or-loss of function证实，过表达DRAM1能增加溶酶体与自噬体融合从而促进心肌细胞自噬，促进缺血后心肌细胞存活，而DRAM1缺失会加重自噬流障碍及心肌细胞凋亡。并从分子水平首次揭示了DRAM1通过直接与ATG7相互作用，从而增加ATG5-ATG12复合体促进自噬流的分子机制。.本项目首次揭示了DRAM1在缺血性心肌病中的生物学作用，并明确其调控心肌细胞自噬流的分子机制。也从一个全新的角度揭示了改善AMI后自噬流受损的新机制。这从一个新的角度阐明AMI心肌重构的发病机制，并为评估DRAM1是否可以作为防治AMI的新靶点提供了重要的实验依据。