SIRT5/ammonia信号通路介导适应性自噬在急性心肌梗死中的作用及其机制研究

Apoptosis of myocardial cells after acute myocardial infarction can cause cardiac dysfunction and cardiac insufficiency. Studies have shown that autophagy has a two-way regulation of hypoxia cardiomyocyte apoptosis, but the specific mechanism is unknown. Applicant's previous mouse and cell studies confirmed that adapted autophagy in early myocardial hypoxia can reduce cardiomyocyte apoptosis; inhibition of autophagy leads to increased apoptosis and ultimately impaired cardiac function. Through bioinformatics analysis, it was found that SIRT5 gene changed significantly in early myocardial hypoxia. Gene Oncology analysis suggested that SIRT5 was directly related to apoptosis. Cardiomyocyte experiments confirmed that SIRT5 expression was down-regulated and ammonia concentration was increased after hypoxia. Recent studies have shown that SIRT5 is involved in the regulation of adapted autophagy by regulating intracellular ammonia metabolism. Based on this, the following scientific hypothesis is proposed: after hypoxia, cardiomyocytes regulate ammonia metabolism through SIRT5 and induce adapted autophagy. In order to verify this hypothesis, the applicant intends to explore the regulation and mechanism of SIRT5 for hypoxic myocardium autophagy from molecular, cellular and animal multifaceted surfaces by establishing a myocardial infarction mouse model and a myocardium hypoxia model using adenovirus transfection and protein fluorescent tracing. The results can provide new ideas for the treatment of myocardial infarction and ischemic cardiomyopathy.

急性心肌梗死（心梗）后心肌细胞凋亡可引起心脏舒缩功能障碍和心功能不全。研究表明自噬对缺氧心肌细胞凋亡有双向调控作用，然而具体机制不明。申请者前期小鼠及细胞研究证实：心肌缺氧早期产生适应性自噬，可减少细胞凋亡；抑制自噬则导致凋亡增加，最终损害心脏功能。通过生物信息学分析，发现心肌缺氧早期SIRT5基因变化显著，GO注释分析提示SIRT5与细胞凋亡直接相关；心肌细胞实验证实缺氧后SIRT5表达下调，氨浓度升高。近期研究发现SIRT5通过调控细胞内氨代谢，参与适应性自噬的调节。据此提出如下科学假说：缺氧后心肌细胞通过SIRT5调控氨代谢，诱导适应性自噬、抑制凋亡。为证实这一假说，申请者拟通过建立心梗小鼠模型及心肌细胞缺氧模型，采用腺病毒转染、蛋白荧光示踪等方法，从分子、细胞及动物多层面探究SIRT5对缺氧心肌自噬的作用，明确调控适应性自噬的机制。研究结果可为心肌梗死及缺血性心肌病治疗提供新思路。

急性心肌梗死（心梗）后心肌细胞凋亡可引起心脏舒缩功能障碍和心功能不全。研究表明自噬对缺氧心肌细胞凋亡有双向调控作用，然而具体机制不明。本次研究证明从生信分析的结果出发，在细胞、小鼠中，从分子、细胞及动物多层面论证SIRT5对于急性心肌缺氧情况下的保护作用及机制。通过生物信息学分析，发现心肌缺氧早期SIRT5基因变化显著；心肌细胞实验证实缺氧后细胞自噬升高、SIRT5表达下调，氨浓度升高。在本人既往研究中发现心肌缺氧早期细胞自噬主要作用为清除细胞中受损的线粒体，从而达到细胞稳态，避免凋亡。而SIRT5主要表达与细胞线粒体中，故SIRT5下降这一现象或为细胞缺氧后线粒体清除后的结果。为证实SIRT5直接参与调控细胞自噬，本人进行心肌细胞SIRT5基因敲除，敲除后细胞自噬水平提高，证明在心肌细胞缺氧后SIRT5表达下降和自噬增高的因果关系，在细胞层面SIRT5参与调控细胞自噬。进一步在体实验中，经小鼠静脉注射SIRT5-/-腺病毒，静脉注射3天后检测小鼠心肌组织中SIRT5表达下降。将小鼠分为假手术组、手术组以及SIRT5-/-手术组三组，其中手术组和SIRT5-/-手术组小鼠行冠状动脉前降支结扎术，术后7天、21天行心脏超声检查，结果显示手术组和SIRT5-/-手术组小鼠较假手术组心功能明显下降，但SIRT5-/-手术组小鼠较手术组小鼠有更多的心功能保留。为进一步明确这种现象是由于存活心肌组织代偿还是由于缺氧心肌组织丢失减少，检测各组小鼠心肌组织中纤维浸润发现SIRT5-/-小鼠心肌梗死后心梗面积较手术组小，心肌组织中纤维侵润和凋亡相关蛋白表达降低。在体实验证明了SIRT5对心肌缺氧后细胞凋亡存在调控作用。实验结果证实SIRT5明确参与调控缺氧心肌自噬及凋亡，并通过这种机制减少细胞凋亡产生。研究结果可为心肌梗死及缺血性心肌病治疗提供新思路。