Sirtuin蛋白家族在衰老心肌缺血/再灌注损伤易损性增加中的机制研究

The mechanism of aging-related vulnerability to myocardial ischemia reperfusion injury remains unclear. Type 1 protein of Sirtuin protein family (SIRT1) has been shown to inhibit myocardial apoptosis. In contrast, Type 2 protein of Sirtuin protein family (SIRT2) has been shown to trigger necroptosis. It is a new issue that how the SIRT1 induced cell survival coexist with the SIRT2 induced cell necroptosis in heart. Our previous studies found that ①AMPK-NAMPT-NAD+ pathway recession may reduce myocardial SIRT1 activity and impaire SIRT1's nucleocytoplasmic shutting in the senescent heart; ② A low level of NAD+ and decline in the activity of SIRT1 have been described in the senescent heart, specific blocking SIRT2 is still can effectively reduce the myocardial infarction. We hypothesize that impaired anti-apoptotic effect cause declined SIRT1 activation and SIRT2 induced necroptosis may jointly result in intolerance of the senescent heart to ischemic insults. Therefore, Transgenic mouse model and selective genetic intervention were utilized to examine the key regulatory mechanisms about impaired SIRT1 activation and SIRT2 induced necroptosis in the ischemic senescent heart. We also would investigate that synergistic inhibition the apotosis/necroptosis signaling cascade is a potential target for ameliorating the intolerance of aged heart to ischemic insults. The aim of the project is to enhance our mechanistic understanding of the relationship between myocardium survival and death during the aging process.

衰老心肌对于缺血损伤的耐受性明显下降，其分子机制仍未探明。Sirtuin 蛋白家族中Type 1蛋白介导抗凋亡，而Type 2蛋白却可引发细胞程序性坏死，这两种蛋白信号如何在心肌中达到动态平衡是阐明心肌损伤机制所需解决的全新课题。我们发现AMPK-NAMPT-NAD+通路衰退可降低衰老心肌Sirtuin type 1蛋白活性；在低NAD+水平和type1蛋白活性受抑情况下，阻断type 2蛋白能减少衰老心肌梗死面积，故设想衰老心肌缺血易损性增加可能由Sirtuin type 1蛋白抗凋亡能力的减退和type 2蛋白的促坏死作用共同导致。本研究将在细胞、分子及动物水平全面探讨衰老心肌Sirtuin type 1蛋白活性减退与type 2蛋白诱发心肌坏死的关键调控机制，并通过对这两种蛋白的选择性基因干预，实现抗凋亡/抑坏死协同提高衰老心肌抗缺血损伤能力，为缺血性心脏病的治疗提供新的思路和方向。

本项目旨在探讨SIRT蛋白对衰老心肌缺血损伤的影响和作用机制。本研究在前期研究基础上，主要围绕SIRT1 在衰老心肌缺血损伤中的作用和衰老心肌自噬和程序性坏死的调节机制探讨衰老心肌易损机制，目前取得的研究结果证实：.1. 与成年对照小鼠相比，老年小鼠MI/R损伤加重，心肌梗死面积显著增加；与之对应，衰老心肌中的SIRT1的活性显著减退，衰老心肌细胞核内的SIRT1水平较成年组相比显著降低，更多的SIRT1停留在衰老心肌细胞浆内。.2. 采用SIRT1的特异性激动剂STR 1720处理老年小鼠可有效减轻老年小鼠MI/R损伤，减小心肌梗死面积，抑制血浆CK活性水平。上述结果证实本研究提出的衰老心肌SIRT1活性受抑导致老年心肌缺血易损性。.3. 进而发现，衰老心肌SIRT1活性减退可显著影响衰老心肌的自噬功能。本研究证实心肌细胞核内的SIRT1可将LC3脱乙酰，促进LC3进入胞浆与Atg7结合形成自噬体；另一方面，心肌细胞核内的SIRT1将FoxO1脱乙酰促进其转录活性，促进Rab7的蛋白表达，Rab7可有效调控自噬体与溶酶体结合形成自噬溶酶体形成功能性自噬流。.4.明确上述机制后，本研究在衰老小鼠整体动物实验中证实：衰老心肌CARM1水平下降造成心肌自噬转录水平下降，自噬流障碍。激动AMPKα-p-FoxO3a抑制Skp2，可以提升心肌细胞核内CARM1水平，改善衰老心肌自噬流功能。.5.本课题发现衰老心肌缺血再灌注中，自噬流发生严重障碍，自噬底物蛋白p62大量堆积。p62结合necroptosis关键蛋白RIP1与RIP3结合，促进RIP1-RIP3坏死复合体形成，并进一步促进RIP3与MLKL的磷酸化激活，造成衰老心肌IR坏死加重，心肌缺血易损性增加。.6. 抑制necroptosis可显著减轻衰老心肌MI/R损伤。.本研究的新发现证实：1）心肌缺血性损伤过程中确有Necroptosis的存在和发生，Necroptosis是衰老心肌缺血易损的新机制；2）心肌SIRT1可通过细胞核内脱乙酰机制调控LC3和FoxO1信号途径控制心肌细胞自噬流，衰老心肌SIRT1活性减退是自噬流障碍的重要原因；3）衰老心肌自噬流障碍导致p62堆积并与necroptosis关键蛋白RIP1与RIP3结合形成坏死复合物，极大促进Necroptosis的发生。