SIRT6对自噬的调控及其与心肌细胞肥大的关系研究

Cardiac hypertrophy, as a common independent risk factor for various cardiovascular diseases, has a close relationship with epigenetic regulation. The sirtuins (SIRT) family belongs to class III histone deacetylases (HDACs) and includes seven members. Previously, we have demonstrated that SIRT6 could protect cardiomyocytes from hypertrophic response, which was dependent on its deacetylase activity. However, the molecular mechanism underlying the anti-hypertrophic effect of SIRT6 remains to be clarified. It's known that autophagy is one of the main processes that allow for the degradation of proteins and organelles, and plays an important role in the maintenance of intracellular environment stabilization. Recent evidences have revealed that the autophagy of cardiomyocytes should be kept at an appropriate level to avoid cardiac malfunction. In cultured neonatal rat cardiomyocytes submitted to isoproterenol stimulation, we observed downregulation of autophagy accompanying with cellular hypertrophy Moreover, our experiments showed that overexpression of SIRT6 in cardiomyocytes could result in increased level of autophagy. It suggests that moderate activation of autophagy may be an effective way to prevent cardiac hypertrophy. Intrigued by these findings, we ask whether the anti-hypertrophic effect of SIRT6 can be attributed to its regulation on autophagy of cardiomyocytes. This study is aimed to search for autophagy-related gene (ATG) proteins directly interacting with SIRT6 or deacetylated by SIRT6, elucidate the relation between SIRT6-mediated autophagy and deacetylase activity of SIRT6, and further investigate the connections between SIRT6,autophagy and cardiac hypertrophy by overexpressing SIRT6 protein through adenovirus injection delivery into hypertrophic rat hearts. Our research will provide a further insight into the mechanism underlying the anti-hypertrophic effect of SIRT6 and support the novel therapeutic strategy against cardiac hypertrophy by targeting SIRT6.

心肌肥大是心血管疾病的独立危险因素，与表观遗传调控关系密切。我们前期发现，Ⅲ类组蛋白去乙酰化酶Sirtuins家族的SIRT6亚型具有依赖于其去乙酰化酶活性的抗心肌肥大功能，但机制有待阐明。自噬是细胞内物质降解的主要途径之一，对保证细胞基本的能量需要、维持内环境稳定具重要意义。我们在异丙肾上腺素诱导乳鼠心肌细胞肥大模型中观察到自噬水平下降，并且发现在心肌细胞中过表达SIRT6可提高自噬水平，认为适度激活自噬可能有助于心肌肥大的防治，SIRT6抗心肌肥大作用可能与调节心肌细胞自噬有关。本课题拟在此基础上重点寻找与SIRT6直接作用或受其去乙酰化修饰的自噬相关基因编码蛋白，探讨SIRT6调控自噬与其去乙酰化酶活性的关系，并利用腺病毒载体心室壁注射技术，在大鼠心脏中特异性过表达SIRT6，研究SIRT6、自噬和心肌肥大之间的关系，分析SIRT6抗心肌肥大的分子机制及作为心肌肥大治疗靶点的可能性。

课题研究工作按计划实施，顺利完成。（1）首先，我们采用异丙肾上腺素（ISO）腹腔注射构建大鼠心肌肥大模型，发现心肌组织SIRT6蛋白表达及酶活性均显著降低，同时心肌细胞自噬水平明显下降。在分离培养的原代乳鼠心肌细胞中，ISO在引起心肌细胞自噬水平降低的同时，可时间和浓度依赖性地抑制SIRT6表达，提示SIRT6功能下降与ISO诱导的心肌肥大有关。（2）在过表达SIRT6或利用siRNA沉默SIRT6基因后，观察了心肌细胞自噬水平的变化，发现SIRT6参与维持细胞基础水平的自噬且此作用依赖于其去乙酰化功能。我们进一步通过构建SIRT6腺病毒表达载体，采用心室室壁注射方式实现了SIRT6在大鼠心脏中的特异性过表达，并在整体水平上明确了SIRT6对心肌自噬具有正向调控作用。（3）为明确SIRT6抑制心肌肥大作用是否与其上调自噬有关，我们在ISO诱导的乳鼠心肌细胞肥大模型中，同时加入自噬抑制剂3-MA，观察心肌细胞自噬的变化。结果表明，3-MA能够明显抑制心肌细胞的自噬水平，SIRT6对ISO所引起的心肌肥大的抑制作用能够被3-MA所逆转。同样，敲低自噬相关基因（Atg）家族关键成员Atg5，可显著抑制心肌细胞自噬，并减弱SIRT6的抗肥大作用。这些实验证实SIRT6可通过激活心肌自噬从而抑制ISO所致心肌肥大。（4）进而我们对SIRT6调控自噬的相关分子机制进行了探索。通过对Atg家族成员的筛选，我们发现过表达SIRT6可显著提高LC3即Atg8的表达，该作用与转录因子FoxO3有关。SIRT6 通过其组蛋白去乙酰化功能抑制Akt表达，降低FoxO3的磷酸化水平，从而促进其在细胞核内的聚集及对自噬相关基因的转录。干扰FoxO3或Akt抑制剂均可阻断SIRT6上调心肌细胞自噬的作用。提示SIRT6对自噬的调控作用与Akt/FoxO3通路有关。. 通过本课题的实施，我们揭示了SIRT6对心肌细胞自噬具有正向调控作用，并且SIRT6的抗心肌肥大作用至少部分与其促进心肌自噬有关。这些发现为进一步理解SIRT6在心血管系统中的作用提供了新的线索。已在SCI收录杂志发表标注本基金号的研究论文4篇，达到或超过了课题的预期目标。