AMPK 通过 Atg4C磷酸化调节心力衰竭自噬与凋亡平衡的机制研究

The impaired balance of myocardial autophagy and apoptosis leads to the occuring and progression of heart failure. My previous work demonstrated that AMPK (AMP-activated protein kinase) activation inhibited the function of ATF4, blocked ER stress and apoptosis, and then attenuated heart failure. Recently, we found that the activated AMPK directly phosphorylated Atg4C at Threonine 452 (T452), then induced LC3B formation and autophagy. Moreover, the phosphorylated- Atg4C bind to ATF4 and inhibit its function, then blocked ER stress and apoptosis. Based on these works, the present proposal aimed to verify that Atg4C is the new substrate of AMPK, to describe the exact site of phosphorylated Atg4C by constructing the mutant plasmid, to test the p-Atg4C function in autophagy through gain of function and loss of function, to demonstrate the role of Atg4C in ATF4 function and ER stress. What’s more, we use AMPKα2-/- and wild type mice to induce heart failure model to further verify the AMPK/Atg4C function and its regulation in autophagy and apoptosis in vivo, which may provide the fundamental molecular basis of heart failure.

心肌细胞自噬和凋亡的失衡导致心力衰竭的发生和进展，本人既往发现AMPK活化可抑制ATF4活性，缓解内质网应激反应及心肌细胞凋亡，减轻心力衰竭。我们最近发现，活化的AMPK可直接磷酸化Atg4C的苏氨酸T452位点，从而促进LC3B的形成，启动自噬反应；另一方面，磷酸化的Atg4C可与ATF4结合并抑制其活性，阻断内质网应激级联反应，减少心肌细胞凋亡。本研究拟在前期的工作基础上，进一步验证Atg4C为AMPK新的底物，构建突变质粒明确Atg4C具体的磷酸化位点，进行功能获得和功能缺失实验充分证明p-Atg4C对自噬功能的作用，探明AMPK调节自噬是否依赖于Atg4C，阐明Atg4C如何抑制ATF4活性及内质网应激，并且利用已有的AMPKα2-/-和野生型小鼠建立心衰模型，进一步在体及离体验证AMPK对Atg4C 磷酸化修饰，及其对自噬/凋亡平衡的调控，为心力衰竭的防治提供分子细胞学基础。

心肌细胞自噬和凋亡的失衡导致心力衰竭的发生和进展。本研究在前期工作的基础上，按照原定计划，较为顺利地完成了实验内容，成功构建突变质粒，确定具体的Atg4C磷酸化位点及其对自噬功能的作用，探明了AMPK调节自噬依赖于Atg4C，阐明了Atg4C如何抑制ATF4活性及内质网应激，同时构建载体及购买干预药物，完成了在体研究。对磷酸化位点进行质谱分析，并且进行动物实验，进行心力衰竭造模，给予干预药物，同时利用AMPKα2-/-小鼠进行在体研究，检测小鼠心脏功能，检测心肌细胞自噬/凋亡的状态，完成在体研究。目前已经完成撰写论文并投稿，进入到总结课题阶段。为心力衰竭的防治提供了细胞生物学基础。