Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fourth Military Medical University, Xi'an 710032, China.
Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Life Sci. 2019 May 15;225:64-71. doi: 10.1016/j.lfs.2019.04.002. Epub 2019 Apr 3.
In myocardial ischemia-reperfusion (MI/R) injury, impaired autophagy function worsens cardiomyocyte death. AMP-activated protein kinase (AMPK) is a heterotrimeric protein that plays an important role in cardioprotection and myocardial autophagic function. AMPKα1 and α2 are localized primarily in the cytoplasm and nucleus, respectively, in cardiomyocytes, but the isoform-specific autophagy regulation of AMPK during MI/R remains unclear.
An MI/R model was built, and the protein expression of AMPKα1/α2, p-AMPK, mTOR, p-mTOR, TFEB, p-FoxO3a, SKP2, CARM1, TBP, Atg5, LAMP2, LC3B, and p62 during ischemia and reperfusion was determined by western blotting. Recombinant adeno-associated virus (serotype 9) vectors carrying tandem fluorescent-tagged LC3 or mRFP-GFP-LC3/GFP-LC3 were used to evaluate the autophagy status. AMPKα2 knockout mice were used for in vivo studies.
Both cytoplasmic AMPKα1 and nuclear α2 subunit expression decreased during the reperfusion period, which led to AMPKα1-mTOR-TFEB and AMPKα2-Skp2-CARM1-TFEB signaling inhibition, respectively. The decreased TFEB level during reperfusion suppressed autophagy. Metformin could activate both the AMPKα1- and α2- mediated pathways, thus restoring autophagy flux during reperfusion. Nevertheless, in AMPKα2 knockout mice, nuclear α2-regulated Skp2-CARM1-TFEB signaling was inhibited, while α1-related signaling was comparatively unaffected, which partially impaired metformin-enhanced autophagy.
Our study suggests that metformin had the dual effects of promoting both cytoplasmic AMPKα1- and nuclear AMPKα2-related signaling to improve autophagic flux and restore cardiac function during MI/R.
在心肌缺血再灌注（MI/R）损伤中，自噬功能受损会加重心肌细胞死亡。AMP 激活的蛋白激酶（AMPK）是一种异三聚体蛋白，在心脏保护和心肌自噬功能中发挥重要作用。AMPKα1 和α2 主要分别位于心肌细胞的细胞质和细胞核中，但 AMPK 在 MI/R 期间对自噬的同工型特异性调节尚不清楚。
构建 MI/R 模型，通过 Western 印迹法测定缺血和再灌注过程中 AMPKα1/α2、p-AMPK、mTOR、p-mTOR、TFEB、p-FoxO3a、SKP2、CARM1、TBP、Atg5、LAMP2、LC3B 和 p62 的蛋白表达。使用携带串联荧光标记 LC3 或 mRFP-GFP-LC3/GFP-LC3 的重组腺相关病毒（血清型 9）载体来评估自噬状态。使用 AMPKα2 敲除小鼠进行体内研究。
再灌注期间细胞质 AMPKα1 和核内α2 亚基表达均减少，分别导致 AMPKα1-mTOR-TFEB 和 AMPKα2-Skp2-CARM1-TFEB 信号抑制。再灌注期间 TFEB 水平降低抑制了自噬。二甲双胍可以激活 AMPKα1 和α2 介导的通路，从而在再灌注期间恢复自噬流。然而，在 AMPKα2 敲除小鼠中，核内α2 调节的 Skp2-CARM1-TFEB 信号被抑制，而α1 相关信号相对不受影响，这部分削弱了二甲双胍增强的自噬作用。
本研究表明，二甲双胍具有促进细胞质 AMPKα1 和核内 AMPKα2 相关信号的双重作用，可改善 MI/R 期间的自噬通量并恢复心脏功能。
