Abstract 16137: Redox Regulation Of AMP-activated Protein Kinase By Thioredoxin-1 During Myocardial Ischemia
AMP-activated protein kinase (AMPK) plays a critical role in mediating adaptation to energy stress. Whether AMPK is regulated by posttranslational oxidative modification (PTOM) remains unknown. Here we show that AMPK α subunit is a direct substrate of thioredoxin-1 (Trx1), a 12kD anti-oxidant. Although Thr-172 phosphorylation of AMPK was increased by 30min of ischemia (2.2 fold, p<0.01) in wild type (WT) mice, ischemia-induced activation of AMPK was inhibited in dominant negative Trx1 mice (Tg-DN-Trx1) (1.1 fold, p<0.01 vs WT), suggesting that Trx1 positively regulates AMPK during ischemia. The size of myocardial infarction (MI)/area at risk after prolonged ischemia (PI) was significantly greater in Tg-DN-Trx1 (62% vs 39% p<0.05) than in WT mice, suggesting that Trx1 is protective against PI. In cultured cardiomyocytes (CMs), phosphorylation of AMPK and ACC, a substrate of AMPK, during glucose deprivation (GD) was significantly attenuated (0.6 and 0.7 fold, p<0.05) when endogenous Trx1 was downregulated. Downregulation of Trx1 inhibited glucose uptake (0.3 fold, p<0.05) and glycolysis (4 fold, p<0.05), decreased ATP content (1.5 fold, p<0.05), and exacerbated GD-induced cell death (60% vs 37% p<0.05). These data suggest that Trx1 plays an essential role in mediating AMPK activation and adaptive responses during GD. H2O2 pretreatment in CMs inhibited AMPK activation in a dose- and time-dependent manner, suggesting that AMPK activity is negatively regulated by oxidative stress. GD induced transient accumulation of reactive oxygen species (ROS) in CMs, as analyzed by CM-H2DCFA staining. GD also induced cysteine oxidation of AMPK through disulfide bond formation, as evaluated with N-ethyl-maleimide labeling with or without β-mercaptoethanol. Downregulation of Trx1 further enhanced AMPK oxidation in response to GD, suggesting that endogenous Trx1 prevents AMPK oxidation in CMs. In vitro kinase assays revealed that AMPK oxidation inhibited its phosphorylation and activation by LKB1, an upstream kinase. These data suggest that Trx1 is essential for mediating activation of AMPK in response to energy stress. Cysteine oxidation prevents phosphorylation of AMPK by upstream kinases, which represents a novel mechanism of AMPK regulation by PTOM in CMs.
- © 2012 by American Heart Association, Inc.