Abstract 93: Sestrin2 Mediates the LKB1-AMPK Signaling Cascade in the Ischemic Heart

Background: AMP-activated Protein Kinase (AMPK) has emerged as a pertinent stress-activated kinase shown to have substantial cardioprotective capabilities against myocardial ischemia/reperfusion (I/R) injury. The regulation of AMPK in the ischemic and reperfused heart is critical to the development of new therapeutic strategies. We hypothesized that a novel stress-inducible protein, sestrin2, mediates AMPK activation in the ischemic heart. Methods and Results: C57BL/6 mice were subjected to left anterior descending coronary artery occlusion for different time points of ischemia and I/R in order to detect the signaling activity in the left ventricle. The kinetics of AMPK phosphorylation at Thr 172 of the α catalytic subunit appears brief and bell-shaped, peaking around 10 min (3.6-fold vs. sham, p <0.01), decreasing with prolonged ischemia, and leveling off to basal levels upon reperfusion. Intriguingly, at 10 min of ischemia, there was also a robust increase in p53 accumulation compared to basal conditions. Consequently, sestrin2 a p53-inducible protein, accumulates at the peak of ischemic AMPK activation (10 min, 2.5-fold vs. sham, p <0.05). Respective left ventricle lysates were immunoprecipitated with sestrin2 and the immunoblots revealed that sestrin2 forms a complex with AMPK during I/R, moreover, AMPKα2 is the predominant isoform that interact with sestrin2 as compared to AMPKα1 isoform during I/R. Interestingly, LKB1, an upstream AMPK kinase becomes significantly associated with sestrin2 (5 min), while there is a dramatic interaction between sestrin2 and p-AMPK (Thr 172 ) (10 min), suggesting the interaction of LKB1 with sestrin2 initiates the phosphorylation of AMPK in this complex. Furthermore, sestrin2 knock out hearts demonstrate impaired ischemic AMPK activation and higher sensitivity to I/R-induced injury as compared to wild type hearts ( p <0.01). Conclusions: Here we show for the first time evidence of a unique mechanism that sestrin2 is a stress-induced scaffold protein that mediates the activation of AMPK in the ischemic myocardium via a time-dependent interaction with LKB1. Moreover, sestrin2 deficiency leads to blunted ischemic AMPK activation and increased sensitivity to ischemic insults.