Abstract 15689: Activation of PKR-Like Endoplasmic Reticulum Kinase is Required for Autophagy Stimulation and Cardiomyocyte Survival During Acute Myocardial Ischemia

Abstract

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-resident serine/threonine kinase that regulates the cellular response to ER stress and energy deprivation. Under stress, PERK activation can be either protective or detrimental. We studied for the first time whether PERK regulates cardiomyocyte (CM) survival and death during myocardial ischemia, which is characterized by both ER and energy stress. We found that PERK is significantly activated after 30 minutes of myocardial ischemia (2.4 ± 0.04-fold, p<0.001), as shown by its increased phosphorylation status. After 3 hours of ischemia, mice with cardiac-specific overexpression of dominant-negative PERK (Tg-DN-PERK) displayed a significant increase in infarct size/area at risk with respect to non-transgenic (NTg) mice (66.4 ± 4.1% vs. 38.5 ± 6.8%, p<0.05), indicating that ischemia-induced PERK activation is cardioprotective. PERK signaling regulates autophagy in cancer cells. Autophagy is an intracellular degradation mechanism that promotes CM survival during ischemia by relieving both energy and ER stress. We found that autophagy activation during ischemia is abrogated in the hearts of Tg-DN-PERK with respect to NTg, as indicated by reduced LC3-II accumulation (0.71 ± 0.05-fold, p<0.01). Consistently, CMs with adenovirus-mediated overexpression of DN-PERK displayed reduced LC3-II accumulation (0.69 ± 0.1-fold vs. control CMs, p<0.05) after 4 hours of glucose deprivation (GD) in vitro, a condition that mimics ischemia-induced energy stress. A similar result was also observed after bafilomycin A1 treatment to test autophagic flux. These data indicate that PERK is required for autophagy activation and flux during CM ischemia and GD. Finally, we saw that DN-PERK overexpression significantly promotes CM death after GD, as shown by propidium iodide assay (2.01 ± 0.25-fold vs. control CMs, p<0.05). However, co-overexpression of Atg7, which activates autophagy, significantly reduced cell death induced by DN-PERK (-61%, p<0.05). These data suggest that autophagy inhibition partly mediates the detrimental effects of PERK inhibition during energy stress. In conclusion, our study demonstrates that PERK activation is required for autophagy activation and CM survival during ischemia.