Abstract 17364: Oxidative Stress Stimulates Autophagic Flux during Ischemia/Reperfusion and Mediates Reperfusion Injury
Autophagy, a bulk degradation process in which proteins are degraded through lysosomes, is activated in the heart during stress, such as ischemia/reperfusion (I/R). Oxidative stress plays an important role in mediating cell death in response to I/R. Whether I/R-induced autophagy is mediated through oxidative stress is unknown. To elucidate the role of oxidative stress in mediating autophagy, cultured cardiomyocytes were treated with H2O2 and N-2-mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species. H2O2 induced autophagy in vitro, which was significantly attenuated in the presence of MPG, as evaluated by LC3-II accumulation (H2O2 1.5, H2O2+MPG 0.2 fold vs control, p<0.01) and the level of p62, a protein degraded by autophagy (H2O2 0.1, H2O2+MPG 0.8 fold vs control, p<0.01). Autophagosome and autolysosome formation was evaluated with fluorescent mRFP-GFP-LC3 puncta. MPG significantly inhibited H2O2 -induced increases in autophagosomes (green and red dots: H2O2 3.6, H2O2+MPG 0.7 fold vs control, p<0.05) and autolysosomes (red dots: H2O2 5.5, H2O2+MPG 0.09 fold vs control, p<0.01). In mice subjected to I/R, MPG attenuated I/R -induced- increases in oxidative stress, evaluated by dihydroethidium staining, and autophagic flux, indicated by expression of LC3-II expression (I/R 2.8, I/R+MPG 7.9 fold vs control, p<0.01), p62 (I/R 0.2, I/R+MPG 0.9 fold vs control, p<0.05), autophagosome (green and red dots: I/R 6.4, I/R+MPG 4.8 fold vs control, p<0.05) and autolysosome formation (red dots: I/R 8.1, I/R+MPG 2.7 fold vs control, p<0.05), and Beclin1 expression (I/R 12.1, I/R+MPG 3.9 fold vs control, p<0.01), and decreased the size of myocardial infarction (MI)/area at risk (AAR) (−20.3% vs I/R, p<0.05), suggesting that oxidative stress plays an important role in mediating autophagy and MI during I/R. MI/AAR after I/R was significantly reduced in beclin1+/− mice (−27.3%, p<0.05), in which autophagy is suppressed, whereas beclin1+/− mice treated with MPG exhibited no significant additional reduction in MI/AAR after I/R (−2.7%). These results suggest that oxidative stress plays an important role in mediating autophagy during I/R, and that activation of autophagy through oxidative stress mediates myocardial injury in response to I/R.
- © 2010 by American Heart Association, Inc.