Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/ reperfusion (I/ R). One feature of I/ R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/ R injury. We employed simulated I/ R of cardiac HL-1 cells as an in vitro model of I/ R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation ( autophagic flux), as determined by steady- state levels of autophagosomes in relation to lysosomal inhibitor- mediated accumulation of autophagosomes. We found that I/ R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/ R and significantly reduced activation of pro- apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/ R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/ R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/ R injury to the heart cell.