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.
Hamacher-Brady
Anne
A
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Brady
Nathan R
NR
Gottlieb
Roberta A
RA
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001343:Autophagy / Q000502:physiology*; D002460:Cell Line; D006325:Heart Atria / Q000166:cytology / Q000473:pathology; D051379:Mice; D017202:Myocardial Ischemia / Q000473:pathology* / Q000517:prevention & control*; D015428:Myocardial Reperfusion Injury / Q000473:pathology* / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000473:pathology* / Q000502:physiology