Impaired Autophagosome Clearance Contributes to Cardiomyocyte Death in Ischemia/Reperfusion Injury

Abstract

Background— In myocardial ischemia, induction of autophagy via the AMP-induced protein kinase pathway is protective, whereas reperfusion stimulates autophagy with BECLIN-1 upregulation and is implicated in causing cell death. We examined flux through the macroautophagy pathway as a determinant of the discrepant outcomes in cardiomyocyte cell death in this setting. Methods and Results— Reversible left anterior descending coronary artery ligation was performed in mice with cardiomyocyte-restricted expression of green fluorescent protein–tagged microtubule–associated protein light chain-3 to induce ischemia (120 minutes) or ischemia/reperfusion (30–90 minutes) with saline or chloroquine pretreatment (n=4 per group). Autophagosome clearance, assessed as the ratio of punctate light chain-3 abundance in saline to chloroquine-treated samples, was markedly impaired with ischemia/reperfusion compared with sham controls. Reoxygenation increased cell death in neonatal rat cardiomyocytes compared with hypoxia alone, markedly increased autophagosomes but not autolysosomes (assessed as punctate dual fluorescent mCherry-green fluorescent protein tandem-tagged light chain-3 expression), and impaired clearance of polyglutamine aggregates, indicating impaired autophagic flux. The resultant autophagosome accumulation was associated with increased reactive oxygen species and mitochondrial permeabilization, leading to cell death, which was attenuated by cyclosporine A pretreatment. Hypoxia-reoxygenation injury was accompanied by reactive oxygen species–mediated BECLIN-1 upregulation and a reduction in lysosome-associated membrane protein-2, a critical determinant of autophagosome-lysosome fusion. Restoration of lysosome-associated membrane protein-2 levels synergizes with partial BECLIN-1 knockdown to restore autophagosome processing and to attenuate cell death after hypoxia-reoxygenation. Conclusion— Ischemia/reperfusion injury impairs autophagosome clearance mediated in part by reactive oxygen species–induced decline in lysosome-associated membrane protein-2 and upregulation of BECLIN-1, contributing to increased cardiomyocyte death.