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 (AMPK) pathway is protective, whereas reperfusion stimulates autophagy with BECLIN-1 upregulation, and is implicated in causing cell death. We examined flux through the macro-autophagy 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 GFP-tagged microtubule associated protein light chain-3 (LC3) to induce ischemia (120 minutes) or ischemia-reperfusion (IR, 30–90 minutes) with saline or chloroquine (CQ) pretreatment (n=4/group). Autophagosome clearance, assessed as the ratio of punctate LC3 abundance in saline to CQ treated samples was markedly impaired with IR as compared with sham controls. Reoxygenation increased cell death in neonatal rat cardiomyocytes (NRCMs) as compared with hypoxia alone; markedly increased autophagosomes but not autolysosomes (assessed as punctate dual fluorescent mCherry-GFP tandem tagged LC3 expression); and impaired clearance of polyglutamine aggregates, indicating impaired autophagic flux. The resultant autophagosome accumulation was associated with increased reactive oxygen species (ROS) and mitochondrial permeabilization leading to cell death, which was attenuated by cyclosporine A pretreatment. Hypoxia-reoxygenation injury was accompanied by ROS-mediated BECLIN-1 upregulation and reduction in Lysosome Associated Membrane Protein-2 (LAMP2), a critical determinant of autophagosome-lysosome fusion. Restoration of LAMP2 levels synergizes with partial BECLIN-1 knockdown to restore autophagosome processing and attenuate cell death following hypoxia-reoxygenation. Conclusions Ischemia-reperfusion injury impairs autophagosome clearance mediated in part by ROS-induced decline in LAMP2 and upregulation of BECLIN-1, contributing to increased cardiomyocyte death.

Autophagic program is regulated by miR-325

“…Beclin 1 is a well-known key regulator of autophagy, 34,35 and recent studies have shown that Beclin 1 mediates the autophagic cell death in cardiomyocytes. 36 Our results also showed that knockdown of Beclin 1 attenuated autophagy (Supplementary Figure 3A) and cell death (Supplementary Figure 3B). Overexpression of Beclin 1 sensitized cells to undergo death, and increased cell death was observed upon both A/R and Beclin 1 treatment in the presence of a lysosomal inhibitor bafilomycin A1 (Supplementary Figure 3C).…”

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

“…We next determined whether repetitive starvation would offer cytoprotection to HR-induced cell death. 36 Interestingly, 2 cycles of repetitive starvation were associated with a modest (and statistically significant) »13% to 15% reduction in HRinduced cell death (Fig. 12E and F).…”

“…Indeed, we have previously found that transcriptional suppression of the autophagy machinery with reperfusion-induced accumulation of BECN1 explains the lack of a protective role of the observed extent of autophagy induction in cardiac ischemia-reperfusion injury. 36,68 Further insights into this process could be gleaned via careful examination of the effects of intermittent fasting on the transcriptional program of the injured myocardium, in future studies.…”

“…26 Ischemia-reperfusion modeling Mice were subjected to reversible left anterior descending artery ligation for 30 min followed by reperfusion, in an openchest procedure, as described. 36 Overall surgical mortality was very low (<3 %). At 24 h post induction of ischemia, cardioplegic solution was injected retrogradely through the aorta in situ, followed by sectioning of the left ventricle in slices; which were incubated in triphenyl tetrazolium chloride for 30 min at 37 C. All surgeries were performed by one surgeon (C.J.W.)…”

“…35]); or the paradoxical effects on autophagy observed with haploinsufficiency of BECN1. 13,17,36 To determine if LAMP2 deficiency impairs autophagic flux with repetitive fasting, we subjected Lamp2 heterozygous null mice and littermate wild types for 6 wk of intermittent fasting followed by assessment of autophagic flux with injection of CQ (or diluent) on a fed day. Lamp2 heterozygous null mice demonstrated impaired autophagic flux on a fed day after being subjected to the intermittent fasting protocol, assessed as CQinduced accumulation of LC3-II (fold change in CQ/dil over nonfasted mice: 0.97 § 0.06 vs. Fig.…”