Mitochondrial oxidative phosphorylation and mitophagy in myocardial ischaemia/reperfusion: effects of chloroquine.

Abstract

The aim of this study was to evaluate the temporal relationship between mitochondrial oxidative phosphorylation and mitophagy in rat hearts subjected to ischaemia/reperfusion. Measurements were made at specific points during the experimental protocol (snapshot approach) and by assessments of mitophagic flux, using chloroquine pre-treatment. Isolated working rat hearts were subjected to 25 or 30 minutes of global ischaemia/10 minutes of reperfusion. Half of each group received chloroquine (10 mg/kg, intraperitoneally) one hour before experimentation. Mitochondria were isolated after stabilisation, ischaemia and reperfusion, and oxidative phosphorylation was measured polarographically. Mitochondrial mitophagy markers were detected by Western blot analysis. Mitochondrial oxygen uptake (state 3) and oxidative phosphorylation rate were reduced by ischaemia and increased by reperfusion. Chloroquine pre-treatment increased both parameters. Using a snapshot approach, exposure to ischaemia ± reperfusion had little effect on mitochondrial PINK1, Parkin and p62/SQSTM1 expression. Ischaemia reduced Rab9 expression, and reperfusion upregulated the phosphor DRP1, phosphor/total DRP1 ratio and Rab9 levels. Chloroquine significantly reduced PINK1, p62/SQSTM1, Rab9 and particularly Parkin expression during reperfusion, without an effect on mitochondrial total and phospho DRP1 levels. Ischaemia/reperfusion-induced changes in mitochondrial oxidative phosphorylation function occurred concomitantly with changes in mitophagic flux. Pre-treatment with chloroquine profoundly affected mitochondrial function as well as the pattern of mitophagy during ischaemia/reperfusion.