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

Abstract

Aim: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: DRP1; Parkin; Rab9; mitochondrial oxidative phosphorylation; mitophagy; myocardial ischaemia/reperfusion; p62/SQSTMI; PINK1.

Fig. 1

Experimental protocol. Mitochondria were prepared after 40 minutes of stabilisation; after 25 minutes of global ischaemia; after 10 minutes of reperfusion following 25 minutes of global ischaemia; after 30 minutes of global ischaemia; after 10 minutes of reperfusion following 30 minutes of global ischaemia.

Fig. 2

Baseline function of working rat hearts during stabilisation: effect of chloroquine pre-treatment (n = 10 hearts/group). CON: control; CQ: chloroquine pre-treatment (10 mg/kg); HR: heart rate (beats/min); W total: work total (mW).

Fig. 3

Effects of ischaemia/reperfusion and chloroquine pre-treatment on mitochondrial function with glutamate/malate as substrates (n = 5 hearts/group). Measurements of mitochondrial function were made after 40 minutes of stabilisation; after 25 minutes of global ischaemia; after 10 minutes of reperfusion following 25 minutes of global ischaemia; after 30 minutes of global ischaemia; after 10 minutes of reperfusion following 30 minutes of global ischaemia. Mitochondria were also prepared from hearts of age-matched control rats for comparison purposes. A. QO2 (state 3) (nAtoms oxygen/mg protein/min); B. QO2 (state 4) (nAtoms oxygen/mg protein/min); C. RCI (state 3/state 4); D. ox-phos rate (nmoles ATP/mg prot/min); E. percentage recovery after re-oxygenation. *p ≤ 0.05 vs corresponding untreated control rats. AMC: age-matched control; CON: control; CQ: chloroquine; STB: stabilisation; ISC: ischaemia; RP: reperfusion; ox-phos: oxidative phosphorylation; RCI: respiratory control index.

Fig. 4

The effects of ischaemia/reperfusion and chloroquine treatment on mitochondrial function with palmitoyl-L-carnitine/malate as substrates (n = five hearts /group). Measurements of mitochondrial function were made at the time points described in Fig. 3. *p ≤ 0.05 vs corresponding untreated control rats. AMC: age-matched control; CON: control; CQ: chloroquine; STB: stabilisation; ISC: ischaemia; RP: reperfusion; ox-phos: oxidative phosphorylation; RCI: respiratory control index.

Fig. 5

Effect of ischaemia/reperfusion and chloroquine on the expression of TOM 70, p62/SQSTM1 (p62), PINK and Parkin (A); pDRP-1/total DRP, total DRP, pDRP and Rab9 (B). Western blot analysis was done on mitochondria (n = four hearts/group) isolated from hearts after 40 minutes of stabilisation, after 25 minutes of global ischaemia; and after 10 minutes of reperfusion following 25 minutes of global ischaemia. *p < 0.05 vs corresponding stabilisation. #p < 0.05 vs corresponding ischaemia. AMC: age-matched controls; STB: stabilisation; ISC: ischaemia: RP: reperfusion; VE: negative control.