Protection against cardiac ischemia-reperfusion injury by hypothermia and by inhibition of succinate accumulation and oxidation is additive

Abstract

Hypothermia induced at the onset of ischemia is a potent experimental cardioprotective strategy for myocardial infarction. The aim of our study was to determine whether the beneficial effects of hypothermia may be due to decreasing mitochondria-mediated mechanisms of damage that contribute to the pathophysiology of ischemia/reperfusion injury. New Zealand male rabbits were submitted to 30 min of myocardial ischemia with hypothermia (32 °C) induced by total liquid ventilation (TLV). Hypothermia was applied during ischemia alone (TLV group), during ischemia and reperfusion (TLV-IR group) and normothermia (Control group). In all the cases, ischemia was performed by surgical ligation of the left anterior descending coronary artery and was followed by 3 h of reperfusion before assessment of infarct size. In a parallel study, male C57BL6/J mice underwent 30 min myocardial ischemia followed by reperfusion under either normothermia (37 °C) or conventionally induced hypothermia (32 °C). In both the models, the levels of the citric acid cycle intermediate succinate, mitochondrial complex I activity were assessed at various times. The benefit of hypothermia during ischemia on infarct size was compared to inhibition of succinate accumulation and oxidation by the complex II inhibitor malonate, applied as the pro-drug dimethyl malonate under either normothermic or hypothermic conditions. Hypothermia during ischemia was cardioprotective, even when followed by normothermic reperfusion. Hypothermia during ischemia only, or during both, ischemia and reperfusion, significantly reduced infarct size (2.8 ± 0.6%, 24.2 ± 3.0% and 49.6 ± 2.6% of the area at risk, for TLV-IR, TLV and Control groups, respectively). The significant reduction of infarct size by hypothermia was neither associated with a decrease in ischemic myocardial succinate accumulation, nor with a change in its rate of oxidation at reperfusion. Similarly, dimethyl malonate infusion and hypothermia during ischemia additively reduced infarct size (4.8 ± 2.2% of risk zone) as compared to either strategy alone. Hypothermic cardioprotection is neither dependent on the inhibition of succinate accumulation during ischemia, nor of its rapid oxidation at reperfusion. The additive effect of hypothermia and dimethyl malonate on infarct size shows that they are protective by distinct mechanisms and also suggests that combining these different therapeutic approaches could further protect against ischemia/reperfusion injury during acute myocardial infarction.

Keywords: Dimethyl malonate; Hypothermia; Ischemia; Myocardial infarction; Reperfusion; Succinate.

Fig. 1

Experimental protocol in rabbits (a), real-time temperature regulation (b) and infarct size after discrete or continuous hypothermia (c). TLV, total liquid ventilation. *p < 0.05 vs Control; †p < 0.05 vs TLV and Control

Fig. 2

Experimental protocol (a) and infarct size (b) measurements in mice. *p < 0.05 vs Control

Fig. 3

Tissue concentrations of succinate at the end of ischemia in non-ischemic and ischemic area in rabbit (a), myocardial concentration of succinate during ischemia and reperfusion in mice, in ischemic and non-ischemic areas (b), experimental protocol for evaluation of succinate inhibition by dimethyl malonate in rabbits with or without discrete hypothermia (c) and subsequent infarct size (d). TLV, total liquid ventilation. *p < 0.05 vs Control; †p < 0.05 vs Malonate group

Fig. 4

Consequence of hypothermia on ATP/ADP preservation at the end of ischemia in rabbits (a), on hypoxanthine and xanthine levels at the end of ischemia in mice (b) and on H₂O₂ production by reverse electron transport at 37 °C or 32 °C (c). *p <0.05 between groups