The cardioprotectant 3',4'-dihydroxyflavonol inhibits the mitochondrial permeability transition pore

Abstract

Purpose: 3',4'-Dihydroxyflavonol (DiOHF) reduces injury caused by myocardial ischaemia and reperfusion (IR) in association with a reduction in oxidative stress. Oxidative stress contributes to the opening of the mitochondrial permeability transition pore (mPTP), a key event in myocardial IR injury. The aim of this study was to determine the effect of DiOHF on mPTP, as well as mitochondrial respiration and the generation of reactive oxygen species (ROS) by cardiac mitochondria after IR in anaesthetised rats. Methods: Male Wistar rats were anaesthetised with pentobarbitone (70 mg/kg iv), intubated and ventilated. Through a left thoracotomy the left main coronary artery was occluded for 30 min and then reperfused for 15 minutes. The heart was then rapidly removed, cooled and the area at risk of ischaemia homogenised and centrifuged for the isolation of mitochondria. In sham experiments the rats were anaesthetised but not subject to ischaemia. DiOHF (10 mg/kg iv) or vehicle (50% DMSO) was injected intravenously 5 minutes before reperfusion. mPTP opening was measured by monitoring mitochondrial Ca2+ retention capacity. Oxygen consumption of isolated mitochondria was measured in the presence of pyruvate (5 mM) and malate (5 mM) with a Clark-type electrode and ROS generation was assessed by measuring the rate of H2O2 production detected by amplex red. Results: Treatment of sham rats with DiOHF significantly increased the concentration of Ca2+ required to stimulate mPTP opening (sham 87±6; sham+DiOHF 120±9 μM). This was accompanied by an increase in state 3 oxygen consumption (sham 352±55; sham+DiOHF 572±21 nmol O2/min/mg protein) and a decrease in H2O2 release (sham 0.028±0.002; sham+DiOHF 0.019±0.002 nmol/min/mg protein). IR significantly decreased the concentration of Ca2+ required to stimulate mPTP opening (IR 44±5 μM), decreased state 3 oxygen consumption (IR 232±15 nmol O2/min/mg protein) and increased H2O2 release (IR 0.034±0.001 nmol/min/mg protein) compared to sham. Treatment with DiOHF prevented IR-induced changes in mPTP opening (IR+DiOHF 78±7 μM), state 3 oxygen consumption (IR+DiOHF 375±30 nmol O2/min/mg protein) and H2O2 release (IR 0.028±0.002 nmol/min/mg protein) so that there was no difference compared to sham. Conclusions: In normal rats DiOHF inhibits mPTP opening and decreases mitochondrial ROS production. Importantly, DiOHF administration before reperfusion prevents IR-induced mPTP opening, impairment of state 3 respiration and increases in ROS production. The beneficial actions of DiOHF on mitochondria are likely to make a major contribution to its cardioprotective actions.