Ischemic Preconditioning Decreases Mitochondrial Proton Leak and Reactive Oxygen Species Production in the Postischemic Heart

Abstract

Background Proton leak (H+ leak) dissipates mitochondrial membrane potential (mΔΨ) through the reentry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H+ leak may affect reactive oxygen species (ROS) production. We measured H+ leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. Materials/Methods Isolated rat hearts (n=6/group) were subjected to either Control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H+ leak is the respiratory rate required to maintain membrane potential at -150mV in the presence of oligomycin-A Mitochondrial complex III ROS production was measured by fluorometry using Amplex-Red. Results IPC improved recovery of RPP at end reperfusion (63±4% vs. 21±2% in Control-IR, p<0.05). Ischemia-reperfusion caused increased H+ leak (94±12 vs. 31±1 nanomoles O/mg protein/min in Non-Ischemic Control, p<0.05). IPC attenuates these increases (55±9 nanomoles O/mg protein/min, p< 0.05 vs. Control-IR). IPC reduced mitochondrial ROS production compared to Control-IR (31±2 vs. 40±3 nanomoles/mg protein/min, p<0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in Control-IR for all mΔΨ and respiration rates. Conclusions Increasing H+ leak is not associated with increased ROS production. IPC decreases both the magnitude of H+ leak and ROS production after ischemia-reperfusion.

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