Differential effects of buffer pH on Ca(2+)-induced ROS emission with inhibited mitochondrial complexes I and III.

Daniel P. Lindsay,Ryan Lubbe,David F. Stowe,Amadou K. S. Camara,Mohammed Aldakkak

doi:10.3389/fphys.2015.00058

Abstract

Excessive mitochondrial reactive oxygen species (ROS) emission is a critical component in the etiology of ischemic injury. Complex I and complex III of the electron transport chain are considered the primary sources of ROS emission during cardiac ischemia and reperfusion (IR) injury. Several factors modulate ischemic ROS emission, such as an increase in extra-matrix Ca2+, a decrease in extra-matrix pH, and a change in substrate utilization. Here we examined the combined effects of these factors on ROS emission from respiratory complexes I and III under conditions of simulated IR injury. Guinea pig heart mitochondria were suspended in experimental buffer at a given pH and incubated with or without CaCl2. Mitochondria were then treated with either pyruvate, a complex I substrate, followed by rotenone, a complex I inhibitor, or succinate, a complex II substrate, followed by antimycin A, a complex III inhibitor. H2O2 release rate and matrix volume were compared with and without adding CaCl2 and at pH 7.15, 6.9, or 6.5 with pyruvate + rotenone or succinate + antimycin A to simulate conditions that may occur during in vivo cardiac IR injury. We found a large increase in H2O2 release with high [CaCl2] and pyruvate + rotenone at pH 6.9, but not at pHs 7.15 or 6.5. Large increases in H2O2 release rate also occurred at each pH with high [CaCl2] and succinate + antimycin A, with the highest levels observed at pH 7.15. The increases in H2O2 release were associated with significant mitochondrial swelling, and both H2O2 release and swelling were abolished by cyclosporine A, a desensitizer of the mitochondrial permeability transition pore (mPTP). These results indicate that ROS production by complex I and by complex III is differently affected by buffer pH and Ca2+ loading with mPTP opening. The study suggests that changes in the levels of cytosolic Ca2+ and pH during IR alter the relative amounts of ROS produced at mitochondrial respiratory complex I and complex III.

Highlights

Ischemic injury is a multifactorial process that predisposes to further injury during reperfusion (Camara et al, 2007; Aldakkak et al, 2008a,b, 2011)
We investigated in a recent isolated mitochondrial study the impact of extreme conditions that might mimic the period of ischemia and reperfusion on reactive oxygen species (ROS) emission (Aldakkak et al, 2013)
We found a large increase in hydrogen peroxide (H2O2) release when complex III electron transfer was blocked by antimycin A (AA) in succinate-energized mitochondria incubated in elevated extra-matrix Ca2+ buffer

Summary

Introduction

Ischemic injury is a multifactorial process that predisposes to further injury during reperfusion (Camara et al, 2007; Aldakkak et al, 2008a,b, 2011). One key aspect of ischemic injury is the increase in mitochondrial production of reactive oxygen species (ROS) above the antioxidant ability of the endogenous ROS scavenging system (Trachootham et al, 2008; Stowe and Camara, 2009; Camara et al, 2010). An increase in ROS emission (more production and less scavenging) during ischemia leads to oxidative stress and apoptosis that leads to cellular dysfunction and death (Trachootham et al, 2008). During IR, these complexes are prone to self-induced oxidative damage (Gadicherla et al, 2012), which impairs their activity, predisposing them to even greater ROS production (Rouslin, 1983; Chen et al, 2007, 2008; Musatov and Robinson, 2012). The damage to complex I may occur abruptly within 20 min of ischemia, whereas damage to complex III may occur more gradually as ischemia proceeds (Rouslin, 1983; Chen et al, 2007)

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