Abstract
Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].
Highlights
Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia
For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinolrelated complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, permeability transition pores (PTP) opening may result in succinate/malatefueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2؉], [NAD؉], and [ADP]
The major finding of this study is that Mg2ϩ, NADϩ, and ADP induce marked acceleration of ROS production when complex III is inhibited by antimycin in permeabilized cardiac mitochondria
Summary
In contrast to succinate-fueled RET-driven ROS production by complex I, ROS production by forward electron transport through complexes I or III requires the flow of electrons out of these complexes to be inhibited. The complex I inhibitor rotenone has dehydrogenase; ⌬⌿, mitochondrial membrane potential; PTP, permeability transition pore; RET, reverse electron transport; Q, ubiquinone; QH2, ubiquinol; SDH, succinate dehydrogenase; I/R, ischemia/reperfusion We report the novel finding that when complex III is inhibited by antimycin in a setting in which succinate and its closely related metabolites are in limited supply, ROS production by complex III is dramatically increased after pore opening in the inner membrane by addition of NADϩ/Mg2ϩ to activate malic enzyme (ME2) These findings may be relevant to the damaging ROS burst during reperfusion after prolonged ischemia when complex III has been damaged, and mitochondrial permeability transition pore opening leads to elevated matrix Mg2ϩ, ADP, and a low NADH/NADϩ ratio
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