Mitochondrial handling of excess Ca 2+ is substrate‐dependent with implications on ROS generation

Mitochondrial complex III is a major source of ROS during ischemia and reperfusion (I/R). Several mechanisms modulate ROS production such as mitochondrial Ca 2+ uptake. Here we sought out the mechanisms of ROS generation from complex III due to excess extra matrix Ca 2+ . Guinea pig heart mitochondria were suspended in respiration media containing different concentrations of Ca 2+ , and either amplex red/horseradish peroxidase, rhodamine 123, or calcium green to measure H 2 O 2 generation, membrane potential (ΔΨ), or [Ca 2+ ], respectively. Either pyruvate or succinate (10 mM) was added next, followed by antimycin A (AA; 5 μM) to inhibit complex III. Mitochondria established a fully polarized ΔΨ under excess extra matrix Ca 2+ with pyruvate, whereas they remained partially depolarized with succinate as the substrate. AA caused Δ Ψ depolarization for both substrates, and matrix Ca 2+ extrusion which was more with succinate than pyruvate and coincided with a larger H 2 O 2 generation that was significantly decreased with cyclosporine A, an inhibitor of permeability transition pore (PTP) opening. Succinate accumulates during I/R and becomes the major substrate utilized by mitochondria. Mitochondrial inability to maintain a polarized Δ Ψ under excess extra matrix Ca 2+ when succinate, but not pyruvate, is the substrate may lead to PTP opening and enhancing ROS formation from complex III during I/R.