Impairment of pH gradient and membrane potential mediates redox dysfunction in the mitochondria of the post-ischemic heart

The mitochondrial electrochemical gradient (Delta p), which comprises the pH gradient (Delta pH) and the membrane potential (Delta psi), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide (O-.(2)-) and H2O2 overproduction leading to oxidative injury. However, the role of Delta pH and Delta W in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced (O-.(2)-) and H2O2 production when compared to state 2 conditions. Oligomycin (increases DpH by inhibiting ATP synthase) increased .(O-.(2)-) and H2O2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial (O-.(2)-) and H2O2 generation is dependent on Delta pH and that IR impaired the DpH of normal mitochondria. Conversely, nigericin (dissipates Delta pH) dramatically reduced (O-.(2)-) and H2O2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced Delta pH impairment in normal mitochondria. Valinomycin, a K+ ionophore used to dissipate Delta W, drastically diminished (O-.(2)-) and H2O2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that Delta W also contributed to O-.(2)- generation by normal mitochondria and that IR mediated Delta W impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by Delta pH restricts electron flow, controls a limited amount of O-.(2)- generation, and results in a more reduced myocardium; however, IR causes Delta pH impairment and prompts a more oxidized myocardium.