Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

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Abstract
ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BKCa) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BKCa channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BKCa channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2−·), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BKCa channel opener 1-(2′-hydroxy-5′-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2−·, or an antagonist of the BKCa channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2−· generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBKCa channels; 2) NS effects are blocked by BKCa antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.