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 (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) 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. BK(Ca) 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 BK(Ca) 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 mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.

We measured the effects of global ischemia and reperfusion on intracellular Na(+), NADH, cytosolic and mitochondrial (subscript mito) Ca(2+), relaxation, metabolism, contractility, and Ca(2+) sensitivity in the intact heart. Langendorff-prepared guinea pig hearts were crystalloid perfused, and the left ventricular (LV) pressure (LVP), first derivative of LVP (LV dP/dt), coronary flow, and O(2) extraction and consumption were measured before, during, and after 30-min global ischemia and 60-min reperfusion. Ca(2+), Na(+), and NADH were measured by luminescence spectrophotometry at the LV free wall using indo 1 and sodium benzofuran isophthalate, respectively, after subtracting changes in tissue autofluorescence (NADH). Mitochondrial Ca(2+) was assessed by quenching cytosolic indo 1 with MnCl(2). Mechanical responses to changes in cytosolic-systolic (subscript sys), diastolic (subscript dia), and mitochondrial Ca(2+) were tested over a range of extracellular [Ca(2+)] before and after ischemia-reperfusion. Both [Ca(2+)](sys) and [Ca(2+)](dia) doubled at 1-min reperfusion but returned to preischemia values within 10 min, whereas [Ca(2+)](mito) was elevated over 60-min reperfusion. Reperfusion dissociated [Ca(2+)](dia) and [Ca(2+)](sys) from contractile function as LVP(sys-dia) and the rise in LV dP/dt (LV dP/dt(max)) were depressed by one-third and the fall in LV dP/dt (LV dP/dt(min)) was depressed by one-half at 30-min reperfusion, whereas LVP(dia) remained markedly elevated. [Ca(2+)](sys-dia) sensitivity at 100% LV dP/dt(max) was not altered after reperfusion, but [Ca(2+)](dia) at 100% LV dP/dt(min) and [Ca(2+)](mito) at 100% LV dP/dt(max) were markedly shifted right on reperfusion (ED(50) +36 and +125 nM [Ca(2+)], respectively) with no change in slope. NADH doubled during ischemia but returned to normal on initial reperfusion. The intracellular [Na(+)] ([Na(+)](i)) increased minimally during ischemia but doubled on reperfusion and remained elevated at 60-min reperfusion. Thus Na(+) and Ca(2+) temporally accumulate during initial reperfusion, and cytosolic Ca(2+) returns toward normal, whereas [Na(+)](i) and [Ca(2+)](mito) remain elevated on later reperfusion. Na(+) loading likely contributes to Ca(2+) overload and contractile dysfunction during reperfusion.

Heinen A, Aldakkak M, Stowe DF, Rhodes SS, Riess ML, Varadarajan SG, Camara AK. Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca 2ϩ -sensitive K ϩ channels. Am J Physiol Heart Circ Physiol 293: H1400-H1407, 2007. First published May 18, 2007; doi:10.1152/ajpheart.00198.2007.-Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O2 concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH2-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O2•Ϫ ); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate ϩ rotenone-dependent H 2O2 production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca 2ϩ -sensitive K ϩ channel (mtBKCa) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O 2 consumption, membrane potential (⌬⌿ m), H2O2 release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 M) increased state 2 and state 4 respiration by 26 Ϯ 4% and 14 Ϯ 4%, respectively; this increase was abolished by the BK Ca channel blocker paxilline (5 M). Paxilline alone had no effect on respiration. NS-1619 did not alter ⌬⌿ m or redox state but decreased H 2O2 production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K ϩ influx through mtBKCa channels reduces mitochondrial H2O2 production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H 2O2 production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K ϩ influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.

We determined in intact hearts whether inhibition of Na(+)/H(+) exchange (NHE) decreases intracellular Na(+) and Ca(2+) during ischemia and reperfusion, improves function during reperfusion, and reduces infarct size. Guinea pig isolated hearts were perfused with Krebs-Ringer solution at 37 degrees C. Left ventricular (LV) free wall intracellular Na(+) concentration ([Na(+)](i)) and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured using fluorescence dyes. Hearts were exposed to 30 min of ischemia with or without 10 microM of benzamide (BIIB-513), a selective NHE-1 inhibitor, infused for 10 min just before ischemia or for 10 min immediately on reperfusion. At 2 min of reperfusion, BIIB-513 given before ischemia decreased peak increases in [Na(+)](i) and [Ca(2+)](i), respectively, from 2.5 and 2.3 times (controls) to 1.6 and 1.3 times pre-ischemia values. At 30 min of reperfusion, BIIB-513 increased systolic-diastolic LV pressure (LVP) from 49 +/- 2% (controls) to 80 +/- 2% of pre-ischemia values. BIIB-513 reduced ventricular fibrillation by 54% and reduced infarct size from 64 +/- 1% to 20 +/- 3%. First derivative of the LVP, O(2) consumption, and cardiac efficiency were also improved by BIIB-513. Similar results were obtained with BIIB-513 given on reperfusion. These data show that Na(+) loading is a marker of reperfusion injury in intact hearts in that inhibiting NHE reduces Na(+) and Ca(2+) loading during reperfusion while improving function. These results clearly implicate the ionic basis by which inhibiting NHE protects the guinea pig intact heart from ischemia-reperfusion injury.