ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria.

Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel. In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 μm, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitochondrial uniporter. Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore. Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers. ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without preventing the action of valinomycin. In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects were inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis. Potassium channel-opening drugs are powerful cardioprotective agents capable of limiting cardiac cell injury following ischaemia-reperfusion (Gross, 1995; Grover, 1997; Kloner et al. 1998). These chemically diverse compounds share the property of promoting K+ current through ATP-sensitive K+ (KATP) channels (Quast, 1992; Terzic et al. 1995; Edwards & Weston, 1997). It has been assumed that the cardioprotective action of potassium channel openers is mediated through sarcolemmal KATP channels which, when open, shorten the action potential duration and limit Ca2+ influx into cardiac cells. Growing evidence, however, indicates that the protective efficacy does not always correlate with the degree of action potential shortening, implying additional cellular sites of drug action (Garlid et al. 1997; Liu et al. 1998). The prime candidates include mitochondria, which also harbour an ATP-sensitive K+ conductance, recognized as the mitochondrial KATP (mitoKATP) channel (Inoue et al. 1991; Paucek et al. 1992; Garlid, 1996; Szewczyk, 1997). Activation of mitoKATP channels by potassium channel openers has been associated with increased survival of cardiac cells following ischaemia and improved post-ischaemic recovery of heart muscle, although the precise mechanism responsible for such an outcome remains partially understood (Garlid et al. 1997; Liu et al. 1998). Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, is an important mediator of ischaemia-reperfusion injury in the heart (Park et al. 1990; Ferrari et al. 1993; Miyamae et al. 1996; Delcamp et al. 1998). Mitochondrial Ca2+ content is determined by the balance between mitochondrial Ca2+ uptake and release (Gunter et al. 1994; Zoratti & Szabo, 1995). It has been proposed that potassium channel openers, by virtue of their ability to dissipate mitochondrial membrane potential (Czyz et al. 1995; Holmuhamedov et al. 1998), may reduce the driving force for Ca2+ accumulation (Liu et al. 1998). Although maintenance of mitochondrial Ca2+ levels is expected to contribute to cardioprotection, direct evidence that potassium channel openers limit mitochondrial Ca2+ overload is lacking. Therefore, we here examined the action of potassium channel openers on mitochondrial Ca2+ in isolated cardiac mitochondria and living cardiomyocytes. We report that prototype potassium channel openers impede mitochondrial Ca2+ uptake and promote mitochondrial Ca2+ release, thereby diminishing the amount of accumulated Ca2+ within the mitochondrial matrix.