Ischemic preconditioning and diazoxide limit mitochondrial Ca2+ overload during ischemia/reperfusion: Role of reactive oxygen species

Ischemic preconditioning (IPC) and pharmacological treatment with mitochondrial ATP-sensitive K+ (mitoKATP) channel openers, such as diazoxide (DZ), appear to protect ischemic myocardium, in part via a reactive oxygen species (ROS)-dependent mechanism (1,2). Although clarification of this protective mechanism remains elusive, it potentially involves activation and translocation of protein kinase C (PKC) (1,3), diminution of ROS production during reperfusion (4) and maintenance of mitochondrial membrane potential (5). It has been demonstrated in selected species that IPC can only be blocked using a combination of PKC and tyrosine kinase blockers, demonstrating the nexus between ROS, kinase activity and IPC (6). The association, and likely causal role, of mitochondrial Ca2+ ([Ca2+]m) overload with increased cellular injury during ischemia/reperfusion (I/R) has been established (7). Specifically, pharmacological inhibition of [Ca2+]m overload with ruthenium red has been shown to limit reperfusion injury (8), and the level of [Ca2+]m has been demonstrated to be inversely related to the degree of functional recovery following I/R (7). In these latter experiments, IPC and DZ were shown to attenuate the increase in [Ca2+]m during I/R, an effect eliminated by coadministration of the mitoKATP channel blocker 5-hydroxydecanoate (5-HD) (7). Given the data regarding the role of ROS in IPC and DZ administration, and the evidence that limitation of [Ca2+]m overload is associated with functional protection, we tested the hypothesis that lower [Ca2+]m on reperfusion and improved functional recovery are dependent on preischemic generation of ROS. To test this hypothesis, an isolated perfused rat heart model of I/R was employed with beat-by-beat fluorescent measurements of intracellular Ca2+ ([Ca2+]i) and [Ca2+]m, and parallel measurements of hemodynamics and creatine kinase (CK), a marker of cellular injury. The ROS scavenger N-2-mercaptopropionyl glycine (2-MPG) was administered only before ischemia, bridging IPC episodes or during administration of DZ, and these results were compared with untreated hearts.