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Abstract: The mitochondrial ATP-dependent K+ channel (mitoKATP) is widely considered by many to play a central role in cardioprotection by ischemic and pharmacological preconditioning and by ischemic postconditioning. Nevertheless, several laboratories have questioned the existence of mitoKATP. This article summarizes the evidence for and against and addresses two key questions: How strong is the evidence for the presence of a KATP channel in mitochondria? Are the pharmacological agents used to modulate mitoKATP activity sufficiently specific to allow the role of these channels in cardioprotection to be established? [Copyright &y& Elsevier]

Abstract: Mitochondria are central players in the pathophysiology of ischemia–reperfusion. Activation of plasma membrane G-coupled receptors or the Na,K-ATPase triggers cytosolic signaling pathways that result in cardioprotection. Our working hypothesis is that the occupied receptors migrate to caveolae, where signaling enzymes are scaffolded into signalosomes that bud off the plasma membrane and migrate to mitochondria. The signalosome–mitochondria interaction then initiates intramitochondrial signaling by opening the mitochondrial ATP-sensitive K+ channel (mitoKATP). MitoKATP opening causes an increase in ROS production, which activates mitochondrial protein kinase C epsilon (PKCɛ), which inhibits the mitochondrial permeability transition (MPT), thus decreasing cell death. We review the experimental findings that bear on these hypotheses and other modes of protection involving mitochondria. [Copyright &y& Elsevier]

Much of cell death from ischaemia/reperfusion in heart and other tissues is generally thought to arise from mitochondrial permeability transition (MPT) in the first minutes of reperfusion. In ischaemic pre-conditioning, agonist binding to Gi protein-coupled receptors prior to ischaemia triggers a signalling cascade that protects the heart from MPT. We believe that the cytosolic component of this trigger pathway terminates in activation of guanylyl cyclase resulting in increased production of cGMP and subsequent activation of protein kinase G (PKG). PKG phosphorylates a protein on the mitochondrial outer membrane (MOM), which then causes the mitochondrial KATP channel (mitoKATP) on the mitochondrial inner membrane to open, leading to increased production of reactive oxygen species (ROS) by the mitochondria. This implies that the protective signal is somehow transmitted from the MOM to its inner membrane. This is accomplished by a series of intermembrane signalling steps that includes protein kinase C (PKCɛ) activation. The resulting ROS then activate a second PKC pool which, through another signal transduction pathway termed the mediator pathway, causes inhibition of MPT and reduction in cell death. [ABSTRACT FROM PUBLISHER]

We showed recently that mitochondrial ATP-dependent K+ channel (mitoKATP) opening is required for the inotropic response to ouabain. Because mitoKATP opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na+-K+-ATPase with the opening of mitoKATP. In Langendorff-perfused rat hearts, 10-80 µM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoKATP opening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoKATP inhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia–reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex. Methods and results: In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 μM followed by an 8-minute washout before 30 min of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cγ1/protein kinase Cε (PLC-γ1/PKCε) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-γ1/PKCε pathway, but also cardiac protection. This protection was also blocked by a PKCε translocation inhibitor peptide (PKCε TIP). Conclusion: Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-γ1 and PKCε. [Copyright &y& Elsevier]

There is considerable evidence that opening the mitochondrial ATP-sensitive potassium channel (mitoKATP) is cardioprotective in ischemia-reperfusion. Two prominent questions surround the role of mitoKATP in the cardiomyocyte: How does opening mitoKATP protect? What is the normal physiological role of mitoKATP in the heart? Before these questions can be addressed, it is necessary to agree on the bioenergetic consequences of opening mitoKATP, and this distills down to a single question – does opening mitoKATP cause significant uncoupling or not? The evidence strongly indicates that it does not and that reports of uncoupling and inhibition of Ca2+ uptake are the result of using toxic concentrations of KATP channel openers. Thus, opening mitoKATP results in increased K+ flux that is sufficient to change mitochondrial volume but is insufficient to cause significant depolarization of membrane potential. The volume changes, however, have significant bioenergetic consequences for energy coupling in the cell. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the effects of HMR1098, a selective blocker of sarcolemmal ATP-sensitive potassium channel (sarcKATP), in Langendorff-perfused rat hearts submitted to ischemia and reperfusion. The recovery of heart hemodynamic and mitochondrial function, studied on skinned fibers, was analyzed after 30-min global ischemia followed by 20-min reperfusion. Infarct size was quantified on a regional ischemia model after 2-h reperfusion. We report that the perfusion of 10 μM HMR1098 before ischemia, delays the onset of ischemic contracture, improves recovery of cardiac function upon reperfusion, preserves the mitochondrial architecture, and finally decreases infarct size. This HMR1098-induced cardioprotection is prevented by 1 mM 2-mercaptopropionylglycine, an antioxidant, and by 100 nM nifedipine, an L-type calcium channel blocker. Concomitantly, it is shown that HMR1098 perfusion induces (i) a transient and specific inhibition of the respiratory chain complex I and, (ii) an increase in the averaged intracellular calcium concentration probed by the in situ measurement of indo-1 fluorescence. Finally, all the beneficial effects of HMR1098 were strongly inhibited by 5-hydroxydecanoate and abolished by glibenclamide, two mitoKATP blockers. This study demonstrates that the HMR1098-induced cardioprotection occurs indirectly through extracellular calcium influx, respiratory chain complex inhibition, reactive oxygen species production and mitoKATP opening. Taken together, these data suggest that a functional interaction between sarcKATP and mitoKATP exists in isolated rat heart ischemia model, which is mediated by extracellular calcium influx. [Copyright &y& Elsevier]

Myocardial infarction is a manifestation of necrotic cell death as a result of opening of the mitochondrial permeability transition (MPT). Receptor-mediated cardioprotection is triggered by an intracellular signaling pathway that includes phosphatidylinositol 3-kinase, endothelial nitric-oxide synthase, guanylyl cyclase, protein kinase G (PKG), and the mitochondrial KATP channel (mitoKATP). In this study, we explored the pathway that links mitoKATP with the MPT. We confirmed previous findings that diazoxide and activators of PKG or protein kinase C (PKC) inhibited MPT opening. We extended these results and showed that other K+ channel openers as well as the K+ ionophore valinomycin also inhibited MPT opening and that this inhibition required reactive oxygen species. By using isoform-specific peptides, we found that the effects of KATP channel openers, PKG, or valinomycin were mediated by a PKC∊. Activation of PKC∊ by phorbol 12-myristate 13-acetate or H2O2 resulted in mitoKATP-independent inhibition of MPT opening, whereas activation of PKC∊ by PKG or the specific PKC∊ agonist ψ∊ receptor for activated C kinase caused mitoKATP-dependent inhibition of MPT opening. Exogenous H2O2 inhibited MPT, because of its activation of PKC∊, with an IC50 of 0.6 (±0.1) μ. On the basis of these results, we propose that two different PKC∊ pools regulate this signaling pathway, one in association with mitoKATP and the other in association with MPT. [ABSTRACT FROM AUTHOR]