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The gap junction plays roles not only in electrical coupling of cardiomyocytes but also in intercellular transport of biologically active substances. Furthermore, the gap junction participates in decision making on cell survival versus cell death in various types of cells, and a part of reperfusion injury in the heart has been indicated to be gap junction mediated. The contribution of gap junction communication (GJC) and/or mitochondrial "hemichannels" to protective signaling during the trigger phase of ischemic preconditioning (IPC) is suggested by observations that IPC failed to protect the heart when GJC was blocked during TPC. Although ischemia suppresses both electrical and chemical GJC, chemical GJC persists for a considerable time after electrical GJC is lost. IPC facilitates the ischemia-induced suppression of chemical GJC, whereas IPC delays the reduction of electrical GJC after ischemia. The inhibition of GJC during sustained ischemia and reperfusion by GJC blockers mimics the effect of IPC on myocardial necrosis. IPC induces distinct effects on the interaction of connexin-43 with protein kinases, and the phosphorylation of connexin-43 at Ser368 by PKCϵ is a primary mechanism of inhibition of chemical GJC by IPC. Several lines of evidence support the notion that the modulation of GJC is a part of the mechanism of IPC-induced protection against myocardial necrosis and arrhythmias, though what percentage of IPC protection is attributable to the inhibition of GJC during ischemia-reperfusion still remains unclear. [ABSTRACT FROM AUTHOR]

Ischemic preconditioning (PC) suppresses chemical coupling of cardiomyocytes via gap junctions (GJs) during ischemia, which is an adjunct mechanism of protection. The aim of this study was to characterize roles of protein kinases in PC-induced GJ modulation. In isolated rat hearts, ventricular tissues were sampled before and after ischemia with or without PC, and intercalated disc-rich fractions were separated for immunoprecipitation and immunoblotting. Levels of protein kinase C (PKC)-ε, p38mitogen-activated protein kinase (MAPK)-α, and Src coimmuno-precipitated with connexin-43 (Cx43) were increased after ischemia, whereas p38MAPKβ was not detected in the Cx43 immunoprecipitates. PC did not modify the level of Cx43-Src complex after ischemia. However, PC enhanced Cx43-PKCε complex formation, which was abolished by PKCε translocation inhibitory peptide (TIP). In contrast, PC reduced Cx43-p38MAPKα complex level and p38MAPK activity in the Cx43 immunoprecipitates after ischemia. The effect of PC on Cx43-p38MAPKα interaction was mimicked by SB-203580, a p38MAPK inhibitor. PC reduced permeability of GJs to Lucifer yellow in the myocardium at 25 mm after ischemia, and this effect was abolished by PKCε-TIP. SB-203580 increased the GJ permeability at 15 mm after ischemia compared with that in untreated controls, but the difference became insignificant 25 mm after ischemia. In conclusion, PC has distinct effects on interaction of GJ Cx43 with PKCε, p38MAPKα, and Src during ischemia. Suppression of GJ permeability during ischemia by PC is primarily achieved by enhanced interaction of Cx43 with PKCε, which overwhelms the counterbalancing effect of reduced Cx43-p38MAPKα interaction. [ABSTRACT FROM AUTHOR]

Ohori K, Miura T, Tanno M, Miki T, Sato T, Ishikawa 8, Horio Y, Shimamoto K. Ser9 phosphorylation of mitochondrial GSK-3β is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis. Am J Physiol Heart Circ Physiol 295: H2079-H2086, 2008. First published September 19, 2008; doi:10.1152/ajpheart.00092.2008.-The aim of this study was to determine the role of GSK-3β in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/mI) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3β and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H[sub2]O[sub2] from 38.3 ± 2.7% to 26.0 ± 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3β (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3β knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3β (K85R). The level of colocalization of intracellular GSK-3β with mitochondria assessed by enhanced green fluorescent proteintagged GSK-3β or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho GSK-3β colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl2-associated X protein (BAX) after exposure to H[sub2]O[sub2] was inhibited by EPO pretreatment and by GSK-3β knockdown. These results suggest that the suppression of GSK-3β activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the hypothesis that δ-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the δ-opioid receptor activation. A δ-opioid receptor agonist, [D-Ala²,D-Leu5]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-ε inhibitor [PKC-ε translocation inhibitory peptide (PKC-ε-TIP)] but was not affected by a PKC-δ inhibitor (rottlerin). After DADLE infusion, PKC-ε, but not PKC-δ, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser368) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-ε-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-ε activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-ε plays a crucial role in δ-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic δ-opioid receptor activation. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine whether erythropoietin (EPO) affords additional cardioprotection to the preconditioned myocardium by enhanced phosphorylation of Akt, STAT3, or glycogen synthase kinase-3β (GSK-3β). Preconditioning (PC) with 5-min ischemia/5-min reperfusion and EPO (5,000 U/kg iv) reduced infarct size (as % of area at risk, %IS/AR) after 20-min ischemia in rat hearts in situ from 56.5 ± 1.8% to 25.2 ± 2.1% and to 36.2 ± 2.8%, respectively. PC-induced protection was significantly inhibited by a protein kinase C inhibitor, chelerythrine (5 mg/kg), and slightly blunted by a phosphatidylinositol-3-kinase inhibitor, wortmannin (15 μg/kg). The opposite pattern of inhibition was observed for EPO-induced protection. The combination of PC and EPO further reduced %IS/AR to 8.9 ± 1.9%, and this protection was inhibited by chelerythrine and wortmannin. The additive effects of PC and EPO on infarct size were mirrored by their effects on the level of phosphorylated GSK-3β at 5 min after reperfusion but not their effects on the level of phospho-Akt or phospho-STAT3. To mimic phosphorylation-induced inhibition of GSK-3β activity, SB-216763 (SB), a GSK-3β inhibitor, was administered before ischemia or 5 rain before reperfusion. Infarct size was significantly reduced by preischemic injection (%IS/AR = 40.4 ± 2.2% by 0.6 mg/kg SB and 34.0 ± 1.8% by 1.2 mg/kg SB) and also by prereperfusion injection (%IS/AR = 32.0 ± 2.0% by 1.2 mg/kg SB). These results suggest that EPO and PC afford additive infarct size-limiting effects by additive phosphorylation of GSK-3β at the time of reperfusion by Akt-dependent and -independent mechanisms. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37°C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18β-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation. [ABSTRACT FROM AUTHOR]

Presents a study that examined whether the mitochondrial adenosine triphosphate-sensitive potassium channel (mitoK[subATP]) is an effector downstream of protein kinase C (PKC)-∈ in the mechanism of preconditioning in isolated rabbit hearts. Surgical preparation and perfusion of isolated hearts; Discussion of the hemodynamic parameters in isolated hearts; Relationship between activation of PKC and opening of mitoK[subATP].

Provides information on a study that examined the function of ecto-5'-nucleotidase activity during ischemic preconditioning of myocardium. Materials and methodology; Experimental findings; Statistical analysis; Results.