δ-Opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-ε-mediated phosphorylation of connexin 43

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.

Protective role of gap junctions in preconditioning against myocardial infarction

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.