Abstract 1605: Contribution of Gap Junction-Dependent and -Independent Mechanisms to Myocardial Salvage by δ-Opioid Receptor Activation
Objective Although some evidence suggests that suppression of the gap junction (GJ) contributes to myocardial salvage by preconditioning (PC), its relative importance among multiple protective mechanisms of PC remains unclear. To obtain a clue to this question, we examined the hypothesis that GJ-dependent and -independent mechanisms in PC are mediated by different protein kinase C (PKC) isoforms, affording different levels of myocardial salvage.
Methods and Results First, we assessed roles of PKC-ϵ and PKC-δ in suppression of GJ permeability by PC with δ-opioid receptor activation in isolated buffer-perfused rat hearts. Interaction of connexin-43 (Cx43), a GJ subunit protein, with PKC-ϵ and PKC-δ was determined by immunoprecipitation and immunoblotting. After activation of the δ-opioid receptor by [D-Ala2, D-Leu5]-enkephalin (DADLE, 300 nM), Cx43 in the ventricular myocardium was co-immunoprecipitated with PKC-ϵ, though such interaction with Cx43 was not detected for PKC-δ. GJ permeability during 25-min ischemia, which was determined by use of Lucifer yellow, was significantly reduced by pretreatment with DADLE to 38.5±6.0% (n=4) of the control level. This effect of DADLE on GJ permeability was abolished by a PKC-ϵ translocation inhibitory peptide (PKC-ϵ-TIP, 100 nM) as well as a non-selective PKC inhibitor, calphostin C (200 μM). Second, the effects of PKC isoform-selective inhibitors on infarct size limitation by DADLE were examined. Myocardial infarction was induced in vitro by 35-min coronary occlusion/2-hr reperfusion, and infarct size was expressed as % of risk area (%IS/RA). DADLE (300 nM) significantly reduced %IS/RA from 36.3±1.9% (n=7) in controls to 12.9±2.3% (n=6). A PKC-δ-selective inhibitor, rottlerin (0.1 μM), inhibited DADLE-induced infarct size limitation by 60% (%IS/RA=27.4±4.6%, n=8). PKC-ϵ-TIP also attenuated DADLE-induced protection by 40% (%IS/RA =21.1±3.7%, n=8). Rottlerin alone or TIP alone did not modify infarct size.
Conclusion In PC with δ-opioid receptor activation, PKC-ϵ-mediated suppression of GJ and a PKC-δ-mediated GJ-independent mechanism contribute to approximately 40% and 60%, respectively, of the myocardial salvage.