Protein kinase C-epsilon is responsible for the protection of preconditioning in rabbit cardiomyocytes

Abstract

The role of protein kinase C (PKC) in the protection of ischemic preconditioning (PC) is still controversial, partly because of the multiple isozymes of PKC and the inability to directly measure PKC activity in vivo. In this study we have used novel peptide inhibitors which correspond to part of the amino acid sequence from the isozyme-specific RACK-binding site on the PKC molecule. The peptides prevent binding of a specific activated PKC isozyme to its RACK, thus halting isozyme translocation and function. The inhibitor peptides are cross-linked to the membrane-translocating antennapedia homeodomain peptide that allows their entry into cells. The effect of inhibitors of PKC-beta, -delta, -epsilon and -eta were evaluated. Rabbit adult ventricular myocytes were obtained by enzymatic dissociation. Ischemia was simulated by centrifuging the myocytes into an oxygen-free pellet for 180 min. PC was induced by 10 min of pelleting followed by resuspension in oxygenated medium for 15 min. During simulated ischemia cells undergo a predictable increase in osmotic fragility as judged by determination of the number of stained cells following their incubation in hypotonic (85 mOsm) trypan blue. The percentage of cells experiencing membrane rupture, and thus cell staining, was considered to be an index of ischemic injury. PC significantly delayed the progression of osmotic fragility during simulated ischemia (P<0.01). The protection of PC was abolished by the peptide inhibitor of PKC-epsilon but not by the peptide inhibitors selective for PKC-beta, PKC-delta, or PKC-eta; each was applied at 100 n N. Protection could also be induced by the PKC activator oleoylacetyl glycerol, and that protection was aborted by the inhibitor selective for PKC-epsilon, but not by the inhibitor for PKC-delta. None of the above peptide treatments affected the osmotic fragility in non-PC cells during simulated ischemia. Our studies further support PKC as a critical part of the signal transduction pathway in PC and indicate that PKC-epsilon alone is responsible for the early phase of PC's protection in rabbit cardiomyocytes.