Protein kinase c-e is responsible for the protection of preconditioning inrabbit cardiomyocytes

The role of protein kinase C (PKC) in the protection of ischemic preconditi oning (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 inhibito r peptides are cross-linked to the membrane-translocating antennapedia home odomain peptide that allows their entry into cells, The effect of inhibitor s of PKC-beta, -delta, -epsilon and -eta were evaluated. Rabbit adult ventr icular myocytes were obtained by enzymatic dissociation. Ischemia was simul ated by centrifuging the myocytes into an oxygen-free pellet for 180 min. P C was induced by 10 min of pelleting followed by resuspension in oxygenated medium for 15 min. During simulated ischemia cells undergo a predictable i ncrease in osmotic fragility as judged by determination of the number of st ained cells following their incubation in hypotonic (85 mOsm) trypan blue. The percentage of cells experiencing membrane rupture, and thus cell staini ng, was considered to be an index of ischemic injury. PC significantly dela yed the progression of osmotic fragility during simulated ischemia (P<0.01) . The protection of PG was abolished by the peptide inhibitor of PKC-epsilo n but not by the peptide inhibitors selective for PKC-beta, PKC-delta, or P KC-eta; each was applied at 100 nM. Protection could also be induced by the PKC activator olcoylacetyl glycerol, and that protection was aborted by th e inhibitor selective for PKC-epsilon, but not by the inhibitor for PKC-del ta, 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. (C) 1999 Academic Press.