Sustained in vivo cardiac protection by a rationally designed peptide that causes ɛ protein kinase C translocation

Abstract

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). Protein kinase C (PKC) has been suggested to mediate preconditioning. Here, we describe an PKC-selective agonist octapeptide, receptor for activated C-kinase (RACK), derived from an PKC sequence homologous to its anchoring protein, RACK. Introduction of RACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased PKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that PKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this PKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.preconditioning ͉ hypoxia ͉ transgenic pseudoreceptors for activated C-kinase ͉ ischemia

εPKC Is Required for the Induction of Tolerance by Ischemic and NMDA-Mediated Preconditioning in the Organotypic Hippocampal Slice

“…Once the peptide crosses the cell membrane, the disulfide bond breaks, the peptide cargo is trapped in the cell, and the peptide carrier can be washed out. These studies already demonstrated that the peptides have highly selective effects; they inhibit or induce translocation of their corresponding isozymes but not that of others (Gray et al, 1997;Dorn et al, 1999). In addition, at concentrations up to ϳ10 M, the peptides or the carrier have no toxic effects on the cell.…”

“…Activation of PKC is associated with translocation from the soluble to the particulate fraction of cells (Kraft and Anderson, 1983;Dorn et al, 1999). Therefore, to confirm that IPC translocates ⑀PKC isozyme in hippocampal organotypic slices, the effect of IPC on subcellular localization of ⑀PKC isozyme was determined by cell fractionation and Western blot analysis.…”

“…The ⑀V1-2 [⑀PKC inhibitor, amino acids 14 -21 (EAVSLKPT)] (Gray et al, 1997) and ⑀RACK [⑀PKC activator, amino acids 85-92 (HDAPI-GYD)] (Dorn et al, 1999) peptides were synthesized at Stanford's Protein and Nucleic Acid facility and conjugated to Tat [carrier peptide, amino acids 47-57 (YGRKKRRQRRR)] (Schwarze et al, 1999) via a cysteine-cysteine bond at their N termini, as described previously (Chen et al, 2001).…”

Adaptor proteins in protein kinase C-mediated signal transduction

“…Because the a nity of pseudo-adaptor peptide is lower than that of the true binding-site on the adaptor, when the pseudo-adaptor peptide leaves the enzyme, the enzyme binds the adaptor (step 3). Using this approach, we have identi®ed isozymeselective activators, or pseudoRACKs, for bPKC and for ePKC (Dorn et al, 1999). For more details see the text and (Souroujon and Mochly- Peptides that induce binding of PKC isozyme to its RACK in the absence of second messenger generation were also identi®ed.…”

“…For more details see the text and (Souroujon and Mochly- Peptides that induce binding of PKC isozyme to its RACK in the absence of second messenger generation were also identi®ed. These activator peptides, collectively termed pseudo-RACK peptides, are derived from a sequence in PKC that shares homology with its corresponding RACK (Dorn et al, 1999;Souroujon and Mochly-Rosen, 1998). However, there is a charge di erence between the pseudo-RACK sequence and the corresponding sequence of the PKC-binding site on RACK.…”