A Selective ε-Protein Kinase C Antagonist Inhibits Protection of Cardiac Myocytes from Hypoxia-induced Cell Death

Abstract

Protein kinase C activation is thought to protect cardiac tissue from subsequent ischemic injury by a process termed preconditioning. The protein kinase C isozyme that mediates preconditioning has not yet been identified. Using a cell culture model of hypoxic preconditioning, we found that cardiac myocyte viability after 9 h of hypoxia was increased by more than 50% over control. Preconditioning activated protein kinase C isozymes as evidenced by translocation from one cell compartment to another as follows: there was a 2.1-fold increase in ⑀-protein kinase C activation, a 2.8-fold increase in ␦-protein kinase C activation, and no increase in ␤ I -protein kinase C activation. 4␤-Phorbol 12-myristate 13-acetate mimicked hypoxic preconditioning, increasing myocyte survival after prolonged hypoxia by 34% compared with control. We previously identified an ⑀-protein kinase C-selective antagonist, ⑀V1-2 peptide, that inhibits ⑀-protein kinase C translocation and function in cardiac myocytes (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962-24966). ⑀V1-2 peptide abolished hypoxic preconditioning and phorbol ester-mediated cardiac protection. Therefore, preconditioning can be induced in this culture model, and activation of ⑀-protein kinase C is critical for cardiac myocyte protection.

Expression of constitutively stable hybrid hypoxia-inducible factor-1α protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury

“…As expected (2,13,28,35,38), subjecting cultured cardiomyocytes to an episode of sublethal hypoxia decreased cell death induced by subsequent simulated lethal ischemiareperfusion, as measured by the MTS and LDH assays (Fig. 1).…”

“…We also measured the cellular protein levels of HIF-1␣ and the mRNA levels of several HIF-1 target genes in a cultured neonatal rat cardiomyocyte model of preconditioning (2,13,23,28,35,38). Our results suggest that expression of a constitutively stable hybrid HIF-1␣ protects cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes.…”

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.…”

“…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).…”

Protein kinase C as a stress sensor

“…Cardiac preconditioning, which has been demonstrated in many species, can be induced through a variety of methods including hypoxia/ischemia [40], anesthetics [41], glucose [42], phorbol esters [43], cannabinoids [44], and heat-stress [45]. It is assumed that these act in a unified manner and activation of PKCε is, most likely, key to this control [46].…”