Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through ε protein kinase C

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Abstract
Objectives: Arachidonic acid is a second messenger which activates protein kinase C (PKC) and is released from the heart during ischaemic preconditioning. The purpose of this study was to examine the effect of arachidonic acid on activation of PKC in cardiac myocytes and the cellular consequences. Methods: Neonatal rat cardiac myocytes were isolated and maintained in culture. Arachidonic acid-induced activation of PKC was examined by cell fractionation and western blot analysis. Contraction frequency was measured by visual inspection under a microscope. Ischaemia was simulated by subjecting cells to an atmosphere of lower than 0.5% oxygen in the absence of glucose and cell damage determined by release of cytosolic lactate dehydrogenase or direct cell viability assay. Results: Arachidonic acid resulted in translocation of δ and ϵPKC but not α, βII, η or ζPKC isozymes, indicating activation of only δ and ϵPKC. Arachidonic acid induced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor of ϵPKC. Pretreatment with arachidonic acid partially protected cardiac myocytes against ischaemia. Down-regulation of PKC with 24 h 4β-phorbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of ϵPKC translocation all decreased the protective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia. Conclusions: These results demonstrate that arachidonic acid selectively activates δ and ϵPKC in neonatal rat cardiac myocytes, leading to protection from ischaemia. We suggest this is a potential mechanism of PKC activation during PC. In addition, our results suggest that different classes of free fatty acid directly exert cardioprotection from ischaemic injury in cardiac myocytes.

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