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

Objectives: Amchidonic acid is a second messenger which activates protein k inase C (PKC) and is released from the heart during ischaemic preconditioni ng. The purpose of this study was to examine the effect of arachidonic acid on activation of PRC in cardiac myocytes and the cellular consequences. Me thods: Neonatal rat cardiac myocytes were isolated and maintained in cultur e. Arachidonic acid-induced activation of PKC was examined by cell fraction ation and western blot analysis. Contraction frequency was measured by visu al inspection under a microscope. Ischaemia was simulated by subjecting cel ls 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 di rest cell viability assay. Results: Arachidonic acid resulted in translocat ion of delta and epsilon PKC but not alpha, beta II, eta or zeta PKC isozym es, indicating activation of only delta and epsilon PKC. Amchidonic acid in duced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor o f epsilon PKC. Pretreatment with arachidonic acid partially protected cardi ac myocytes against ischaemia. Down-regulation of PKC with 24 h 4 beta -pho rbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of epsilon PKC translocation all decreased the pro tective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia. Conclusio ns: These results demonstrate that arachidonic acid selectively activates d elta and epsilon PKC in neonatal rat cardiac myocytes, leading to protectio n from ischaemia. We suggest this is a potential mechanism of PKC activatio n during PC. In addition, our results suggest that different classes of fre e fatty acid directly exert cardioprotection from ischaemic injury in cardi ac myocytes. (C) 2001 Elsevier Science B.V. All rights reserved.