Activation of protein kinase C (PKC) has been associated with the cardioprotective effect of ischaemic preconditioning (IP). We recently showed that neonatal rat cardiac myocytes overexpressing constitutively active PKC-δ were resistant to ischaemia. The signalling molecules situated downstream of PKC in this protective signalling cascade remain to be characterised. One group of key regulatory proteins that control cellular responses to stress signals are the mitogen-activated protein kinases (MAPKs). To investigate the role of this kinase family in myocardial protection, we used genetic manipulation to examine the interplay between PKC and MAPKs in a surrogate model of IP. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning with 90 min simulated ischaemia and 30 min re-oxygenation. To specifically investigate the contribution of PKC, MAPK phosphorylation was assessed before and during IP in myocytes overexpressing constitutively active PKC-δ. Although there was no significant increase in basal level MAPK activation, transfection with active PKC-δ completely abolished the phosphorylation observed during IP. In agreement, PKC-δ also caused a marked reduction in p38 activation during ischaemia alone. Moreover preconditioning, which gives rise to a similar level of protection, caused a strikingly similar inhibition of ischaemia-induced p38 activation (54.5±16.3%, n=3). To demonstrate that p38 inhibition was the cause of protection and not simply a consequence of attenuation of ischaemia, we used pharmacological inhibition of p38 during ischaemia. Addition of SB203580, during ischaemia alone, was sufficient to protect myocytes against cell death (CK = 38.0±3.1%, LDH = 77.3±3.9%, MTT = 127.1±4.8% vs control, n=20, p<0.05). Therefore in summary, these data show that p38 activation during ischaemia is detrimental to myocyte survival. Moreover, by using adenoviral mediated expression of individual p38 isotypes, we showed that p38a is activated during ischaemia (250.8±60.3% vs control, n=3), whereas p38β is unchanged (71.4±32.0% vs control, n=3), suggesting that the deleterious effect may be as a result of differential p38α activation. In conclusion, active PKC-δ and IP inhibit p38 activation during SI which, at least in part, accounts for protection. This negative regulation of p38 by PKC during ischaemia may represent a common mechanism for IP-induced cardioprotection.
|Issue number||SUPPL. 1|
|Publication status||Published - 1 May 1999|