The temporal relationship between p38MAPK and HSP27 activation in ischaemic andpharmacological preconditioning

An ischaemic preconditioning protocol and subsequent sustainedischaemia were characterized by activation and attenuation of p38MAPK phosphorylation, respectively. However, the significance of eventsdownstream of p38 MAPK needs investigation. Therefore the temporal relationshipbetween phosphorylation of p38 MAPK and its downstream substrateHSP27 was studied during either an ischaemic or –adrenergic preconditioningprotocol and during sustained ischaemia.Isolated rat hearts were preconditioned (with or without a p38 MAPKinhibitor, SB203580) with 1 × 5 min or 3 × 5 min global ischaemia or 5 min–adrenergic stimulation (10^–7 M isoproterenol), followed by 25 min sustainedischaemia and 30 min reperfusion. Hearts were freeze–clamped at differenttime intervals and fractionated to determine p38 MAPK and HSP27phosphorylation, via Western blotting.Significant phosphorylation of cytosolic p38 MAPK and membrane (myo–fibrillar) HSP27 occurred at the end of the first preconditioning episode.However, p38 MAPK phosphorylation disappeared during subsequent preconditioningepisodes, while HSP27 phosphorylation was maintained for theduration of the protocol. Similar changes in p38 MAPK and HSP27 occurredwith 5 min –adrenergic preconditioning. After 25 min ischaemia, significantphosphorylation of cytosolic and membrane HSP27 was observed, while p38MAPK phosphorylation was attenuated in ischaemic and –adrenergic preconditionedcompared to non–preconditioned hearts. SB203580–inducedabolishment of p38 MAPK and HSP27 phosphorylation during the triggeringphase of both preconditioning protocols reversed the changes in these parametersseen after sustained ischaemia.The results suggest that p38 MAPK activation triggers HSP27 phosphorylationduring both the preconditioning protocols and during sustainedischaemia. Protection of preconditioned hearts during sustained ischaemiawas characterized by phosphorylation of both cytosolic and myofibrillarHSP27.