Ischaemic preconditioning : an investigation of the patterns of kinase activation and protein expression profiles during reperfusion in the rat heart

[摘要] ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of deathworldwide with 3.8 million men and 3.4 million women dying globally each year.Although existing myocardial reperfusion strategies such as thrombolysis andpercutaneous coronary intervention (PCI), if applied in a timely manner, limitmyocardial infarct size, the mortality and morbidity remains significantly high.Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardialischaemia/reperfusion injury through cardioprotective signaling pathways which isrecruited at the time of myocardial reperfusion, thereby improving clinicaloutcomes in patients with coronary artery disease.Ischaemic preconditioning is a phenomenon whereby short intermittent episodesof coronary occlusion followed by reperfusion protect the myocardium against asubsequent period of sustained ischaemia. This protection is reflected in thelimitation of infarct size and improved functional recovery of the ischaemic heartduring reperfusion. Despite intensive research efforts, the promise of an effectivecardioprotective strategy using the endogenous protective mechanisms of theheart which underlies IPC, has not yet been materialized. Although progress hasbeen made in terms of signaling mechanisms in the preconditioned heart, theidentification of the myocardial reperfusion phase as the critical 'window forcardioprotection, requires the elucidation of the signal transduction pathwaysduring the reperfusion phase after IPC.In view of the above, the aims of the present study were to investigate:i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPCduring early and late reperfusionii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70(HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB inthe context of IPC during early reperfusioniii. the involvement of autophagy and apoptosis during early and latereperfusion after IPCiv. the correlation of the protein kinases with the hemodynamic parameters ofthe heartv. the mechanism of IPC by means of two-dimensional (2D) proteomicsMethods: The isolated perfused working rat heart model was used withfunctional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 minglobal ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion whenhearts were snap-frozen for western blotting analysis. Alternatively, hearts werereperfused for 30 min to record hemodynamic parameters and measurefunctional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC heartswere reperfused for 30 min to serve as control for the 30 min reperfused IPCgroup. Activation of the protein kinases was determined by western blottinganalysis.For the proteomic study mitochondrial and cytosolic proteins were isolated fromheart tissue and separated in the first dimension by isoelectric focusing, followedby separation in the second dimension by two dimensional gel electrophoresis.The PD Quest software programme was used to identify significantly expressedprotein spots. Protein spots of interest were excised and subjected to in-geldigestion and the resulting peptides were analysed by mass spectrometry.Proteins were identified by Mascot and the Swiss Prot database.Results: Western blotting analysis demonstrated that the RISK pathway and p38MAPK are activated very early in reperfusion, but the activation is not sustainedduring the reperfusion period. Autophagy is also upregulated during this earlyreperfusion phase; it is attenuated in the middle reperfusion phase and increasefor a second peak of upregulation in the late reperfusion phase. In addition, weidentified CAMKII as a novel marker of functional recovery in IPC afterreperfusion.The proteomic analysis identified twenty differentially expressed mitochondrialand thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins weredirectly related to cardiac metabolism.Conclusion: Activation of the majority of the protein kinases investigated in thepresent study is associated with the hemodynamic parameters of the heartinstead of functional recovery. Results indicated that the variable signalingpatterns could be attributed to differences in heart rate and the effect thereof(ejection fraction, minimum and maximum rate of contraction), as a result ofsympathetic stimulation due to psychological stress in the animals beforeslaughtering. Proteomics results demonstrated that IPC hearts which failed afterischaemia /reperfusion are metabolically compromised and 'worse off comparedto non-IPC hearts.

[发布机构] Stellenbosch University