The signaling pathways involved in the cardioprotection offered by insulin to the global low flow ischaemic/reperfused myocardium

[摘要] ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection underischaemic stress. In the past it was believed that the protective effects of insulin, suchas the (a) recruitment of glucose transporters to enhance glucose entry into the cell, (b)stimulation of glycolysis, (c) enhancement of glycogen synthesis, (d) improved proteinsynthesis, and (e) positive inotropic and chronotropic properties, were metabolic oforigin, but lately the emphasis has shifted towards the diverse signal transductionpathways elicited by insulin. Although these beneficial effects of insulin onischaemia/reperfusion induced injury have been studied for many years, the exactprotective mechanism is still not resolved. Aim: To investigate the influence of insulinon the signaling pathways as a possible protective mechanism againstischaemia/reperfusion and therefore to investigate the possible roles and crosssignaling of cyclic adenosine monophosphate (cAMP), protein kinase B (PKB) and p38mitogen activated protein kinase (p38 MAPK) in the cardioprotection offered by insulinto the reperfused, ischaemic myocardium. Materials and methods: Isolated rat heartswere perfused retrogradely in accordance with the Langendorff technique (95%02, 5%C02). After 30 min of stabilization, hearts were subjected to 30 min global low flowischaemia (0,2 ml/min), followed by 30 min of reperfusion. Hearts perfused withstandard Krebs Henseleit solution containing 5 mM glucose were compared to heartsperfused with a perfusion solution containing 5 mM glucose and 0,3 IlIU/ml insulin.Wortmannin was added during either ischaemia or reperfusion. Left ventriculardeveloped pressure (LVDP), rate pressure product (RPP), tissue cAMP and PKB andp38 MAPK activation were measured. Results: Insulin treated hearts showedimproved functional recovery (P<0.05) during reperfusion after ischaemia vs. non-insulin treated hearts (85.5±4.6% vs. 44.8±4.9%). However, the addition ofwortmannin (a Pl3-kinase inhibitor) to the perfusion solution during either ischaemia orreperfusion abolished the improved recovery. At the end of ischaemia, cAMP levels ofthe insulin treated hearts were elevated significantly, while the cAMP content in thenon-insulin treated hearts returned to control levels. Addition of wortmannin duringischaemia abolished this rise in cAMP. Wortmannin added during reperfusion only didnot alter the levels of cAMP at the end of reperfusion. Activation of p38 MAPK wastransient during ischaemia for both insulin and non-insulin treated hearts. Addition ofwortmannin during ischaemia did not alter p38 MAPK levels at the end of ischaemia.P38 MAPK was activated significantly (P<0.001) in the non-insulin treated hearts vs.insulin treated hearts during reperfusion. Wortmannin, added at the onset ofreperfusion, could partially abolish the effects of insulin to suppress p38 MAPKactivation after 30 min of reperfusion. Activation of PKB in insulin treated hearts wassignificantly higher than non-insulin treated hearts during stabilization and earlyischaemia. This activity was depressed by 30 min of ischaemia in both presence andabsence of insulin. Wortmannin, when added before induction of ischaemia did notfurther lower this. The presence of insulin resulted in occurrence of strong PKBactivation during reperfusion, peaking at 15 minutes and diminishing at 30 minutes.Wortmannin, added at the onset of reperfusion, abolished PKB activity measured at theend of reperfusion. Conclusion: Insulin exerted a positive inotropic effect and delayedthe onset to ischaemic contracture. Inhibition of Pl3-kinase by wortmannin abolishedthe protective effects of insulin, arguing for an insulin stimulated PKB involvement incardiac protection. Insulin also increased cAMP production and attenuated activationof p38 MAPK, both associated with improved recovery. This evidence suggestedpossible cross signaling between different signaling pathways.

[发布机构] Stellenbosch University