Mechanisms Underlying Leptin- induced Cardioprotection

Introduction Activation of the Reperfusion Injury SalvageKinase (RISK) pathway, which incorporates phosphatidyli-nositol-3-OH kinase (PI3K)-Akt/protein kinase B (PKB) andp44/42 mitogen-activated protein kinase (MAPK), underliesprotection against ischemia–reperfusion (I/R) injury. Thetemporal nature of the activation of these RISK pathwaycomponents during reperfusion is, however, uncertain. Weexamined Akt and p44/42 phosphorylation in heartssubjected to ischemia and varying periods of reperfusionin the absence or presence of the putative cardioprotectant,apelin-13. Akt activity was also measured.Materials and methods Langendorff perfused C57Bl/6Jmouse hearts were subjected to 35 min global ischemiafollowed by 0, 2.5, 5 or 10 min reperfusion with or without1μMapelin-13. Basal and apelin-induced phosphorylation ofAkt (at both the threonine 308 and serine 473 phosphory-lation sites) and p44/42 during the reperfusion phase wasdetermined by Western blotting and Akt activity measuredusing an Enzyme-Linked ImmunoSorbent Assay (ELISA).Results Basal phosphorylation of both Akt and p44/42increased progressively with time of reperfusion. Apelinenhanced Akt and p44/42 phosphorylation at all reperfu-sion time points. Akt activity did not change under basalconditions but was increased by apelin at 5 min (NS) and10 min (p<0.05) reperfusion.Discussion We conclude that under basal conditions Aktand p44/42 phosphorylation increases with time of reperfu-sion but that this is not accompanied by increased kinase(Akt) activity. On application of a cardioprotectant, however,kinase phosphorylation and activity are enhanced suggestingthat it is the combination of these two mechanisms that mayunderly the tissue preserving actions of such agents.