Role of Akt Signaling in Mitochondrial Survival Pathway Triggered by Hypoxic Preconditioning

Abstract

Background— The signaling pathways that control ischemia/reperfusion-induced cardiomyocyte apoptosis in heart have not been fully defined. In this study, we investigated whether Akt signaling has a role in the antiapoptotic pathways of preconditioning against hypoxia/reoxygenation (H/R). Methods and Results— Primary cultures of adult rat ventricular myocytes (ARVMs) were subjected to preconditioning (PC) by exposing the cells to 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Non-PC and PC myocytes were subjected to 90 minutes of hypoxia followed by 120 minutes of reoxygenation. Hypoxic-PC protected the myocytes from subsequent H/R injury, as evidenced by decreased apoptosis and LDH release and increased cell viability. H/R-induced cytochrome c release and activation of caspase-3 and -9 were blocked by PC. This protective effect was inhibited by treating the cells with LY294002 (50 μmol/L), a PI3 kinase inhibitor, for 10 minutes before and during PC. PC also induced phosphorylation of Akt and BAD. Protein levels of Bcl-2 in mitochondria were maintained in PC. ARVMs were infected with either a control adenovirus (Adeno lac-Z), an adenovirus expressing dominant-negative Akt, or an adenovirus expressing constitutively active Akt. Ectopic overexpression of constitutively active Akt protected ARVMs from apoptosis induced by hypoxia/reoxygenation compared with Adeno lac-Z. In contrast, dominant negative Akt overexpression abolished the antiapoptotic effect of PC. Conclusions— Our data demonstrated that in adult cardiomyocytes, the antiapoptotic effect of PC against H/R requires Akt signaling leading to phosphorylation of BAD, inhibition of cytochrome c release, and prevention of caspase activation.