Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning

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 mumol/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 dominantnegative 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.