Adenoviral gene transfer of activated phosphatidylinositol 3′-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro

Background-The intracellular signaling pathways that control cardiomyocyte apoptosis have not been fully defined. Because insulin-like growth factor-1 (IGF-1) prevents cardiomyocyte apoptosis, we examined the role of its downstream signaling molecules in an in vitro model of hypoxia-induced cardiomyocyte apoptosis. Methods and Results-Treatment of rat neonatal cardiomyocytes with IGF-1 increased activity of both phosphatidylinositol 3' (PI 3)-kinase and its downstream target, Akt (also known as protein kinase B or PKB). Cardiomyocytes were subjected to hypoxia for 24 hours, and apoptosis was assessed by DNA laddering, TUNEL staining, and ELISA for histone-associated DNA fragments, IGF-1 treatment (100 nmol/L) reduced cardiomyocyte apoptosis, and this effect was inhibited by simultaneous treatment with a PI 3-kinase inhibitor. Cardiomyocytes were infected with either a control adenovirus (Ad.EGFP) or adenoviruses carrying constitutively active forms of PI 3-kinase (Ad.BD110) or Akt (Ad.myr-Akt-HA), Ad.BD110 significantly inhibited apoptosis of hypoxic cardiomyocytes compared with Ad.EGFP (61.0 +/- 4.6% less DNA fragmentation than in Ad.EGFP-infected cells, P < 0.0001). Ad.myr-Akt-HA even more dramatically inhibited apoptosis of hypoxic cardiomyocytes (90.9 +/- 1.4% less DNA fragmentation than in controls, P < 0.0001), Conclusions-IGF-1 activates PI 3-kinase and Akt in cardiomyocytes. Activated PI 3-kinase and Akt are each sufficient to protect hypoxic cardiomyocytes against apoptosis in vitro. Adenoviral gene transfer provides a useful tool for investigating the role of these signaling pathways in cardiomyocyte apoptosis.