Abstract 360: Autophagy Is Stimulated by Energy Starvation through Activation of AMPK and Promotes Survival of Cardiac Myocytes
Autophagy is an intracellular bulk degradation process by which damaged proteins and organelles can be removed. Autophagy is stimulated by acute and chronic ischemia in the heart. Although autophagy promotes survival of organisms under starvation through reutilization of amino acids, it also promotes programmed cell death. Thus, the goals of this study were to elucidate the causative role of autophagy in survival of cardiac myocytes under stresses and the signaling mechanism mediating autophagy. Glucose deprivation (GD) strongly induced autophagy in cultured cardiac myocytes as evidenced by increases in LC3-II/LC-3I (control: 1.0±0.1, GD: 6.9±0.8, p<0.05), punctate GFP-LC3 dots, and detection of autophagosomes by electron microscopy. These results were accompanied by a significant reduction in intracellular ATP (control: 1.00±0.06, GD: 0.58±0.09, p<0.05). Survival of cardiac myocytes under GD was decreased by inhibition of autophagy with 3-methyladenine (3-MA), and increased by stimulation of autophagy with Atg5, an autophagic gene (cell survival rate, control: 77.8±5.6%; 3-MA: 59.6±7.1%, p<0.05; Atg5: 86.2±6.0%, p<0.05), suggesting that autophagy is protective against GD. Induction of autophagy by GD coincided with activation of AMPK as evidenced by increased Thr172 phosphorylation (2.5 fold, p<0.01) and inactivation of mTOR as evidenced by decreases in Thr389 phosphorylation of p70S6K (0.4 fold, p<0.05). Inhibition of AMPK by adenine 9-β-D-arabinofuranoside or dominant negative (DN)-AMPK significantly reduced GD-induced autophagy, suggesting that AMPK plays an essential role in mediating autophagy caused by GD. Furthermore, inhibition of mTOR by rapamycin mimicked the effects of GD upon autophagy. Autophagy was also induced by 20 min ischemia (IS) in the mouse heart in vivo (LC3-II/LC3-I control: 1.00±0.09, IS: 3.39±0.78, p<0.05), and was accompanied by activation of AMPK (2.3 fold). Induction of autophagy by myocardial ischemia was inhibited in transgenic mice with cardiac-specific overexpression of DN-AMPKα2. These results suggest that autophagy is cell protective in the heart against energy starved conditions, such as GD and ischemia, and that AMPK is a critical upstream mediator of autophagy in cardiac myocytes.