Abstract 1368: Postconditioning Attenuates Cardiac Muscle Cell Apoptosis viaTranslocation of Survival Kinases and Opening of KATP Channels in Mitochondria
Activation of survival kinases by postconditioning (Postcon) has been linked to a reduction in infarct size. This study tested the hypothesis that Postcon prevents apoptosis by activating survival kinases and opening KATP channels in mitochondria. Cultured H9c2 cardiac muscle cells were exposed to 8 h hypoxia followed by 3 h of reoxygenation (Re) and mitochondria were isolated at the end of experiment. Relative to hypoxia/Re, Postcon with three cycles of 5 min Re and 5 min hypoxia applied at the onset of Re up-regulated expression of phospho-AKT (116±8* vs. 92±6) and ERK (140±12* vs. 55±2) in isolated mitochondria. Consistent with an attenuation in release of cytochrome c (78±5* vs. 172±6) from mitochondria by Postcon, loss of mitochondrial membrane potential ()Ψm) and opening of mitochondrial permeability transition pore (mPTP) detected by dual fluorescence TMRE and calcein-AM staining was markedly inhibited. Postcon down-regulated Bax (95±3* vs. 132±11) and up-regulated Bcl-2 (108±58* vs. 81±14) in mitochondria. These data were consistent with a reduction in the number of apoptotic cells (20±3%* vs. 43±3% total nuclei)) and DNA fragmentation. To examine the role of KATP channels in activation of survival kinases, H9c2 cells were treated with a mitochondrial KATP channel blocker, 5-hydroxydecanoate (5-HD) at 5 min before Postcon. 5-HD significantly blocked activation of phospho-AKT (66±2†) and ERK (38±1†) as well as reduction in cytochrome c release (78±5†) by Postcon, consistent with an increase in loss of )Ψm and mPTP opening by TMRE and Calcein-AM staining and induction of apoptotic cells (44±3†). However, protection by Postcon was not altered by a sarcolemmal-KATP channel blocker, HMR1098, suggesting that activation of survival kinases is a downstream event after opening of mitochondrial KATP channels. These data suggest that 1) translocation of survival kinases from the cytosol to mitochondria by Postcon is mediated by opening mitochondrial KATP channels; 2) activation of survival kinases by Postcon is responsible for an inhibition of apoptosis through modulating mitochondria-mediated signaling pathways. Values (Arb. U) are mean ± SEM, n=5 for each group. *P<0.05 Postcon vs. hypoxia/Re; †P<0.05 5-HD vs. Postcon.