The Role Of AMPK In Cardiomyocytes Anoxia/Reoxygenation Injury Mediated BY C1-

Objective: AMPKα2plays a very important role in regulating myocardialenergy metabolism. We established the anoxia/reoxygenation injury model andCl--free A/R injury model that Cl-in perfusion solution was replaced with equimolargluconate-during the A/R protocol of H9c2myocytes to explore the role of AMPKα2low expression in acute A/R injury mediated by Cl-. And in order to further study themechanism of A/R injury.Methods: The pSuper.Retro plasmid considered as vector was used to constructthe AMPKα2shRNA recombinant plasmid by RNA interference technique. Weestablished the normal expression and low expression levels of AMPKα2protein inH9c2cells by gene transfection technique. The expression of AMPKα2was assayedby Western Blot analyses. The H9c2cells were randomly divided into five groups:(1)Control group;(2) A/R group;(3) removal of extracellular Cl-+A/R group (Cl--freeA/R group);(4) pSuper+Cl--free A/R group;(5) pS-AMPKα2+Cl--free A/R group.Every group repeats six times. Then Cell viability was assayed by MTT, and SOD,LDH, GSH-Px activity in H9c2were detected. The level of intracellular ROS, thepercentage of apoptosis and the mitochondria membrane potential were measured byflow cytometry.Results:1. We successed to construct AMPKα2shRNA recombinant plasmidwhich was identified by DNA sequencing;2. Western Blot confirmed that thedownregulation of AMPKα2expression by its gene-silencing decreased theexpression levels of AMPKα2protein absolutely;3. A/R injury significantlydecreased H9c2cell viability, activity of SOD and GSH-Px. Cl--free A/R group hasbeen shown to produce a protective effect against A/R injury by increasingantioxidant enzyme. However in RNAi group, the protective effect against A/R injurymediated by Cl-apparently disappeared. Its cell viability, activities of SOD andGSH-Px, the mitochondria membrane potential were decreased while LDH activityand the level of ROS and apoptosis were remarkably increased in H9c2cells compared with Cl--free A/R group (p<0.01). There was no significant differencebetween Cl--free A/R group and pSuper+Cl--free A/R group.Conclusion: AMPKα2participated in the protective effect against A/R injuryproduced by administration with Cl--substitution. The protective effect was abolisheddue to silencing of AMPKα2gene in H9c2cells.