Abstract 607: Degradation of Metabolic Post-translational Modification Sensitizes Cardiac Myocytes to Hypoxia
We have recently discovered an endogenous mechanism of cytoprotection in the heart that involves the post-translational modification of proteins with a metabolic signal. Although this stress-responsive modification (beta-O linkage of N-acetylglucosamine, i.e. O-GlcNAc) exerts profound salubrious effects on the myocardium in vivo, the details regarding the pathobiology of this modification in the cardiac myocyte remain unexplored. We hypothesized that O-GlcNAc levels decrease following hypoxia and that manipulation of these levels would affect cardiac myocyte survival. We subjected isolated cardiac myocyte cultures (n >/= 4/ all groups) to hypoxia (3 hours) and reoxygenation (up to 1 hour). We measured O-GlcNAc levels and determined that they begin decreasing within 15 minutes, and continue to decrease through one hour, of reoxygenation in cardiac myocytes. Such a significant decrement (p < 0.05 vs. baseline) in O-GlcNAc levels was associated with a reduction in mitochondrial membrane potential (according to TMRM via fluorescence microscopy) and release of LDH (via spectrophotometric assay) into the media. To further investigate the regulation of O-GlcNAc levels following hypoxia, we targeted the enzymes controlling the presence of O-GlcNAc on proteins. Blocking the addition of O-GlcNAc (with TT04) profoundly sensitized myocytes to cell death while blocking the removal of O-GlcNAc (with PUGNAc) preserved myocyte survival (according to TMRM microscopy and LDH assay). We conclude that augmentation of O-GlcNAc levels attenuates, while diminution of O-GlcNAc levels exacerbates post-hypoxic injury in cardiac myocytes.