O‐GlcNAcase Exacerbates Post‐Hypoxic Cardiac Myocyte Death

Abstract

We have recently found that hypoxia-reoxygenation reduces global levels of a cytoprotective metabolic signal (O-linked β-N-acetylglucosamine, i.e. O-GlcNAc). Such observations may indicate a net increase in O-GlcNAcase (GCA, removes O-GlcNAc) activity. Because O-GlcNAc exerts protective effects on the myocardium, we hypothesized that gene transfer of GCA further reduces O-GlcNAc levels and sensitizes cardiac myocytes to post-hypoxic injury. Isolated cardiac myocytes (n=4–5/group) were infected with adenovirus overexpressing GCA (AdGCA), or treated with GCA inhibitors, and subjected to hypoxia and reoxygenation. Whole cell lysates were immunoblotted for O-GlcNAc levels, cell injury assessed via post-hypoxic LDH release, and post-hypoxic loss of mitochondrial membrane potential evaluated with tetramethylrhodamine methyl ester (TMRM). Overexpression of GCA significantly reduced (p<0.05) O-GlcNAc levels, exacerbated post-hypoxic LDH release, and favored the loss of mitochondrial membrane potential. GCA inhibition (via PUGNAc, streptozotocin, or alloxan) significantly enhanced (p<0.05) O-GlcNAc levels, reduced post-hypoxic LDH release, and preserved mitochondrial potential. We conclude that hypoxia favors the net loss of the O-GlcNAc post-translational modification reflected by the hypoxia-sensitizing effects of GCA in cardiac myocytes. Funded by NIH (HL0833220) and AHA (0535270N).