Increased levels of
protein O-linked
N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in
protein O-GlcNAcylation resulted in improved survival and viability following
ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of
ischemia and 16 h of reperfusion, and cell viability,
necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with
glucosamine,
hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of
O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both
necrosis and apoptosis compared with untreated cells following I/R.
Alloxan, an inhibitor of
O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with
hyperglycemia was attenuated by
azaserine, which inhibits
glucose metabolism via the
hexosamine biosynthesis pathway. Reperfusion in the absence of
glucose reduced O-GlcNAc levels on reperfusion compared with normal
glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of
glucosamine and
PUGNAc on cellular viability were associated with reduced
calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca(2+). These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection.