Abstract Body: Diabetes Mellitus (DM) is a metabolic disease characterized by elevated plasma glucose levels due to a deficit in the secretion or action of insulin. DM is also associated with heart failure (HF). The Na+/H+ exchanger 1 (NHE1) is implicated in the regulation of intracellular pH and, its activity is increased during HF. The cytoplasmatic domain of NHE1 regulates its activity and could be targeted for O-GlcNAcylation. This modification consists in the transfer of a UDP-GlcNAC, a product of the Hexosamine Biosynthetic Pathway (HBP), to a Ser or Thr by OGT and can be removed by OGA. In DM the flux through HBP is increased, leading to elevated O-GlcNAcylation. The cytosolic tail of the NHE1 is subject to different post-translational modifications and could be a target for O-GlcNacylation. Here, we explored the effect of high glucose (HG) on the NHE1 activity in a murine and in vitro model. H9C2 cells were cultured in DMEM media supplemented with low glucose (LG) (5 mM) and HG (30 mM) and the NHE1 activity was measured by the ammonium prepulse technique. We found that the NHE1 activity was increased in the group HG compared with the cells cultured in LG media (HG:3.7±0.47vsLG:1.142±0.15: p<0.001). The same result was obtained when isolated cardiomyocytes of C57bl/6 (WT) mice were perfused with O-GlcNAc (5 mM) in HEPES buffer and HEPES buffer alone for 5 minutes (GlcNAc: 0.07±0.02 vs HEPES: 0.02±0.01; p<0.05). Also, we measured the O-GlcNAc levels in H9C2 and heart tissue homogenates by Western Blot, and we observed that O-GlcNAc is increased in cells cultured in HG compared with those cultured in LG (HG: 189.4±20.38 vs LG: 100±12.49; p<0.01) and the same tendency was observed in diabetic mice (Ob/Ob) compared with WT mice (Ob/Ob: 10.60±1.19 vs WT: 7.17±1.02). Finally, to study whether the increase in NHE1 activity could be related to the increased O-GlnNacylation, we immunoprecipitated NHE-1 and measured O-GlnAcylation by Western Blot. NHE-1 immunoprecipitated from Ob/Ob mice had increased O-GlcNAcylation compared with WT mice (Ob/Ob: 1.52±0.022vsWT: 1.00±0.17). These results suggest that O-GlcNAcylation of NHE1 increases its activity, contributing to HF progression in diabetes.