Detection of Cardiac O-GlcNAcylation via Subcellular Fractionation and Dual Antibody Analysis in Pressure Overload Cardiac Hypertrophy.

Protein O-GlcNAcylation is a dynamic post-translational modification with emerging roles in cardiac pathophysiology. The availability of differenct pan-specific antibodies to assess global O-GlcNAc levels, and variability in Western blot results has hindered cross-study reproducibility and interpretation. In this study, we applied optimized immunoblotting protocols using both CTD110.6 and RL2 O-GlcNAc antibodies, alongside subcellular fractionation, to investigate temporal and sex-specific changes in cardiac O-GlcNAcylation during pressure overload hypertrophy (POH) from transverse aortic constriction (TAC) during early (1-week POH, 1wTAC) and chonic (6-weeks POH, 6wTAC) POH in mice. Global O-GlcNAc levels were elevated in early POH and returned to baseline in chronic POH, consistent across both antibodies and sexes. Subcellular fractionation revealed persistent O-GlcNAc elevations in cytoplasmic and membrane fractions in chronic POH for both sexes, which were not detected in unfractionated samples. Female mice exhibited significantly higher O-GlcNAc levels than males during POH, particularly at early POH, highlighting sex-specific regulation. OGT and OGA protein levels also varied by compartment and sex, suggesting differential enzymatic control. In conclusion, our findings underscore the importance of methodological rigor in O-GlcNAc detection and demonstrate that fractionation enhances sensitivity to subtle changes in cardiac O-GlcNAcylation. Our principal new findings are protein O-GlcNAcylation dysregulation continues from early POH (1wTAC) into chronic POH (6wTAC groups) along with showing differences in O-GlcNAc levels between males and females during POH. These results provide new insights into the temporal and sex-dependent dynamics of O-GlcNAc signaling in POH and support its potential as a therapeutic target in cardiovascular disease.