Abstract Body: Diabetic cardiomyopathy is characterized by an abnormal force–frequency response, yet the signaling mechanisms linking metabolic disease to defective cardiac adaptation remain poorly understood. O-GlcNAcylation and phosphorylation both modify serine and threonine residues and have been proposed to interact through “yin–yang” regulation, but the extent of this relationship remains unclear.
We performed integrated phosphoproteomic and O-GlcNAc proteomic profiling of cardiac tissue from wild-type and ob/ob mice. Isolated hearts were perfused using the Langendorff system and challenged with increasing pacing frequencies (7–12 Hz) over 15 minutes before rapid tissue preservation. Mass spectrometry identified 5,983 phosphorylation sites and 1,354 O-GlcNAcylation sites. Integration revealed 237 proteins containing both post-translational modifications, including 183 matched phospho–O-GlcNAc sites across 95 proteins. Correlation analysis showed a substantial relationship between phosphorylation and O-GlcNAcylation changes at identical residues (r≈0.48). However, only a minority of sites exhibited classical reciprocal “yin–yang” behavior. Instead, most relationships reflected coordinated regulation between nearby sites within the same protein, with correlations decreasing as the distance between modification sites increased.
Notably, global O-GlcNAcylation was decreased in ob/ob hearts, with 134 sites reduced and only 7 increased (p<0.05). Differentially modified proteins were enriched in metabolic and mitochondrial pathways, including oxidative phosphorylation, the TCA cycle, and fatty acid oxidation. Six phosphosites reached significance (q<0.05), including Nqo1, Limch1, Xirp2, Mapk9, and Syne1. Titin exhibited extensive PTM regulation during stress, with 138 O-GlcNAcylation and 268 phosphorylation sites. In addition, key cardiac regulatory proteins carried both modifications, including phospholamban, myosin binding protein C, desmin, troponins I, T, and C, myosin light chains, and myosin heavy chain 7.
These findings demonstrate that O-GlcNAcylation–phosphorylation crosstalk is remodeled at the network level and contributes to altered cardiac signaling underlying impaired contractile adaptation during the force–frequency response.