The post-translational modification of intracellular proteins by monosaccharides of O-linked -N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia-reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc-cycling in response to I/R injury have not been comprehensively assessed. While numerous approaches exist for the detection of O-GlcNAc in cells and tissues, immunoblotting using GlcNAc-specific antibodies is common. Therefore, my initial goal was to optimize the detection of O-GlcNAc in heart lysates. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, we demonstrated that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The aforementioned improvements in O-GlcNAc detection were accompanied by optimization of approaches for assessing the activity of the enzymes that cycle O-GlcNAc, the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), and detection of UDP-GlcNAc in the heart. Overall, the aforementioned techniques aimed to provide a comprehensive assessment of O-GlcNAc-cycling during heart injury.
As baseline heart physiology and pathophysiology are impacted by sex, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation basally and in ischemic hearts. To address this unprecedented question, male and female wild type murine hearts were subjected to ischemia or I/R injury and assessed for protein O-GlcNAcylation, abundance, and activity of OGT and OGA, abundance of GFAT2, the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP), and levels of UDP-GlcNAc, the product of the HBP and a substrate of OGT. Our data demonstrated elevated O-GlcNAcylation in female hearts both basally and during ischemia. Providing a mechanism for these observations, OGT activity was enhanced in females in all treatments. Ischemia also targets OGT, with reduced O-GlcNAcylation and OGT specific activity detected in this treatment.
Collectively, these findings enhance our understanding of molecular mechanisms regulating O-GlcNAcylation in the heart. They also propose the involvement of O-GlcNAc-mediated regulation in sex-dependent cardioprotection. Future investigations into the molecular mechanisms regulating the sex-dependent and injury-associated changes to OGT activity may aid in combating I/R injury in both males and females
