Abstract 20245: Specific Intracellular Glycosylation Directly Modulates CaMKII Activity During Hyperglycemia
GlcNAcylation, a post-translational modification in which an O-GlcNAc moiety is added to a serine or threonine residue, is emerging as an important regulator of protein function. Though O-GlcNAc modification is particularly prevalent during conditions of hyperglycemia, as is observed in diabetes, little is known regarding the effects of GlcNAcylation on the function of cardiac signaling proteins. Our lab and others have previously demonstrated that the multifunctional Ca2+/calmodulin dependent protein kinase II (CaMKII), a key regulator of apoptosis and functional remodeling in structural heart disease, is subject to acute, persistent activation by post-translational modification. Here, we test the hypothesis that O-GlcNAc modification modulates CaMKII activity and is upregulated in the failing human heart. We cultured rat cardiomyocytes in either high glucose (4.5g/L) or glucose-free media for 24 hours, followed by lysis and immunoprecipitation using antibodies against CaMKII. Immunoblot of the resulting fraction with a general O-GlcNAc antibody showed an increased fraction of O-GlcNAc modified CaMKII for cells exposed to high glucose conditions compared to control myocytes (n=3, p<0.01). We then assessed the effects of GlcNAcylation on CaMKII activity using a direct kinase assay and the FRET-based biosensor Camui, which detects conformational shifts in CaMKII associated with kinase activation. Treatment of CaMKII/Camui with high glucose in the presence of purified O-GlcNAc transferase (the enzyme that catalyzes GlcNAcylation) resulted in an eight-fold increase in kinase activity (n=6, p<0.01) and a significant change in FRET similar to that observed during CaMKII phosphorylation (n=6, p<0.01). Finally, we used antibodies against both CaMKII and O-GlcNAc to probe human heart samples from patients with and without both heart failure and Type II diabetes. GlcNAcylation of cardiac CaMKII was increased in patients with heart failure compared to control hearts, and even greater in patients with both heart failure and Type II diabetes (n=3, p<0.01). Taken together, these data suggest a role for O-GlcNAc modification in regulation of CaMKII function and transition from hyperglycemia to heart failure.
- © 2010 by American Heart Association, Inc.