Abstract 16695: O-GlcNAc Cycling Enzymes and Myofilaments in Diabetic Cardiomyopathy: An Increased Association Leading to Myofilament Ca2+ Desensitization
Protein O-GlcNAcylation, the addition of a single β-D-N-acetylglucosamine (GlcNAc) to serine/threonine residues, by OGT (O-GlcNAc transeferase) and removal by OGA (O-GlcNAcase), is a post-translational modification that regulates cardiovascular function. Regulation of O-GlcNAcylation serves as a metabolic/nutrient sensor, and is disrupted in diabetes. We hypothesize that altered O-GlcNAcylation of myofilament proteins is an important molecular mechanism that contributes to myofilament Ca2+ desensitization in diabetes. To test the role of O-GlcNAc in this process we enzymatically removed endogenous O-GlcNAcylation in cardiac skinned fibers from normal and diabetic type 1 rats via a homologue of human OGA (CpNagJ). We found that removing endogenous O-GlcNAc restored myofilament response to Ca2+ only in streptozotocin (STZ)-induced type 1 diabetic rat muscles (ECa50 4.17±0.48 µM STZ Pre- CpNagJ vs. 2.73±0.22 µM STZ Post- CpNagJ, n=5 vs. n=4, p=0.029), whereas in control muscles it had no effect. Similarly, maximal Ca2+ activated force (Fmax) and myofilament cooperativity (n Hill), remained unchanged after CpNagJ treatment in both groups. Immunoprecipitation experiments in normal and diabetic hearts uncovered OGT and OGA interactions with MyBP-C, α-actin, tropomyosin, and myosin light chain 1. Except for MyBP-C, these protein interactions were increased in the diabetic hearts. In normal myocytes, confocal and immuno-electron microscopy analyses, illustrated OGT and OGA compartmentalization at the Z-disk and A-band, respectively. Strikingly, analysis of their ultra-structural distribution reveal that in the diabetic myocardium OGT and OGA compartmentalization was lost, with both proteins exhibiting a more diffuse localization and increased immuno-staining. Moreover, OGT and OGA were readily detected within mitochondria, and OGT immuno-staining was elevated in diabetic mitochondria. Our data demonstrate that removal of endogenous myofilament O-GlcNAcylation, specifically from diabetic muscles, restores myofilament Ca2+ sensitivity to normal. This suggests that altered GlcNAc “cycling” plays a key mechanistic role in the abnormal myofilament response to Ca2+ in diabetic cardiac muscle.
- © 2011 by American Heart Association, Inc.