Abstract P250: Reversal of Excessive O-GlcNAcylation Restores Myofilament Function in Diabetic Cardiomyopathy: Potential Role of OGT/OGA Enzymes Localization
Diabetes Mellitus impacts the heart contractile apparatus, as well as cellular ion homeostasis, and energy production systems. Diabetic cardiomyopathy is accompanied by extensive changes in protein post-translational modifications (PTMs), including phosphoylation and O-GlcNAcylation. The cellular and molecular mechanisms of diabetic cardiomyopathy are only partially understood. We previously identified 32 total O-GlcNAcylation sites on MHC, Actin, MLC 1, MLC2, and TnI from normal hearts, and showed that exposure of skinned muscles to GlcNAc induces myofilament Ca2+ desensitization. In addition, Actin O-GlcNAcylation was increased when exposed to GlcNAc in vitro or to hyperglycemia in two models of Diabetes Mellitus (DM) type 1 and type 2. Our hypothesis is that hyperglycemia directly alters the cycling of O-GlcNAcylation on key myofilament regulatory proteins, and that excessive O-GlcNAcylation is responsible for defective myofilament Ca2+-activated response, thus contributing to diabetic cardiomyocyte dysfunction. To test this hypothesis, we have identified OGT localization at the Z-disks where other relevant signaling molecules reside, whereas OGA is localized along the M-band. OGT and OGA affinity for myofilament proteins (Actin, Tm, MLC1 and MLC2) is dramatically up-regulated in the diabetic heart, as assessed by co-immunoprecipitation and Western Blot. We also investigated the effect ofO-GlcNAc removal from normal and diabetic skinned muscles by an engineered hexosaminidase (CPJ). We found that 1 hour exposure to CPJ restored myofilament Ca2+ sensitivity exclusively in diabetic cardiac muscles (EC50 4.17±0.48 µM pre-CPJ vs 2.73±0.22 µM post-CPJ, n=5 vs n=4, p=0.029 ). These results establish a direct link between cardiac protein hyper-O-GlcNAcylation and diabetic altered cardiac myofilament Ca2+ sensitivity. Furthermore, OGT and OGA are abundant, and located at strategic signaling compartments, such as the Z-Disk. Overall these data suggest that a specific increase of OGT/OGA affinity towards key myofilament regulatory proteins, and subsequent hyper-O-GlcNAcylation may lead to dysfunctional regulation of myocardial contractility.
- © 2011 by American Heart Association, Inc.