Abstract P250: Reversal of Excessive O -GlcNAcylation Restores Myofilament Function in Diabetic Cardiomyopathy: Potential Role of OGT/OGA Enzymes Localization

Abstract

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 Ca 2+ 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 Ca 2+ -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 of O -GlcNAc removal from normal and diabetic skinned muscles by an engineered hexosaminidase (CPJ). We found that 1 hour exposure to CPJ restored myofilament Ca 2+ sensitivity exclusively in diabetic cardiac muscles (EC 50 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 Ca 2+ 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.