Diabetes is a major risk factor for cardiovascular disease, culminating in myocardial infarction, and heart failure. Prolonged hyper-0-GlcNAcylation, due to nutrient excess and hyperglycemia, is a major molecular cause of glucose toxicity and insulin resistance. Increased 0-GlcNAcylation directly contributes to diabetic cardiomyopathy and to dysfunctional mitochondria, perhaps contributing to excessive production of reactive oxygen species (ROS). Even though 0-GlcNAcylation clearly plays an important role in diabetic cardiovascular disease, virtually nothing is known about 0-GlcNAcylation in the cardiomyocyte. This project will elucidate the roles of 0-GlcNAc in diabetic cardiomyopathy and will define the "0-GlcNAcome" of the cardiomyocyte at the site-specific level.
Specific Aims :
Aim 1 : Quantify the Site-Specific Crosstalk Between 0-GlcNAcylation and Phosphorylation in the cardiomyocyte proteome and in purified cardiomyocyte mitochondria from Normal and Diabetic Rats. Using chemico-enzymatic photocleavable tag enrichment combined with electron transfer dissociation (ETD) tandem mass spectrometry, we will quantify site occupancy for both 0-GlcNAc and phosphate in cardiomyocyte contractile and mitochondrial proteins from normal and diabetic rats.
Aim 2 : Determine the Specific Roles of 0-GlcNAcylation in normal cardiomyocyte mitochondria, and the sites of action and mechanisms of diabetes-induced dysfunction, leading to ROS production. We wilt specifically alter 0-GlcNAcylation using methods developed during the past 20-years, and correlate alterations with specific mitochondrial function.
Aim 3 : Elucidate the properties and regulation of cardiomyocyte mitochondrial isoforms of O-GlcNAc Transferase and 0-GlcNAcase. Virtually nothing is known about the mitochondrial isoforms of 0-GlcNAc Transferase (OGT) or 0-GlcNAcase (OGA). We will elucidate their localization, activities, molecular associations and kinetic activities in mitochondria from normal and diabetic rats.
Aim 4 : Evaluate the affects and roles of diabetes-induced mitochondrial dysfunction and increased O- GlcNAcylation of cardiomyocyte contractile machinery on cardiac physiology and function. Working closely with Core D we will systematically evaluate the importance of the crosstalk between 0-GlcNAcylation and phosphorylation of cardiomyocyte contractile and mitochondrial proteins on the physiological functions of cardiomyocytes These studies will open a new paradigm for understanding the regulation of cardiac functions and in diabetic cardiomyopathies. They will lead to totally unexplored avenues of possible therapeutic interventions

Public Health Relevance

Diabetes is a major epidemic and contributes to cardiovascular disease, which ultimately results in heart failure or myocardial infarction. Increased 0-GlcNAcylation, a sugar post-translational modification, underlies molecular events contributing to diabetic cardiomyopathies by affecting the functions of contractile and mitochondrial proteins within the cardiomyocyte. These studies will elucidate the importance of O- GlcNAc in both normal and diabetic cardiomyocyte physiology, and will possibly lead to novel treatments.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL107153-04
Application #
8669125
Study Section
Special Emphasis Panel (ZHL1-CSR-H)
Project Start
Project End
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
4
Fiscal Year
2014
Total Cost
$329,004
Indirect Cost
$124,915
Name
Johns Hopkins University
Department
Type
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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