Novel quantitative proteomic approaches to define the altered interplay between OGlcNAcylation and Phosphorylation in myofilament dysfunction of diabetic hearts

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Project Abstract/Summary The candidate I am a Mexican-American MD/PhD who works as a basic scientist. I am also an Assistant Professor in the Johns Hopkins School of Medicine, Division of Pediatric Cardiology where I also completed my post- doctoral fellowship training. My interest in and commitment to a translational and basic research career started as a 4th year medical student. I am convinced that funding through the NIH/NHLBI Mentored Career Development Award to Promote Faculty Diversity in Biomedical Research will be instrumental to achieve my goal, which is to become an independent investigator and a future leader in the field of molecular cardiology and its translation into the advancement of therapies of diabetic cardiomyopathy and heart failure.  Research Proposal: Novel quantitative proteomic approaches to define the altered interplay between O-GlcNAcylation and Phosphorylation in myofilament dysfunction of diabetic hearts In North America, the 2010 prevalence of diabetes was 37.4 million (10.2%) and is on a steady rise16. Diabetic patients are 2 to 4 times more at risk of dying from heart disease than the general population17. Among cardiovascular complications, diabetic cardiomyopathy refers to a progressive diastolic and systolic dysfunction due to a contractile deficit of the cardiac muscle that develops independently from coronary artery disease. While it is present in 60% of diabetic patients, no therapy is currently available to halt or significantly alter the course of diabetic cardiomyopathy18. Post-translational modifications of the sarcomere regulate cardiac function and when dysregulated contribute to cardiac dysfunction. Recent work in our group has focused on the identification, quantification and functional characterization of myofilament O-GlcNAcylation and Phosphorylation1-8. The goal of this proposal is to use state of the art quantitative proteomic approaches to extensively map and perform site-specific quantification of all potentially O-GlcNAcylated and Phosphorylated myofilament proteins of normal and diabetic hearts during baseline cardiac function and during β-adrenergic and force-frequency stimulation. By comparing O-GlcNAc/Phosphate stoichiometry changes between baseline and enhanced workload we will identify key sites for abnormal myofilament function in diabetic cardiomyopathy. By using gene transfer techniques, the present proposal also will perform in vivo and in vitro functional work to define the role of the interplay between O-GlcNAcylation and Phosphorylation and the mechanisms that lead to impaired cardiac contractile reserve in diabetes. Advances in this field can potentially generate early diagnostic tools for diabetic cardiomyopathy and open new therapeutic venues to fix the molecular motors of a failing diabetic heart. The specific aims of this proposal are Aim 1: To perform global myofilament site-specific O- GlcNAcylation and Phosphorylation mapping and quantification in normal and type 2 diabetic hearts. Aim 2: To identify O-GlcNAcylated and Phosphorylated sites with the greatest stoichiometric variation during β-adrenergic and force-frequency stimulation in normal and type 2 diabetic heart myofilaments. Aim 3: To validate the functional impact of altered balance of O-GlcNAcylation and Phosphorylation competing sites on cardiac contractility by manipulating myofilament proteins with gene transfer. The environment The Johns Hopkins School of Medicine possesses an excellent environment to perform basic and translational research. Johns Hopkins University has a strong foundation and facilities in research focused on diseases of adults and children. For example, the Department of Pediatrics presently has 27 million dollars in NIH research dollars. The medical campus will enable the candidate to access numerous state-of-the-art core facilities. The mentors, advisors and collaborators outlined in this application will assist in a successful completion of the candidate career and research goals. We have assembled a superb team of fine scientist and established faculty with many years of experience and great success mentoring young scientists. My main mentor is Dr. Anne M Murphy, co-mentor is Dr. Jennifer Van Eyk. Dr. Gerald W. Hart and Dr. Brian O'Rourke form the advisor committee.