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

项目摘要/摘要 候选人 我是墨西哥裔美国医学博士，从事基础科学工作。我也是一名助理教授 约翰霍普金斯医学院，儿科心脏病学部，我也在那里完成了我的职位- 博士研究生奖学金培训。我开始了对翻译和基础研究事业的兴趣和承诺 作为一名四年级的医科学生。我相信，通过NIH/NHLBI指导的职业生涯提供资金 促进生物医学研究教师多元化的发展奖将有助于实现我的 目标是成为分子心脏病学领域的独立研究者和未来的领导者 并将其转化为糖尿病心肌病和心力衰竭治疗的进展。 研究建议：用新的定量蛋白质组学方法来确定 O-GlcN酰化和磷酸化在糖尿病心脏肌丝功能障碍中的作用 在北美，2010年糖尿病患病率为3740万人(10.2%)，而且还在稳步上升。 糖尿病患者死于心脏病的风险是普通人群的2到4倍。 在心血管并发症中，糖尿病心肌病指的是进行性的舒张期和收缩期 独立于冠状动脉发育的心肌收缩缺陷所致的功能障碍 疾病。虽然它存在于60%的糖尿病患者中，但目前还没有治疗方法可以停止或显著 改变糖尿病心肌病的病程18。 肌节的翻译后修饰调节心脏功能，并在失调时 会导致心脏功能不全。我们小组最近的工作集中在识别、量化和 肌丝O-GlcN酰化和磷酸化1-8的功能特征。这项提议的目标是 使用最先进的定量蛋白质组学方法广泛地定位和执行特定的位点 所有可能的O-GlcN酰化和磷酸化的肌丝蛋白的定量 糖尿病心脏在基础心功能期间以及在β肾上腺素能和力频刺激期间。通过 比较基线和增强工作负载之间O-GlcNAc/磷酸化学计量比的变化，我们将 找出糖尿病心肌病肌丝功能异常的关键部位。通过使用基因转移 技术，本提案还将在体内和体外进行功能工作，以确定 O-GlcN酰化和磷酸化的相互作用及其导致心脏损害的机制 糖尿病患者的收缩储备。这一领域的进展可能会产生糖尿病的早期诊断工具 心肌病和开辟新的治疗场所来修复 糖尿病心脏衰竭的分子马达。的具体目标 这项建议是 目标1：进行全局肌丝部位特异性O- 谷氨酰胺酰化和磷酸化定位及定量 正常心脏和2型糖尿病心脏。 目的2：鉴定O-GlcN酰化和磷酸化位点 β-肾上腺素能和 力频刺激对正常和2型糖尿病心脏的影响 肌丝。 目的3：验证O-GlcN酰化和磷酸化竞争平衡改变对功能的影响 通过基因转移操纵肌丝蛋白对心肌收缩能力的影响。 环境问题 约翰霍普金斯医学院拥有良好的环境，可以进行基础和 翻译研究。约翰霍普金斯大学在研究方面拥有雄厚的基础和设施 成人和儿童的疾病。例如，儿科目前有2700万美元 国立卫生研究院的研究经费。医学园将使候选人能够访问许多最先进的核心 设施。本申请中概述的导师、顾问和协作者将帮助成功 完成应聘者的职业生涯和研究目标。我们已经组建了一支优秀的科学家团队 拥有多年经验并在指导年轻科学家方面取得巨大成功的知名教师。我的Main 导师是安妮·M·墨菲博士，共同导师是詹妮弗·范·埃克博士。杰拉尔德·W·哈特博士和布莱恩·奥鲁尔克博士 组建顾问委员会。

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.