Role of titin in age-associated diastolic dysfunction

描述(申请人提供)：申请人的长期职业目标是发展成为一名独立的研究人员，研究氧化应激在老化心肌中的作用。在目前的研究计划中，申请人已提议在波士顿大学医学院学习单细胞力学和质谱学的新技术。为了他早期的职业发展，申请人组建了一支由氧化应激、蛋白质组学研究、肌细胞力学和生物老年学领域的领先专家组成的团队。在获奖期结束时，申请者将获得宝贵的培训，这将使他能够开发和批判性地测试以生物老年学研究为中心的新假设。衰老的标志之一是心脏被动僵硬增加，导致舒张性心力衰竭。随着年龄的增长，细胞的氧化还原状态向增加形成活性氧物种(ROS)的方向转变。巨大的弹性蛋白Titin是心肌细胞被动机械特性的主要决定因素。随着年龄的增长，肌动蛋白弹性结构域的翻译后氧化修饰会降低心肌细胞的顺应性，从而导致衰老心肌的舒张期功能障碍。这项研究提案的总体目标是验证这样一个假设，即翻译后氧化的Titin的年龄相关积累有助于随着年龄的增长而导致舒张期功能障碍。第一个目标是测试心肌细胞的舒张期特性是否对氧化还原敏感。新鲜分离的成年和老化小鼠心脏的心肌细胞将被用或不用ROS生成器处理，去皮和Titin机制将被评估。第二个目的是测试老化是否会导致Titin弹性结构域的翻译后氧化。Titin将从成年和老化的小鼠心脏中分离出来，并进行质谱分析。第三个目标是测试老年小鼠的药理抗氧化治疗是否逆转肌动蛋白的氧化修饰并改善舒张期功能。第四个目标将测试参与肌动蛋白蛋白分解过程的机制是否会随着年龄的增长而受损。

DESCRIPTION (provided by applicant): The applicant's long-term career objective is to develop into an independent investigator studying the role of oxidative stress in the aging myocardium. In the current research plan, the applicant has proposed to learn new techniques in single myocyte mechanics and mass spectrometry at Boston University School of Medicine. For his early career development, the applicant has assembled a team of leading experts in the field of oxidative stress, proteomics research, myocyte mechanics, and biogerontology. At the end of the award period, the applicant will have acquired invaluable training which will allow him to develop and critically test new hypotheses central to biogerontology research. One of the hallmarks of aging is an increase in the ventricular passive stiffness leading to diastolic heart failure. With advancing age, the cellular redox state is shifted towards increased formation of reactive oxygen species (ROS). The giant elastic protein titin is the major determinant of passive mechanical properties of the cardiomyocyte. With progressive aging, post-translational oxidative modification of the elastic domain of titin could decrease cardiomyocyte compliance, and thus contribute to the diastolic dysfunction seen in aging myocardium. The overall goal of this research proposal is to test the hypothesis that age-associated accumulation of posttranslationally oxidized titin contributes to diastolic dysfunction with age. The first aim will test if myocyte diastolic properties are redox sensitive. Freshly isolated myocytes from adult and aging mouse hearts will be treated with or without ROS generators, skinned, and titin mechanics will be assessed. The second aim will test if aging induces post-translational oxidation of the elastic domain of titin. Titin will be isolated from adult and aging mouse hearts and processed for mass spectrometry. The third aim will test if pharmacological antioxidant treatment in aging mice reverses oxidative modification of titin and improves diastolic function. The fourth aim will test if mechanisms involved in proteolytic processing of titin are impaired with aging.