Role of titin in age-associated diastolic dysfunction

描述（由申请人提供）：申请人的长期职业目标是发展成为一名独立的研究者，研究氧化应激在老化心肌中的作用。在目前的研究计划中，申请人已提出到波士顿大学医学院学习单细胞力学和质谱新技术。为了他早期的职业发展，申请人在氧化应激，蛋白质组学研究，肌细胞力学和生物老年学领域组建了一个领先的专家团队。在奖励期结束时，申请人将获得宝贵的培训，这将使他能够发展和批判性地测试对生物老年学研究至关重要的新假设。衰老的标志之一是心室被动僵硬增加导致舒张性心力衰竭。随着年龄的增长，细胞氧化还原状态转向增加活性氧（ROS）的形成。巨弹性蛋白titin是心肌细胞被动力学特性的主要决定因素。随着年龄的增长，titin弹性结构域的翻译后氧化修饰可降低心肌细胞的顺应性，从而导致衰老心肌的舒张功能障碍。本研究计划的总体目标是验证与年龄相关的翻译后氧化titin积累导致舒张功能障碍的假设。第一个目的是测试心肌细胞的舒张特性是否对氧化还原敏感。从成年和衰老小鼠心脏中分离的新肌细胞将使用或不使用ROS发生器，剥皮，并评估titin机制。第二个目的是测试老化是否会引起titin弹性区域的翻译后氧化。将从成年和衰老小鼠的心脏中分离Titin并进行质谱分析。第三个目的是测试衰老小鼠的药物抗氧化治疗是否能逆转titin的氧化修饰并改善舒张功能。第四个目标将测试titin蛋白水解过程中涉及的机制是否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.