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个独立的研究者，研究氧化应激在衰老心肌中的作用。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肌细胞力学和质谱学方面的新技术。为了他早期的职业发展，申请人组建了一个由氧化应激，蛋白质组学研究，肌细胞力学和神经生物学领域的领先专家组成的团队。在奖励期结束时，申请人将获得宝贵的培训，这将使他能够开发和批判性地测试新的假设中心的植物学研究。衰老的标志之一是心室被动僵硬度增加，导致舒张性心力衰竭。随着年龄的增长，细胞氧化还原状态向活性氧（ROS）形成增加的方向转变。巨弹性蛋白肌联蛋白是心肌细胞被动力学性质的主要决定因素。随着年龄的增长，肌联蛋白弹性结构域的翻译后氧化修饰可降低心肌细胞的顺应性，从而导致衰老心肌中观察到的舒张功能障碍。这项研究的总体目标是检验以下假设：随着年龄的增长，与年龄相关的氧化后肌联蛋白的积累会导致舒张功能障碍。第一个目标将测试肌细胞舒张特性是否是氧化还原敏感的。将用或不用ROS发生器处理来自成年和老化小鼠心脏的新鲜分离的肌细胞，去皮，并评估肌联蛋白机制。第二个目的是测试老化是否诱导肌联蛋白弹性结构域的翻译后氧化。将从成年和衰老小鼠心脏中分离肌联蛋白，并进行质谱分析。第三个目的是测试药物抗氧化剂治疗是否能逆转肌联蛋白的氧化修饰并改善舒张功能。 第四个目标是测试肌联蛋白的蛋白水解加工机制是否会随着衰老而受损。

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.