Cardiomyocyte stretch and intracellular calcium release with advancing age

描述(由申请人提供)：近八分之一(12.5%)的美国人年龄在65岁以上，65岁及以上的人数预计在未来20年将增加到~20%。心脏病在老年人群中很普遍，因此在美国是一个主要的公共卫生问题。心脏随着年龄的增长而重塑，这些重塑事件往往会导致心脏在每次跳动之间放松的能力受损。这个舒张期是心脏的舒张期，在此期间，心脏充满血液和 在收缩期间为下一次收缩做准备。随着年龄的增长，舒张期功能障碍的特点是“僵硬”增加。考虑到较大的硬度使其更难填充，心脏作为泵的效率较低，在较高的充盈压力和充盈容量下运行，更容易发生心律失常。由于随着年龄增长而导致心功能不全的机制(S)尚不清楚，因此为患者开发新的治疗方法需要新的机制洞察。心肌细胞内钙离子浓度的升高和降低分别控制心肌细胞的收缩和松弛，并受肌浆网(“SR”)的调节。因此，本项目致力于了解衰老如何改变舒张期充盈(即心肌细胞的被动伸展)和肌浆网钙释放之间的关系。为了直接观察钙释放的明确功能元件(例如，钙“火花”和“波”)，高分辨率共聚焦成像将与荧光钙指示剂(Fluo-4)一起使用，以参考明确定义的收缩蛋白的间距(即肌节长度)。更具体地说，本研究项目将比较年轻(3个月)、中年(14个月)和衰老(24个月)C57BL/6小鼠完整的灌流心脏和酶分离的完整心肌细胞，以验证随着年龄的增长，心肌细胞在拉伸和SR钙释放之间具有高度敏感的耦合的被动僵硬增加的中心假设。目的1确定年轻、中年和衰老心脏的细胞和肌节长度随着左心室充盈压力的明确变化而发生的实际变化。在目标2中，将控制单个分离的心肌细胞的长度，以确定细胞被动拉伸如何改变钙释放，从而确定细胞僵硬和钙调节方面的关键年龄差异。在目标3中，目标1和目标2的结果将通过研究完整的工作心脏内的钙释放和压力发展来整合。总体而言，该项目利用创新的方法来研究单个心肌细胞在高度受控的实验条件下以及它们在完整心脏内的自然环境中的功能。该指导奖还将在密苏里大学高度一体化的环境中提供重要的技术培训，这将使申请者在与老龄化人口相关的心脏生理学方面拥有独特的实验技能和宝贵的视角。预计该项目的结果将深入了解老龄化期间心脏病的机制，最终目标是将这些发现转化为对老龄化人口的治疗。 与公共健康相关：健康的心脏表现出高度协调的细胞内钙释放，以发挥有效泵的作用。舒张期充盈过程中钙释放的变化(即被动拉伸)使心脏容易出现充盈受损和心律失常，这通常与衰老有关。这项研究计划调查衰老如何影响拉伸诱导的细胞内钙释放，目的是将我们的发现转化为改善老龄化人群心脏功能的治疗方法的开发。

DESCRIPTION (provided by applicant): Nearly 1 in 8 (12.5%) of Americans are older than 65 years and the number of individuals 65 and over is expected to increase to ~20% over the next 2 decades. Cardiac disease is prevalent in the aged population and therefore represents a major public health concern in America. The heart remodels with advancing age and these remodeling events often result in the impaired ability of the heart to relax between each beat. This period of relaxation is the cardiac diastole, during which time the heart fills with blood and prepares for the next contraction during systole. With aging, diastolic dysfunction is characterized by increased "stiffness". Given that greater stiffness makes it harder to fill, the heart is less effective as a pump and operates at elevated filling pressures and volumes with a greater propensity for arrhythmia. Because the mechanism(s) underlying cardiac dysfunction with advancing age are poorly understood, developing new therapeutic treatments for affected individuals requires new mechanistic insight. The rise and fall of calcium concentration within cardiac cells controls their contraction and relaxation, respectively, and is regulated by the sarcoplasmic reticulum ("SR"). Therefore, this project is focused on understanding how aging alters the relationship between diastolic filling (i.e., passive stretching of cardiac myocytes) an calcium release from the SR. To directly observe well-defined functional elements (e.g., calcium "sparks" and "waves") of calcium release, high resolution confocal imaging will be used with fluorescent calcium indicators (Fluo-4) with reference to the well-defined spacing of contractile proteins (i.e., sarcomere length). More specifically, this research project will compare intact perfused hearts and enzymatically dissociated intact ventricular cardiomyocytes from young (3 month), middle-aged (14 month), and senescent (24 month) C57BL/6 mice to test the central hypothesis that with advancing age cardiomyocytes have increased passive stiffness with highly sensitive coupling between stretch and SR calcium release. Aim 1 will determine the actual changes that occur in cell and sarcomere lengths of young, middle-aged, and senescent hearts in response to defined changes in left ventricular filling pressure. In Aim 2, the length of individual isolated cardiomyocytes will be controlled to determine how passive stretch of the cell alters calcium release in order to identify key age-associated differences in cell stiffness and calcium regulation. In Aim 3, findings from Aims 1 and 2 will be integrated by investigating calcium release and pressure development within the intact, working heart. Overall, this project utilizes innovative methods to study the function of individual cardiomyocytes under highly controlled experimental conditions as well as in their native environment within the intact heart. This mentored award will also provide important technical training in a highly integrative environment at the University of Missouri, which will give the applicant unique experimental skills and valuable perspective into cardiac physiology as it relates to aging populations. It is anticipated that results from this project will yield insight into the mechanisms of cardiac diseas during aging, with the ultimate goal of translating these findings into treatments for the aging population. PUBLIC HEALTH RELEVANCE: The healthy heart exhibits highly coordinated intracellular calcium release in order to function as an effective pump. Alterations in calcium release during diastolic filling (i.e., passive stretch) predispose the heart to impaired filling and arrhythmia, which are commonly associated with aging. This research proposal investigates how aging affects stretch-induced intracellular calcium release with the goal of translating our findings int the development of therapies to improve cardiac function in the aging population.