Cardiomyocyte stretch and intracellular calcium release with advancing age

描述（由申请人提供）：近八分之一（12.5%）的美国人年龄超过65岁，预计在未来20年内，65岁及以上的人数将增加到约20%。心脏病在老年人群中普遍存在，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，这些重塑事件通常导致心脏在每次跳动之间放松的能力受损。这段放松的时间是心脏舒张期，在此期间，心脏充满血液， 为心脏收缩期的下一次收缩做准备随着年龄的增长，舒张功能障碍的特征是增加的“僵硬”。鉴于更大的刚度使其更难填充，心脏作为泵的效率较低，并且在升高的填充压力和容量下操作，具有更大的心律失常倾向。由于随着年龄的增长，心功能不全的潜在机制知之甚少，为受影响的个体开发新的治疗方法需要新的机制见解。心肌细胞内钙浓度的上升和下降分别控制它们的收缩和舒张，并且由肌浆网（“SR”）调节。因此，该项目的重点是了解衰老如何改变舒张充盈（即，心肌细胞的被动拉伸）从SR释放钙。为了直接观察明确定义的功能元件（例如，钙释放的钙“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，参考收缩蛋白的明确定义的间距（即，肌节长度）。更具体地说，该研究项目将比较年轻（3个月）、中年（14个月）和衰老（24个月）C57 BL/6小鼠的完整灌流心脏和酶促分离的完整心室心肌细胞，以测试中心假设，即随着年龄的增长，心肌细胞的被动僵硬度增加，牵拉和SR钙释放之间高度敏感的耦合。目的1将确定年轻、中年和衰老心脏的细胞和肌节长度对左心室充盈压变化的反应。在目标2中，将控制单个分离的心肌细胞的长度，以确定细胞的被动拉伸如何改变钙释放，以确定细胞硬度和钙调节中与年龄相关的关键差异。在目标3中，将通过研究完整工作心脏内的钙释放和压力发展来整合目标1和2的发现。总的来说，该项目利用创新方法在高度受控的实验条件下以及在完整心脏内的天然环境中研究单个心肌细胞的功能。该指导奖还将在密苏里州大学的高度综合环境中提供重要的技术培训，这将为申请人提供独特的实验技能和与老龄化人口相关的心脏生理学的宝贵视角。预计该项目的结果将深入了解老龄化过程中心脏疾病的机制，最终目标是将这些发现转化为老龄化人口的治疗方法。

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.