Mechanobiology of Cardiomyocytes

描述(申请人提供)：心肌肥大是一种适应性生长机制，它由几个信号通路介导，包括那些通过细胞外基质(ECM)蛋白耦合到细胞黏附位置的信号通路。整合素是细胞外基质蛋白的一类重要受体，既可以介导力传递，也可以介导信号转导。因此，整合素被认为是心肌细胞中一种重要的机械感受器，在正常发育和对与生理和病理生理状态相关的机械力做出反应时，作为机械感受器发挥核心作用。然而，定义整合素如何参与感知物理力量并将其转化为细胞信号的确切机制尚不清楚。 我们的长期目标是了解心肌中基于整合素的机械信号转导机制。我们的中心假设是，整合素(α3beta1和alpha5beta1)在心肌细胞中启动机械诱导的细胞信号，涉及整合素与ECM蛋白、纤维连接蛋白(FN)、胶原(CN)和层粘连蛋白(LN)之间的键形成、键应力和/或键解离。我们假设，心肌细胞中整合素介导的细胞信号事件将涉及细胞内钙浓度([Ca~(2+)]j)、细胞硬度/弹性或细胞机械反应的变化。在这项探索性/发育性研究中，我们将建立一种方法来研究小鼠心肌细胞的机械转导过程。这项研究的一个独特方面是使用原子力显微镜(AFM)作为一种工具来探索整合素的功能和细胞力学。这项建议的具体目的是：1)确定整合素-ECM键的形成、键应力或强迫解离是否会导致心肌细胞膜上[Ca~(2+)]i的变化；以及2)确定[Ca~(2+)]j的变化是否会导致可检测到的皮质肌细胞僵硬/弹性或机械反应的变化。细胞外基质与其整合素受体之间的结合力将使用原子力显微镜在心肌细胞中进行测量。心肌细胞对整合素-ECM相互作用的反应将通过测量细胞内钙离子、细胞硬度和细胞对机械刺激的机械反应来研究。这些研究将首次测量单个心肌细胞中整合素-细胞外基质结合的许多关键定量参数。这将为了解心肌细胞的转导机制提供新的见解。

DESCRIPTION (provided by applicant): Cardiac hypertrophy is an adaptive growth mechanism that is mediated by several signaling pathways including those coupled through extracellular matrix (ECM) proteins to sites of cell adhesion. Integrins are an important class of receptors for the ECM proteins that can mediate both force transmission and signal transduction. Consequently, integrins have been proposed to be an important mechanosensor in myocytes and to play a central role as mechanotransducers during normal development and in response to mechanical forces associated with physiological and pathophysiological states. However, the precise mechanisms that define how integrins are involved in sensing and transducing physical forces into cellular signals are unclear. Our long-term goal is to understand the mechanisms involved in integrin-based mechanical signaling in the myocardium. Our central hypothesis is that initiation of mechanically induced cell signaling in cardiac myocytes by integrins (alpha3beta1 and alpha5beta1) involves either bond formation, bond stressing and/or bond dissociation between integrins and ECM proteins, fibronectin (FN), collagen (CN) and laminin (LN). We hypothesize that the integrin-mediated cell signaling events in cardiac myocytes will involve alterations in cytosolic calcium concentration ([Ca2+]j), cell stiffness/elasticity or a cellular mechanical response. In this exploratory/developmental study, we will establish a method to study the mechanotransduction processes in mouse cardiac myocytes. A unique aspect of this study is the use of Atomic Force Microscopy (AFM) as a tool to probe integrin function and cellular mechanics. The specific aims of this proposal are: 1) to determine if integrin-ECM bond formation, bond stressing or forced bond dissociation on the myocyte membrane result in changes of [Ca2+]i; and 2) to determine if the [Ca2+]j changes result in detectable alterations of cortical myocyte stiffness/elasticity or mechanical responses. The binding forces between ECM and their integrin receptors will be measured in the myocytes using the AFM. The myocytes responses to integrin-ECM interactions will be investigated by measuring cytosolic Ca2+, cell stiffness and cellular mechanical responses to a mechanical stimulus. These studies will provide some of the first measurements of many critical quantitative parameters of integrin-ECM bonding in single cardiac myocytes. This will provide novel insight into transduction mechanisms of myocytes.