Mechanobiology of Cardiomyocytes

描述（由申请人提供）：心脏肥大是一种适应性生长机制，由几种信号通路介导，包括通过细胞外基质（ECM）蛋白偶联至细胞粘附位点的信号通路。整合素是一类重要的ECM蛋白受体，可以介导力传递和信号转导。因此，整合素已被提出是一个重要的mechanosensor在肌细胞和发挥核心作用，作为mechanotransducers在正常发育过程中，并在响应与生理和病理生理状态的机械力。然而，定义整合素如何参与感知和将物理力转换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，机械诱导的细胞信号在心肌细胞中的整合素（α 3 β 1和α 5 β 1）的启动涉及键形成，键应力和/或键之间的整合素和ECM蛋白，纤连蛋白（FN），胶原蛋白（CN）和层粘连蛋白（LN）的解离。我们假设，整合素介导的细胞信号转导事件在心肌细胞将涉及改变胞浆钙浓度（[Ca 2 +]j），细胞刚度/弹性或细胞的机械反应。在本探索性/发展性研究中，我们将建立一种方法来研究小鼠心肌细胞中的机械转导过程。这项研究的一个独特的方面是使用原子力显微镜（AFM）作为探测整合素功能和细胞力学的工具。该提议的具体目的是：1）确定肌细胞膜上的整合素-ECM键形成、键应力或强制键解离是否导致[Ca 2 +]i的变化;和2）确定[Ca 2 +] i的变化是否导致皮质肌细胞刚度/弹性或机械响应的可检测改变。将使用AFM在肌细胞中测量ECM与其整联蛋白受体之间的结合力。将通过测量细胞溶质Ca 2+、细胞硬度和对机械刺激的细胞机械反应来研究肌细胞对整合素-ECM相互作用的反应。这些研究将提供一些在单个心肌细胞中整合素-ECM结合的许多关键定量参数的第一次测量。这将为研究心肌细胞的信号转导机制提供新的思路。

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.