Mechanical Activity and Myocyte Remodeling

在正常和病理情况下，机械应变是心肌细胞形状和大小重塑的强有力刺激。事实上，人类心力衰竭之前的主要形状变化是扩张型心肌病中细胞伸长的进展。本项目的总体目标是测试的假设，即纵向机械应变调节细胞延长的差异磷酸化的粘着斑激酶（FAK）在costamere导致差异肌动蛋白帽的CapZ在Z-光盘和细丝添加。 具体目标是： 目的#1：确定各向异性Rho家族G蛋白磷酸化导致肌细胞伸长的机制。我们定义的亚细胞事件负责应变诱导的PKCE信号转导使用PKCE过表达（OE）小鼠，对齐的三维培养的新生大鼠心室肌细胞（NRVM）进行突然静态应变作为模型系统。 目标二：为了验证这一假设，PKC依赖性FAK丝氨酸磷酸化是需要的costameric机械感觉器检测纵向应变。我们研究了PKC依赖性FAK丝氨酸磷酸化响应纵向与横向应变在三维NRVM文化。 目的#3：确定延长的肌细胞是否改变了CapZ磷酸化。我们确定正常小鼠的CapZ磷酸化是否与心室肌细胞的延长不同，以及是否存在响应NRVM对齐3D培养的各向异性机械输入的差异CapZ磷酸化。 目的#4：检验CapZ磷酸化和PIP 2结合改变肌动蛋白加帽并且是长度重塑所需的假设。我们测量肌动蛋白加帽动力学的绿色荧光标记的CapZ，以确定各向异性的机械刺激的影响。我们通过磷酸化、PIP 2和其他CapZ伙伴蛋白调节CapZ结合来确定细胞长度重塑的机制。 在这些实验中，我们使用正常肌细胞延长的验证条件，并用特定的分子干预来挑战这些过程，以确定长度重塑的机制。

Mechanical strain is a powerful stimulus for shape and size remodeling of cardiac myocytes in normal and pathological situations. Indeed, the major change in shape that precedes heart failure in humans is progression to cellular elongation in dilated cardiomyopathy. The overall objective of this project is to test the hypothesis that longitudinal mechanical strain regulates cell lengthening by differential phosphorylation of focal adhesion kinase (FAK) at the costamere leading to differential actin capping by CapZ at the Z-disc and thin filament addition. The Specific Aims are: Aim #1: To determine the mechanisms of anisotropic Rho family G protein phosphorylation leading to myocyte elongation. We define the subcellular events responsible for strain-induced PKCE signaling using the PKCE-over expressing (OE) mouse, and aligned 3D cultured neonatal rat ventricular myocytes (NRVM) subjected to sudden static strain as model systems. Aim #2: To test the hypothesis that PKC-dependent FAK serine phosphorylation is required for the costameric mechanosensory apparatus to detect longitudinal strain. We examine the PKC dependence of FAK serine phosphorylation in response to longitudinal vs. transverse strain in 3D NRVM cultures. Aim #3: To determine whether elongating myocytes have altered CapZ phosphorylation. We determine whether CapZ phosphorylations in normal mice differ from ventricular myocytes that are lengthening and whether there is differential CapZ phosphorylation in response to anisotropic mechanical inputs to NRVM aligned 3D culture. Aim #4: To test the hypothesis that CapZ phosphorylation and PIP2 binding alter actin capping and are required for length remodeling. We measure actin-capping dynamics of green fluorescent tagged-CapZ to determine the effect of anisotropic mechanical stimuli. We determine the mechanism of cell length remodeling by regulation of CapZ binding via phosphorylation, PIP2 and other CapZ partnering proteins. In these experiments, we use validated conditions of normal myocyte lengthening and challenge these processes with specific molecular interventions to determine the mechanisms of length remodeling.