Mechanical Activity and Myocyte Remodeling

在正常和病理情况下，机械应变对心肌细胞的形状和大小重构都是一种强有力的刺激。事实上，在人类心力衰竭之前，主要的形状变化是扩张型心肌病进展为细胞延长。这个项目的总体目标是验证这样的假设，即纵向机械应变通过在胞壁对粘着斑激酶(FAK)的差异磷酸化来调节细胞的延长，从而导致在Z-Disc和细丝添加时CAPZ对不同的肌动蛋白封顶。 具体目标是： 目的1：探讨各向异性Rho家族G蛋白磷酸化导致心肌细胞伸长的机制。我们使用PKCE过度表达(OE)小鼠定义了与应变诱导PKCE信号有关的亚细胞事件，并以3D培养的新生大鼠心室肌细胞(NRVM)在突然静态应变下为模型系统。 目的#2：验证一种假说，即腔体机械感觉装置需要依赖于PKC的FAK丝氨酸磷酸化来检测纵向应变。在3D NRVM培养中，我们研究了对纵向应变和横向应变响应的FAK丝氨酸磷酸化对PKC的依赖性。 目的#3：确定延长的心肌细胞是否改变了CAPZ的磷酸化。我们确定正常小鼠的CAPZ磷酸化是否不同于延长的心室肌细胞，以及对NRVM对齐的3D培养的各向异性机械输入是否存在不同的CAPZ磷酸化。 目的#4：验证Capz磷酸化和PIP2结合改变肌动蛋白封顶和长度重塑所必需的假说。我们测量绿色荧光标记的CAPZ的肌动蛋白封顶动力学，以确定各向异性机械刺激的影响。我们通过磷酸化、PIP2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，我们使用正常心肌细胞延长的有效条件，并通过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.