ACTA2, MYH11, and MYLK Mutations Affecting Smooth Muscle Contraction

家族性胸主动脉瘤和夹层（TAAD）与平滑肌突变有关 肌球蛋白重链、肌动蛋白和肌球蛋白轻链激酶（MLCK）。我们将检验一个总体假设， 致病突变降低平滑肌收缩功能。目标1：检验假设， 导致TAAD的MYH 11突变损害平滑肌细胞的收缩输出。我们将分析主动脉 来自遗传修饰小鼠的平滑肌组织，用于（1）表达中的年龄依赖性适应性变化 （2）[Ca ~（2+）]i与血管收缩性能的关系。 对激动剂作出反应的组织环，和（3）激活收缩性肌球蛋白（RLC）的磷酸化，或 粘着斑（桩蛋白）信号传导模块。目的2：检验MLCK杂合缺失 活动由于平滑肌细胞中减弱的RLC磷酸化而损害收缩输出， 导致升主动脉中TAAD的发展。目的3：检验ACTA 2突变 导致TAAD损害平滑肌收缩输出。我们将分析粘附和收缩 目的1中所述的信号传导模块，以确定特定的肌动蛋白突变是否促进选择性 一个模块或两个模块的功能障碍。目的4：检验ACTA 2突变干扰肌成纤维细胞的假设 宫缩MRTF-A将用于诱导肌成纤维细胞表型表达高量的平滑的肌成纤维细胞。 在来自携带ACTA 2突变的患者的人真皮成纤维细胞中的肌肉α-肌动蛋白。 这些研究将提供深入了解收缩性能缺陷相关的细胞基础 在研究项目1和2中在分子水平上检查ACTA 2和MYH 11突变， 与突变细胞中粘着斑重组相关的通路（项目4）。我们还将继续 MLCK与项目4的合作研究。我们的研究互动的协同作用将提供一个 了解影响血管疾病的分子机制，基于以下假设， ACTA 2、MYH 11和MYLK突变导致TAAD和闭塞性血管疾病， 收缩过程功能障碍。 相关性（参见说明）： 我们计划建立定量的重要性，负责收缩反应的特异性蛋白质， 健康的血管中的平滑肌细胞可能与以下相关的突变紊乱： TAAD。关键信号蛋白的表征将为新的抗肿瘤药物的临床策略提供前景。 药理学靶点，以管理TAAD，并可能对细胞适应，可能有助于 其他血管疾病中涉及平滑肌的收缩反应紊乱。

Familial thoracic aortic aneurysms and dissection (TAAD) are linked to mutations in smooth muscle myosin heavy chain, actin and myosin light chain kinase (MLCK. We will test the overarching hypothesis that disease-causing mutations reduce smooth muscle contractile function. Aim 1: Test the hypothesis that MYH11 mutations that cause TAAD impair contractile output of smooth muscle cells. We will analyze aortic smooth muscle tissues from genetically modified mice for age-dependent adaptive changes in (1) expression of proteins in distinct adhiesion and contractile signaling modules, (2) [Ca2+]i and vasomotor performance in tissue rings in response to agonists, and (3) phosphorylation that activates the contractile myosin (RLC) or focal adhesion (paxillin) signaling modules. Aim 2: Test the hypothesis that heterozygous loss of MLCK activity impairs contractile output because of attenuated RLC phosphorylation in smooth muscle cells and leads to development of TAAD in the ascending aorta. Aim 3: Test the hypothesis that ACTA2 mutations that cause TAAD impair contractile output of smooth muscles. We will analyze the adhesion and contractile signaling modules as described in Aim 1 to determine if specific actin mutations promote selective dysfunction in one module or both. Aim 4: Test the hypothesis that ACTA2 mutations perturb myofibroblast contractions. MRTF-A will be used to induce myofibroblast phenotype expressing high amounts of smooth muscle a-actin in human dermal fibroblasts from patients harboring mutations in ACTA2. These studies will provide insights into the cellular basis of contractile performance defects associated with ACTA2 and MYH11 mutations examined at a molecular level in research Projects 1 and 2, and cellular pathways associated with reorganization of focal adhesions in mutant cells (Project 4). We also will continue collaborative studies on MLCK with Project 4.The synergy of our research interactions will provide an understanding of the molecular mechanisms that influence vascular disease based on the hypothesis that ACTA2, MYH11 and MYLK mutations lead to TAAD and occlusive vascular diseases due to selective dysfunctions of the contractile process. RELEVANCE (See instructions): We plan to establish the quantitative importance specific proteins responsible for the contractile responses of smooth muscle cells in blood vessels in health which may be deranged with mutations associated with TAAD. Characterization of key signaling proteins will provide perspectives on clinical strategies for novel pharmacological targets to manage TAAD, and potentially on cellular adaptations that may contribute to derangement of contractile responses involving smooth muscle in other vascular diseases.