Role of ACTG2 Mutations in Visceral Myopathy

项目总结：肌病性慢性假性肠梗阻（锡波）和巨囊型小结肠 肠功能减退综合征（MMIHS）是一种危及生命的肠动力障碍，其特征在于： 内脏（肠、膀胱和子宫）平滑肌无力。症状包括肠道和膀胱 腹胀、腹痛、呕吐、便秘和生长障碍。许多患有锡波和MMIHS的人需要 静脉内营养至少间歇性和受影响的个人往往有重复的手术和花费 几个月在医院。目前的治疗在很大程度上是无效的，所以一些患有锡波或MMIHS的人经历了小的 肠移植没有一种医学疗法能解决潜在的疾病机制， 治疗使肠平滑肌更强壮。此外，导致内脏肌肉的分子机制 弱点几乎没有调查。在过去的几年里，人们发现杂合子点突变 在γ平滑肌肌动蛋白（肌动蛋白，γ 2; ACTG 2）中，内脏平滑肌中的主要肌动蛋白亚型， 肌肉，发生在几乎一半的人与锡波和MMIHS。这项工作的重点是最常见的 鉴定的ACTG 2突变（精氨酸257变为半胱氨酸，R257 C）。我们的目标是确定这种突变如何影响 肌动蛋白细胞骨架结构，肌动蛋白动力学和平滑肌细胞产生的力。研究还将测试 ACTG 2 R257 C改变平滑肌分化的假说。该模型系统采用人类 肠平滑肌细胞和将人多能干细胞转化为内脏干细胞的新范例 平滑肌样细胞将采用不同的实验方法，包括使用 肌动蛋白丝结合蛋白，荧光标记肌动蛋白的活细胞成像，固定细胞中肌动蛋白细胞骨架分析 细胞，肌动蛋白细胞骨架的电子显微镜，牵引力显微镜测量收缩强度， 平滑肌细胞，以及几种分子方法来评估相关的mRNA和蛋白质水平培养 内脏平滑肌这些研究应该确定ACTG 2 R257 C如何导致破坏性平滑肌 并可能导致新的机制为基础的治疗策略。特别是，体外缺陷， ACTG 2突变的潜在病理生理学，将为高通量药物筛选提供平台 发现锡波/MMIHS的新疗法。

Project Summary: Myopathic chronic intestinal pseudo-obstruction (CIPO) and Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) are life-threatening bowel motility disorders characterized by visceral (bowel, bladder, and uterine) smooth muscle weakness. Symptoms include bowel and bladder distension, abdominal pain, vomiting, constipation, and growth failure. Many people with CIPO and MMIHS need intravenous nutrition at least intermittently and affected individuals often have repeated surgery and spend months in the hospital. Current therapy is largely ineffective so some people with CIPO or MMIHS undergo small bowel transplantation. None of the medical therapies address underlying disease mechanisms and no current therapy makes bowel smooth muscle stronger. Furthermore, molecular mechanisms causing visceral muscle weakness are barely investigated. Within the past few years, it was discovered that heterozygous point mutations in gamma smooth muscle actin (actin, gamma 2; ACTG2), the predominant actin isoform in visceral smooth muscle, occur in almost half of people with CIPO and MMIHS. This work is focused on the most commonly identified ACTG2 mutation (arginine 257 to cysteine, R257C). The goal is to determine how this mutation affects actin cytoskeletal structure, actin dynamics, and force generation by smooth muscle cells. Studies will also test the hypothesis that ACTG2 R257C alters smooth muscle differentiation. The model system employs human intestinal smooth muscle cells and a novel paradigm for converting human pluripotent stem cells to visceral smooth muscle-like cells. Diverse experimental approaches will be employed including live cell imaging using actin filament binding proteins, live cell imaging of fluorescently tagged actin, actin cytoskeleton analysis in fixed cells, electron microscopy of the actin cytoskeleton, traction force microscopy to measure contractile strength of smooth muscle cells, and several molecular methods to assess relevant mRNA and protein levels in cultured visceral smooth muscle. These studies should define how ACTG2 R257C causes devastating smooth muscle weakness and may lead to novel mechanism-based treatment strategies. In particular, in vitro defects that define the underlying pathophysiology of ACTG2 mutations, will provide a platform for high-throughput drug screening to discover new treatments for CIPO/MMIHS.