Role of ACTG2 Mutations in Visceral Myopathy

ACTG2 突变在内脏肌病中的作用

• 批准号: 9904604
• 负责人: Sohaib Khalid Hashmi
• 金额: $ 3.26万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904604
• 关键词: Abdomen; Abdominal Pain; Actins; Acute; Address; Affect; Alleles; Arginine; Binding Proteins; Biochemical; Biochemistry; Biological Models; Bladder; CRISPR/Cas technology; Catheterization; Cell Differentiation process; Cells; Chimeric Proteins; Chronic; Colon; Constipation; Contracts; Cysteine; Cytoskeletal Proteins; Cytoskeleton; Defect; Deterioration; Disease; Dominant-Negative Mutation; Electron Microscopy; Failure; Family; Fibroblasts; Fibrosis; Fluorescence Recovery After Photobleaching; Fluorescent Probes; Food; Functional disorder; Generations; Genes; Genetic Transcription; Goals; Growth; Heterozygote; Hospitals; Human; Impairment; In Vitro; Individual; Infant; Intestinal Pseudo-Obstruction; Intestines; Intravenous; Lead; Life; Measures; Medical; Messenger RNA; Methods; Microfilaments; Missense Mutation; Molecular; Morbidity - disease rate; Movement; Muscle Weakness; Muscle function; Mutation; Myosin ATPase; Nonsense Mutation; Operative Surgical Procedures; Pathogenicity; Pharmaceutical Preparations; Point Mutation; Process; Protein Isoforms; Proteins; Repeat Surgery; Role; Severities; Severity of illness; Small Intestines; Smooth Muscle; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; Structure; Symptoms; Syndrome; System; Testing; Time; Traction Force Microscopy; Transplantation; Uterus; Visceral; Visceral Myopathies; Vomiting; Work; base; fluorescence imaging; high-throughput drug screening; human disease; human pluripotent stem cell; live cell imaging; motility disorder; mutant; myocardin; novel; nutrition; prevent; response to injury; transcription factor; treatment strategy

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.

项目摘要：肌病型慢性假性肠梗阻(CIPO)和巨结肠 肠动力不足综合征(MMIHS)是一种危及生命的肠动力障碍，其特征是 内脏(肠、膀胱和子宫)平滑肌无力。症状包括大便和膀胱。 腹胀、腹痛、呕吐、便秘和生长障碍。许多患有CIPO和MMIHS的人需要 静脉营养至少是间歇性的，受影响的人往往需要重复手术和花费 在医院待了几个月。目前的治疗在很大程度上是无效的，所以一些患有CIPO或MMIHS的人接受了 肠道移植。没有一种医学疗法解决潜在的疾病机制，也没有目前的治疗方法 治疗使肠道平滑肌变得更强壮。此外，引起内脏肌的分子机制 弱点几乎没有被调查过。在过去的几年里，人们发现杂合点突变 在肌动蛋白(肌动蛋白，又称肌动蛋白2；ACTG2)中，内脏肌动蛋白的主要亚型 肌肉，几乎发生在一半的CIPO和MMIHS患者中。这项工作主要集中在最常见的 发现ACTG2突变(精氨酸257变为半胱氨酸，R257C)。目标是确定这种突变是如何影响 肌动蛋白细胞骨架结构、肌动蛋白动力学和平滑肌细胞的作用力。研究还将测试 ACTG2 R257C改变平滑肌分化的假说。模型系统雇佣了人类 肠平滑肌细胞与人类多能干细胞向内脏转化的新范式 平滑肌样细胞。将采用各种实验方法，包括使用 肌动蛋白细丝结合蛋白，荧光标记肌动蛋白的活细胞成像，固定的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 以及几种分子方法评估培养的血管内皮细胞中相关的mRNA和蛋白水平 内脏平滑肌。这些研究应该确定ACTG2 R257C如何导致毁灭性的平滑肌 并可能导致新的基于机制的治疗策略。特别是，在体外缺陷定义 ACTG2突变的潜在病理生理学，将为高通量药物筛选提供平台 探索治疗CIPO/MMIHS的新方法。