Role of ACTG2 Mutations in Visceral Myopathy

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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.