Intestinal smooth muscle phenotypic class switching in myopathic pediatric intestinal pseudo-obstruction

The National Institute of Diabetes and Digestive and Kidney Diseases awarded The Children's Hospital of Philadelphia $160,056 on September 1, 2026, under the Diabetes, Digestive, and Kidney Diseases Extramural Research program (CFDA 93.847) to investigate intestinal smooth muscle phenotypic switching in myopathic pediatric intestinal pseudo-obstruction (MPIPO).

MPIPO is a rare, life-threatening disorder characterized by profound bowel muscle weakness, causing massive abdominal distension, vomiting, and severe constipation, with 55 percent mortality by age 20. Most cases result from mutations in smooth muscle contractile genes, particularly ACTG2. The research addresses why children with MPIPO experience unpredictable fluctuations in disease severity, with seemingly minor infections triggering weeks to months of complete intestinal failure requiring intravenous nutrition. The central hypothesis holds that phenotypic switching—a reversible process where contractile smooth muscle cells convert to pro-fibrotic, weakly contractile synthetic cells—drives these symptom fluctuations through a two-hit mechanism: pathogenic ACTG2 mutations create baseline dysfunction while inflammatory mediators like TNFα trigger additional switching via NFκB activation.

Specific Aim 1 will define molecular mechanisms underlying inflammation-induced phenotypic switching in human intestinal smooth muscle cells using advanced culture techniques, serum-free muscularis medium, aligned nanofibrous scaffolds, and Fourier transform traction microscopy to quantify single-cell contractility changes. Subsequent experiments will test whether NFκB inhibition using FDA-approved tacrolimus and genetic knockdown prevents or reverses TNFα-induced phenotypic switching. Work is performed in Philadelphia, Pennsylvania, with a period of performance extending from September 1, 2026, through May 31, 2031.

Abstract

Myopathic pediatric intestinal pseudo-obstruction (MPIPO) is a rare, life-threatening disorder characterized by profound bowel muscle weakness, causing massive abdominal distension, vomiting, and severe constipation, with 55% mortality by age 20. Most cases result from mutations in smooth muscle contractile genes, particularly ACTG2. Children with MPIPO experience puzzling fluctuations in disease severity, with seemingly minor infections triggering weeks to months of complete intestinal failure requiring intravenous nutrition. The central hypothesis of this research is that phenotypic switching-a reversible process where contractile smooth muscle cells convert to pro-fibrotic, weakly contractile synthetic cells-drives these symptom fluctuations through a two-HIT mechanism: pathogenic ACTG2 mutations create baseline dysfunction while inflammatory mediators like tnfα trigger additional switching via nfκb activation, leading to progressive loss of contractile function. Specific aim 1 will define molecular mechanisms underlying inflammation-induced phenotypic switching in human intestinal smooth muscle cells (HISMC). Preliminary RNA sequencing data demonstrate that tnfα dramatically alters HISMC gene expression, increasing synthetic markers and matrix metalloproteinases while decreasing contractile genes. These studies will employ advanced culture techniques, including serum-free muscularis medium and aligned nanofibrous scaffolds, to maintain mature contractile phenotypes. Fourier transform traction microscopy will quantify single-cell contractility changes following tnfα exposure. Subsequent experiments will test whether nfκb inhibition using FDA-approved tacrolimus and genetic knockdown prevents or reverses tnfα-induced phenotypic switching, establishing proof-of-concept for translational therapies. Specific aim 2 will determine whether the MPIPO-causing ACTG2 R257C mutation promotes phenotypic switching using induced pluripotent stem cell (IPSC)-derived human intestinal organoids grown via a confined culture system that generates centimeters of full-thickness, functional human bowel. Organ bath contractility studies, histology, and electron microscopy will characterize muscle function in mutant versus corrected isogenic organoids. Xenium spatial transcriptomics will profile gene expression in archived human intestinal tissue from MPIPO patients, neuropathic pseudo-obstruction controls, and healthy subjects, validated by rnascope in situ hybridization. These studies will generate the first comprehensive human MPIPO model systems, test whether FDA- approved nfκb inhibitors could prevent disease flares, and provide foundational knowledge about smooth muscle phenotypic plasticity in visceral myopathy. This work positions DR. Wolfson to pursue an independent physician-scientist career developing novel therapeutics for this devastating pediatric disease.