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MBNL loss of function in visceral smooth muscle as a model of myotonic dystrophy type 1

MBNL 内脏平滑肌功能丧失作为 1 型强直性肌营养不良模型

基本信息

批准号：
10605562
负责人：
Janel Ann Merkel Peterson
金额：
$ 4.77万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-15 至 2025-12-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10605562
关键词：
3&apos Untranslated Regions Adult Affect Affinity Alleles Alternative Splicing Anatomy Area Atrophic Binding Sites Biological Assay Blood Pressure Blood Vessels Brain Calcium Calcium Channel Cardiopulmonary Cell Line Clinical Research Cognitive Colorectal Complex Constipation Data Defect Deglutition Development Disease Embryo Esophagus Evaluation Event Exhibits Family Feces Fellowship Fetal Proteins Frequencies Functional disorder Gastrointestinal Motility Gastrointestinal Transit Gastrointestinal tract structure Genes Genetic Transcription Goals Heart High Prevalence Histologic Human Hyperplasia In Vitro Individual Intestines Investigation Knock-out Knockout Mice Large Intestine Life LoxP-flanked allele Maps Measurement Measures Messenger RNA Modeling Molecular Movement Mus Muscle Muscle Contraction Muscle Tonus Muscle Weakness Muscle function Muscular Dystrophies Myocardium Myotonia Myotonic Dystrophy Myotonic dystrophy type 1 Nutrient Obstruction Ontology Oral Pathogenesis Pathogenicity Pathologic Pathology Patients Pattern Periodicity Personal Satisfaction Phenotype Physiological Poly A Polyadenylation Protein Family Protein Isoforms Protein Kinase Proteins RNA RNA Interference RNA Processing RNA Splicing RNA-Binding Proteins Regulation Research Reverse Transcriptase Polymerase Chain Reaction Role RyR1 Severities Skeletal Muscle Smooth Muscle Smooth Muscle Myocytes Spliced Genes Structure Surveys Symptoms Tamoxifen Therapeutic Studies Thick Time Tissue Sample Tissue-Specific Gene Expression Tissues Trinucleotide Repeat Expansion Vascular Smooth Muscle Visceral Water affective disturbance autosome cell motility differential expression fluorescein isothiocyanate dextran gain of function gastrointestinal gastrointestinal system insight knock-down live cell imaging loss of function mRNA Stability motility disorder mouse model novel paralogous gene postnatal prevent release of sequestered calcium ion into cytoplasm skeletal muscle wasting spatiotemporal transcriptome sequencing transcriptomics uptake

项目摘要

ABSTRACT One in 8500 individuals are affected by myotonic dystrophy type 1 (DM1), the most common adult onset muscular dystrophy. Those affected suffer from multisystemic symptoms affecting the brain, heart, and skeletal muscle, which are active areas of investigation. DM1 patient surveys have identified gastrointestinal (GI) disturbances as a predominant patient complaint that affects daily life and well-being and include difficulty swallowing, pseudo-obstruction, and constipation. The cause of DM1 GI pathology is currently unknown and evidence supports a role for visceral smooth muscle dysfunction. The goal of this proposal is to determine the specific molecular mechanisms by which loss of MBNL function affects smooth muscle activity. DM1 is caused by a CTG trinucleotide repeat expansion, ranging between 50 to >4000 repeats, in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. DM1 pathology results from a toxic gain of function of the expanded DMPK RNA [(CUG)exp RNA]. (CUG)exp RNA sequesters and prevents activity of the muscleblind-like (MBNL) family of RNA binding proteins that regulate postnatal RNA processing networks and maintain adult RNA expression patterns. In DM1, loss of MBNL activity predominantly affects alternative splicing, leading to the expression of fetal protein isoforms in adult tissues, causing disease features. The role of two Mbnl paralogs in DM1 pathology is demonstrated by mouse Mbnl1 and/or Mbnl2 knockout (KO) that recapitulate DM1 phenotypes in brain, heart, and skeletal muscle. I am using tamoxifen inducible, conditional double KO of Mbnl1 and Mbnl2 in mice to determine the impact of MBNL loss of function in visceral smooth muscle. This will be the first investigation into the molecular mechanisms of DM1 GI features utilizing MBNL loss as previously established for other DM1 affected tissues. The sponsor’s lab generated homozygous floxed Mbnl1 and Mbnl2 alleles that were combined with a smooth muscle specific CreERT2 (smoCRE;dHOM mice). Five- week old smoCRE;dHOM mice exhibit delayed upper GI motility and known DM1-associated alternative splicing events after Mbnl knock out. Optimized dKO mice will be used to: (1) determine what GI phenotypes result from loss of MBNL, then (2) identify molecular mechanisms for smooth muscle dysfunction. In aim 1, lower GI motility will be assessed by bead expulsion assays and ex vivo assays will identify perturbations of muscle contractility and peristaltic patterning across intestinal segments. In aim 2, RNA sequencing data from both experimental mice and tissues from DM1 affected individuals will be used to identify conserved alternative splicing events, differentially expressed genes, and predominately affected gene ontologies. I anticipate identifying transcriptomic changes affecting calcium dynamics and will perform in vitro investigations of calcium handling in mouse primary smooth muscle cells and immortalized human smooth muscle cells. Together, these aims will address the myogenic basis for DM1 GI pathogenesis and drive DM1 smooth muscle research forward.

摘要