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

• 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
• 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' 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

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.

抽象的 8500人中有一个人受1型肌发育症（DM1）的影响，这是最常见的成人发作 肌肉营养不良。受影响的人患有多个系统的症状，影响大脑，心脏和骨骼 肌肉，是活跃的投资领域。 DM1患者调查已确定胃肠道（GI） 骚乱是影响日常生活和福祉的主要患者投诉，并包括困难 吞咽，伪obstruction和便秘。 DM1 GI病理学的原因目前尚不清楚，并且 证据支持内脏平滑肌功能障碍的作用。该提议的目的是确定 MBNL功能丧失的特定分子机制会影响平滑肌活性。 DM1是由CTG三核苷酸重复膨胀引起的，在3'中介于50至> 4000之间。 肌酸肌张力型蛋白激酶（DMPK）基因的未翻译区域。 DM1病理学来自有毒 扩展的DMPK RNA [（CUG）Exp RNA]的功能增益。 （CUG）Exp RNA隔离器并防止活性 调节产后RNA加工网络的RNA结合蛋白的肌肉闪光（MBNL）家族 并保持成人RNA表达模式。在DM1中，MBNL活性的丧失主要影响替代 剪接，导致成人组织中胎儿蛋白同工型的表达，从而导致疾病特征。角色 小鼠MBNL1和/或MBNL2敲除（KO）证明了DM1病理学中两个MBNL旁系同源物的 概括大脑，心脏和骨骼肌中的DM1表型。我正在使用他莫昔芬诱导的条件 小鼠MBNL1和MBNL2的双KO确定MBNL功能在内脏光滑中的影响 肌肉。这将是使用MBNL丢失对DM1 GI特征的分子机制的首次研究 如先前针对其他DM1影响的组织所建立的。赞助商的实验室产生了纯合子的MBNL1 与平滑肌特异性creert2（smocre; dhom小鼠）结合的MBNL2等位基因。五- 一周大的smocre; dhom小鼠暴露了延迟的上胃肠运动和已知DM1相关的替代剪接 MBNL淘汰后的事件。优化的DKO小鼠将用于：（1）确定从 MBNL的丧失，然后（2）确定平滑肌功能障碍的分子机制。在AIM 1中，GI运动降低 将通过珠子驱动测定法和体内测定法进行评估，将确定肌肉收缩力的扰动 和跨肠部段的蠕动图案。在AIM 2中，来自两个实验的RNA测序数据 来自DM1受影响个体的小鼠和组织将用于识别组成的替代剪接事件， 差异表达的基因，主要影响基因本体。我预计会识别 转录组变化影响钙动力学，并将在体外研究钙处理 小鼠初级平滑肌细胞和永生的人类平滑肌细胞。这些目标将在一起 解决DM1 GI发病机理的肌源基础，并推动DM1平滑肌研究。