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 每 8500 人中就有 1 人患有 1 型强直性肌营养不良 (DM1)，这是最常见的成人发病 肌营养不良症患者会出现影响大脑、心脏和骨骼的多系统症状。 肌肉，这是 DM1 患者调查的活跃领域，已确定胃肠道 (GI)。 干扰是患者的主要主诉，影响日常生活和健康，包括困难 吞咽、假性梗阻和便秘 DM1 胃肠道病理的原因目前尚不清楚。 有证据支持内脏平滑肌功能障碍的作用。该提案的目标是确定内脏平滑肌功能障碍的作用。 MBNL 功能丧失影响平滑肌活动的特定分子机制。 DM1 是由 CTG 三核苷酸重复扩增引起的，范围在 3' 中 50 至 >4000 次重复 肌强直性营养不良蛋白激酶 (DMPK) 基因的非翻译区是由有毒物质引起的。 扩展的 DMPK RNA [(CUG)exp RNA] 的功能获得，(CUG)exp RNA 隔离并阻止其活性。 调节出生后 RNA 处理网络的 RNA 结合蛋白肌盲样 (MBNL) 家族 并维持成体 RNA 表达模式 在 DM1 中，MBNL 活性的丧失主要影响替代性。 剪接，导致胎儿蛋白亚型在成人组织中表达，引起疾病特征。 小鼠 Mbnl1 和/或 Mbnl2 敲除 (KO) 证明了 DM1 病理学中两个 Mbnl 旁系同源物的作用 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型、有条件的。 小鼠中 Mbnl1 和 Mbnl2 的双重 KO 以确定 MBNL 功能丧失对内脏平滑肌的影响 这将是首次利用 MBNL 损失来研究 DM1 GI 特征的分子机制。 正如之前针对其他 DM1 受影响的组织所建立的那样，申办者的实验室生成了纯合 floxed Mbnl1。 和 Mbnl2 等位基因与平滑肌特异性 CreERT2 组合（smoCRE；dHOM 小鼠）。 周龄 smoCRE;dHOM 小鼠表现出上消化道运动延迟和已知的 DM1 相关选择性剪接 Mbnl 敲除后的事件将用于：(1) 确定 GI 表型的结果。 MBNL 丧失，然后 (2) 确定平滑肌功能障碍的分子机制 目标 1，降低胃肠道运动。 将通过珠子排出测定进行评估，离体测定将识别肌肉收缩力的扰动 在目标 2 中，来自两个实验的 RNA 测序数据。 小鼠和来自 DM1 受影响个体的组织将用于识别保守的选择性剪接事件， 我预计会识别出差异表达的基因，并主要影响基因本体。 影响钙动态的转录组变化，并将在体外进行钙处理研究 小鼠原代平滑肌细胞和永生化人类平滑肌细胞将共同实现这些目标。 解决 DM1 胃肠道发病机制的肌源性基础并推动 DM1 平滑肌研究向前发展。