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.

摘要 每8500人中就有一人患有强直性肌营养不良1型(DM1)，这是最常见的成人发病 肌肉营养不良症。那些受影响的人患有影响大脑、心脏和骨骼的多系统症状 肌肉，这是调查的活跃领域。DM1患者调查发现胃肠道(GI) 困扰是患者的主要主诉，影响日常生活和福祉，包括困难 吞咽、假性梗阻和便秘。DM1胃肠道病变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。这项提案的目标是确定 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，重复范围在50到4000个重复之间，在3‘ 肌营养不良症蛋白激酶(DMPK)基因的非翻译区。DM1病理结果是一种有毒的 扩增的DMPK RNA[(CUG)EXP RNA]的功能增强。(Cug)EXP RNA隔离和防止活性 肌盲样蛋白(MBNL)家族的RNA结合蛋白，调节出生后的RNA加工网络 并维持成人RNA的表达模式。在DM1中，MBNL活性的丧失主要影响替代 剪接，导致胎儿蛋白亚型在成人组织中表达，导致疾病特征。角色 通过小鼠MBNL1和/或Mbnl2基因敲除(KO)证实了DM1病理中的两个MBNL同源基因 总结一下大脑、心脏和骨骼肌中的DM1表型。我正在使用他莫昔芬，可诱导的，有条件的 MBNL1和Mbnl2双KO测定MBNL对内脏功能丧失的影响 肌肉。这将是第一次利用MBNL丢失来研究DM1 GI特征的分子机制 与之前为其他受DM1影响的组织建立的一样。赞助商的实验室产生了纯合子的带牙的MBNL1 和Mbnl2等位基因与平滑肌特异性CRERT2(平滑CRE；dHOM小鼠)相结合。五- 周龄的SmoCRE；dHOM小鼠表现出上胃肠道运动延迟和已知的DM1相关的选择性剪接 MBNL淘汰赛后的事件。优化的dKO小鼠将用于：(1)确定GI表型 MBNL的丢失，然后(2)确定平滑肌功能障碍的分子机制。在目标1中，降低胃肠动力 将通过排珠试验进行评估，而体外试验将确定肌肉收缩能力的扰动 以及肠段间的蠕动模式。在目标2中，来自两个实验的RNA测序数据 来自DM1感染个体的小鼠和组织将被用来识别保守的替代剪接事件， 差异表达的基因，以及主要受影响的基因本体。我期待着辨认 影响钙动力学的转录变化，并将在体外研究钙的处理 小鼠原代平滑肌细胞和永生化的人平滑肌细胞。这些目标加在一起，将 阐明DM1 GI发病机制的肌源性基础，推动DM1平滑肌研究向前发展。