Functional and mechanistic analysis of FSHD myocytes

FSHD 肌细胞的功能和机制分析

基本信息

批准号：
10287407
负责人：
Anna Grosberg
金额：
$ 37.99万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-22 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10287407
关键词：
3-Dimensional Address Affect Alleles Architecture Biological Models Biology Cell Death Cell Line Cell Nucleus Cells Characteristics Chromosomes Coculture Techniques D4Z4 Defect Development Disease Drug Screening Electric Stimulation Environment Event Exhibits Facioscapulohumeral Facioscapulohumeral Muscular Dystrophy Frequencies Future Gene Expression Gene Expression Profile Gene Expression Profiling Generations Genes Genetic Transcription Goals Heterochromatin Human In Situ In Vitro Individual Investigation Knowledge Lead Link Measurement Measures Metabolic stress Modeling Morphology Mus Muscle Cells Muscle Fibers Muscular Dystrophies Mutation Nature Outcome Outcome Study Oxidative Stress Pathogenesis Pathogenicity Pathology Pathway interactions Patients Phenotype Physiologic pulse Primates Procedures Process RNA Reagent Reproducibility Severity of illness Skeletal Myoblasts Stress Structure System Tissue-Specific Gene Expression Validation cell killing cell type cytotoxic cytotoxicity differential expression effective therapy experimental study functional disability in vivo Model insight monolayer mouse genome new therapeutic target novel overexpression response self assembly therapeutic development three dimensional cell culture transcription factor transcriptome sequencing

项目摘要

ABSTRACT Facioscapulohumeral dystrophy (FSHD) is one of the most common muscular dystrophies in the U.S. Contraction and loss of heterochromatin at the D4Z4 macrosatellite repeat cluster in the subtelomeric region of chromosome 4q resulting in expression of the DUX4 transcription factor gene within the repeat is linked to the disease. Currently, there is no effective treatment, and the pathogenic process is still not completely understood. Since artificial overexpression of DUX4 is cytotoxic in human myocytes and mice, it is thought that DUX4-induced cytotoxicity is the main mechanism of dystrophy. However, only ~0.1% of patient muscle cells appear to express DUX4, and DUX4 expression can occasionally be observed in muscle cells from unaffected individuals without dystrophic consequence, raising the possibility that FSHD myocytes undergo pathogenic changes beyond DUX4-induced cell death. It has been challenging to accurately model and study the disease in mice because the disease locus and some of the downstream genes affected are primate-specific. Thus, patient myocytes remain important reagents. It is, however, difficult to perform any functional studies in these cells in culture. As a result, it has never been explicitly determined whether FSHD myocytes exhibit any cell- intrinsic functional defect. We obtained preliminary evidence for functional impairment of FSHD myocytes providing the scientific premise to further investigate the correlation between gene expression and functional phenotype changes in FSHD myocytes. In this proposal, therefore, we aim to utilize in vitro 2D and 3D culture systems that allow quantitative structural and functional measurements of FSHD myocyte activity. Specific Aim are (1) establishment and structural/functional analyses of conventional 2D, aligned 2D sheet, and 3D myobundles, using immortalized control, FSHD1 and FSHD2 myocytes; and (2) integrative bulk and single cell/nucleus RNA expression analyses and functional validation to identify an altered gene pathway(s) associated with the functional FSHD phenotype. If successful, the system will provide important platforms to further our understanding of the disease mechanism and discovery of potential new therapeutic targets.

抽象的面肩肱营养不良症 (FSHD) 是美国最常见的肌肉营养不良症之一。亚端粒区 D4Z4 大卫星重复簇异染色质的收缩和丢失导致 DUX4 转录因子基因在重复序列中表达的染色体 4q 与疾病。目前尚无有效治疗方法，致病过程仍未完全彻底明白了。由于 DUX4 的人工过度表达对人类肌细胞和小鼠具有细胞毒性，因此认为 DUX4诱导的细胞毒性是营养不良的主要机制。然而，只有约 0.1% 的患者肌细胞似乎表达 DUX4，并且偶尔可以在未受影响的肌肉细胞中观察到 DUX4 表达没有营养不良后果的个体，增加了 FSHD 肌细胞发生致病性的可能性 DUX4 诱导的细胞死亡之外的变化。准确地建模和研究这种疾病一直具有挑战性在小鼠中，因为疾病位点和一些受影响的下游基因是灵长类动物特有的。因此，患者肌细胞仍然是重要的试剂。然而，很难在这些方面进行任何功能研究培养中的细胞。因此，从未明确确定 FSHD 肌细胞是否表现出任何细胞- 内在功能缺陷。我们获得了 FSHD 肌细胞功能损伤的初步证据为进一步研究基因表达与功能的相关性提供科学前提 FSHD 肌细胞的表型变化。因此，在本提案中，我们的目标是利用体外 2D 和 3D 培养允许对 FSHD 肌细胞活性进行定量结构和功能测量的系统。具体的目标是 (1) 传统 2D、对齐 2D 板材和 3D 的建立和结构/功能分析肌束，使用永生化对照、FSHD1 和 FSHD2 肌细胞； (2) 综合散装和单一细胞/细胞核 RNA 表达分析和功能验证，以确定改变的基因途径与功能性 FSHD 表型相关。如果成功，该系统将为进一步加深我们对疾病机制的理解并发现潜在的新治疗靶点。