Functional and mechanistic analysis of FSHD myocytes

摘要 面肩肱营养不良（FSHD）是美国最常见的肌营养不良症之一。 端粒下区D4 Z4大卫星重复簇异染色质的收缩和丢失 导致DUX 4转录因子基因在重复序列内表达的染色体4 q与 疾病目前尚无有效的治疗方法，其发病过程尚不完全清楚 明白由于DUX 4的人工过表达在人肌细胞和小鼠中具有细胞毒性，因此认为 DUX 4诱导的细胞毒性是营养不良的主要机制。然而，只有约0.1%的患者肌肉细胞 似乎表达DUX 4，并且DUX 4表达偶尔可以在未受影响的肌肉细胞中观察到。 没有营养不良后果的个体，提高了FSHD肌细胞发生致病性 除了DUX 4诱导的细胞死亡之外的变化。准确建模和研究这种疾病一直是一个挑战 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，在本发明中， 患者肌细胞仍然是重要的试剂。然而，很难在这些组织中进行任何功能研究。 细胞培养。因此，从未明确确定FSHD心肌细胞是否表现出任何细胞- 内在功能缺陷我们获得了FSHD心肌细胞功能损害的初步证据 为进一步研究基因表达与功能的相关性提供了科学前提 FSHD心肌细胞的表型变化。因此，在本提案中，我们的目标是利用体外2D和3D培养 允许FSHD肌细胞活性的定量结构和功能测量的系统。具体 目的是（1）建立和结构/功能分析的传统的二维，对齐的二维片，和三维 肌束，使用永生化对照、FSHD 1和FSHD 2肌细胞;和（2）整合的整体和单个肌细胞。 细胞/细胞核RNA表达分析和功能验证，以鉴定改变的基因途径 与功能性FSHD表型相关。如果成功，该系统将提供重要平台， 进一步了解疾病机制和发现潜在的新治疗靶点。

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.