Functional and mechanistic analysis of FSHD myocytes

摘要 面肩肱骨营养不良(FSHD)是美国最常见的肌营养不良症之一。 亚端粒区D4Z4大卫星重复簇异染色质的收缩和丢失 染色体4Q导致DUX4转录因子基因在重复序列中的表达与 疾病。目前还没有有效的治疗方法，致病过程还不完全 明白了。由于人工过表达DUX4对人类心肌细胞和小鼠具有细胞毒性，因此人们认为 DUX4诱导的细胞毒性是营养不良的主要机制。然而，只有~0.1%的患者肌肉细胞 似乎表达DUX4，并且DUX4偶尔可以在未受影响的肌肉细胞中观察到表达 没有营养不良后果的个体，增加了FSHD肌细胞经历致病的可能性 DUX4诱导的细胞死亡以外的变化。准确地模拟和研究这种疾病一直是一个挑战。 因为疾病基因和一些受影响的下游基因是灵长类动物特有的。因此， 患者的心肌细胞仍然是重要的试剂。然而，很难在这些细胞中进行任何功能研究 培养中的细胞。因此，从来没有明确确定FSHD肌细胞是否表现出任何细胞- 固有的功能缺陷。我们获得了FSHD心肌细胞功能受损的初步证据 为进一步研究基因表达与功能之间的相关性提供了科学前提 FSHD心肌细胞表型变化。因此，在这项建议中，我们的目标是利用体外2D和3D培养 允许对FSHD肌细胞活动进行定量结构和功能测量的系统。特定的 目标是(1)常规2D、对齐2D薄片和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；以及(2)整合的块状和单个 细胞/细胞核核糖核酸表达分析和功能验证以确定一条改变的基因途径(S) 与功能性FSHD表型相关。如果成功，该系统将提供重要的平台 进一步加深我们对疾病机制的理解，并发现潜在的新的治疗靶点。

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.