Functional and mechanistic analysis of FSHD myocytes

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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.