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Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy.
Shu, Cynthia; Kaxon-Rupp, Ariana N; Collado, Judd R; Damoiseaux, Robert; Crosbie, Rachelle H.
Afiliação
  • Shu C; Molecular Biology Institute, University of California Los Angeles, Los Angeles, USA.
  • Kaxon-Rupp AN; Department of Integrative Biology and Physiology, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, Los Angeles, CA, 90095, USA.
  • Collado JR; Center for Duchenne Muscular Dystrophy, University of California Los Angeles, Los Angeles, USA.
  • Damoiseaux R; Department of Integrative Biology and Physiology, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, Los Angeles, CA, 90095, USA.
  • Crosbie RH; Department of Integrative Biology and Physiology, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, Los Angeles, CA, 90095, USA.
Skelet Muscle ; 9(1): 32, 2019 12 12.
Article em En | MEDLINE | ID: mdl-31831063
ABSTRACT

BACKGROUND:

Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion.

METHODS:

Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting.

RESULTS:

We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation.

CONCLUSIONS:

We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Distrofia Muscular de Duchenne / Mioblastos / Bibliotecas de Moléculas Pequenas / Descoberta de Drogas / Ensaios de Triagem em Larga Escala / Proteínas de Membrana / Proteínas de Neoplasias Limite: Animals Idioma: En Revista: Skelet Muscle Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Distrofia Muscular de Duchenne / Mioblastos / Bibliotecas de Moléculas Pequenas / Descoberta de Drogas / Ensaios de Triagem em Larga Escala / Proteínas de Membrana / Proteínas de Neoplasias Limite: Animals Idioma: En Revista: Skelet Muscle Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Estados Unidos