ArticleSkeletal muscle2019
Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy.
Article in Skeletal muscle, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 7 citations in OpenAlex.
- The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research.International journal of molecular sciences · 2024Article
- Peripheral modulation of antidepressant targets MAO-B and GABAAR by harmol induces mitohormesis and delays aging in preclinical models.Nature communications · 2023Article
- High-Throughput Screening to Identify Modulators of Sarcospan.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Sarcospan increases laminin-binding capacity of α-dystroglycan to ameliorate DMD independent of Galgt2.Human molecular genetics · 2022Article
- C-X-C motif chemokine ligand 12: a potential therapeutic target in Duchenne muscular dystrophy.Bioengineered · 2021Article
- Loss of sarcospan exacerbates pathology in mdx mice, but does not affect utrophin amelioration of disease.Human molecular genetics · 2021Article
- High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy.Skeletal muscle · 2020Article
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Authors and funding
5 authors at 3 institutions in 1 country.
Funding
Abstract
backgroundDuchenne 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.
methodsStandard 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.
resultsWe 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.
conclusionsWe 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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