Molecular Biology Institute, University of California Los Angeles, Los Angeles, USA.
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. 2019 Dec 12;9(1):32. doi: 10.1186/s13395-019-0218-x.
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.
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.
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.
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.
杜氏肌营养不良症（DMD）是由于细胞膜连接到细胞外基质的丧失引起的。跨膜蛋白 sarcospan（SSPN）在 DMD mdx 小鼠模型中的转基因过表达通过恢复膜附着显著降低疾病病理学。鉴定基于 SSPN 的治疗方法有可能使 DMD 和其他由肌肉细胞附着缺陷引起的肌肉营养不良患者受益。
使用标准克隆方法在荧光报告基因的转染的 C2C12 成肌细胞中产生用于人 SSPN 启动子活性的荧光报告基因。使用专门用于 384 孔微孔板格式的液体处理机和成像系统在核心设施中进行测定开发和筛选。使用定量 PCR 分析药物处理的细胞的靶基因表达，并使用免疫印迹分析靶蛋白表达。
我们研究了 SSPN 及其相关蛋白在成肌细胞分化为肌管期间的基因表达谱，发现分化后 3 天表达增加。我们创建了表达 SSPN 启动子活性的 EGFP 报告基因的 C2C12 肌肉细胞，并观察到在分化过程中报告基因水平的可比增加。针对高通量筛选优化了 384 孔微孔板格式和高内涵成像仪的条件，用于报告基因水平的可视化。我们对 3200 种化合物进行了筛选，并鉴定出 7 种命中化合物，其中包括 L 型钙通道拮抗剂的过度表达，表明 SSPN 基因活性对钙敏感。对选定命中化合物的进一步验证表明，钙通道抑制剂非洛地平增加了野生型和肌营养不良缺陷肌管中的 SSPN 转录物和蛋白水平，而不增加分化。
我们开发了一种包含人 SSPN 蛋白启动子区域融合到荧光报告基因的稳定肌肉细胞系。使用报告细胞，我们创建并验证了一种可扩展的基于细胞的测定法，该测定法能够鉴定增加野生型和肌营养不良缺陷肌肉细胞中 SSPN 启动子报告基因、转录物和蛋白水平的化合物。
