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.

中文翻译：

---

高通量筛选技术的开发，以鉴定肌膜小分子增强剂来治疗杜氏肌营养不良症。

杜氏肌营养不良症（DMD）是由于肌膜与细胞外基质的连接丧失所致。在DMD mdx小鼠模型中，跨膜蛋白sarcospan（SSPN）的转基因过表达通过恢复膜粘着力显着降低了疾病病理。鉴定基于SSPN的疗法有可能使DMD和其他形式的肌营养不良症患者受益，这是由于肌肉细胞粘附缺陷引起的。使用标准的克隆方法来生成C2C12成肌细胞，该细胞稳定地被人类SSPN启动子活性的荧光报告基因转染。分析开发和筛选是在核心设施中使用专门用于384孔微孔板格式的液体处理器和成像系统进行的。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的表达。我们调查了成肌细胞分化成肌管过程中SSPN及其相关蛋白的基因表达谱，揭示了分化3天后表达的增加。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，并观察到分化过程中报告基因水平的可比提高。高通量筛选的分析条件已针对384孔微孔板格式和高含量成像仪进行了优化，以可视化报告基因水平。我们进行了3200种化合物的筛选，确定了7个匹配项，其中包括L型钙通道拮抗剂的过量代表，这表明SSPN基因活性对钙敏感。进一步验证所选命中结果表明，钙通道抑制剂非洛地平在野生型和肌营养不良蛋白缺陷型肌管中均增加了SSPN转录和蛋白质水平，而没有增加分化。我们开发了一种稳定的肌肉细胞系，其中包含与荧光报告基因融合的人SSPN蛋白的启动子区域。使用报告细胞，我们创建并验证了可扩展的，基于细胞的测定方法，该方法能够鉴定出能增加野生型和抗肌营养不良蛋白缺乏的肌肉细胞中SSPN启动子报告基因，转录物和蛋白质水平的化合物。我们开发了一种稳定的肌肉细胞系，其中包含与荧光报告基因融合的人SSPN蛋白的启动子区域。使用报告细胞，我们创建并验证了可扩展的，基于细胞的测定方法，该方法能够鉴定出能增加野生型和抗肌营养不良蛋白缺乏的肌肉细胞中SSPN启动子报告基因，转录物和蛋白质水平的化合物。我们开发了一种稳定的肌肉细胞系，其中包含与荧光报告基因融合的人SSPN蛋白的启动子区域。使用报告细胞，我们创建并验证了可扩展的，基于细胞的测定方法，该方法能够鉴定出能增加野生型和抗肌营养不良蛋白缺乏的肌肉细胞中SSPN启动子报告基因，转录物和蛋白质水平的化合物。