Dystrophin, the protein product of the largest known human gene, plays a critical role in muscle integrity and function by stabilizing the sarcolemma during contraction and relaxation. Due to its large size, the dystrophin gene is vulnerable to a large variety of mutations that may result in total ablation, reduction, or truncation of the protein, leading to Duchenne and Becker muscular dystrophies. Efforts to characterize these dystrophin mutations and develop gene therapies that enable higher production of functional dystrophin in these patients depend on precise and reproducible assessments of dystrophin expression at the sarcolemma in patient muscle biopsies.
We have developed an automated and unbiased approach for detailed quantification of dystrophin expression by immunofluorescence in muscle tissue sections. This novel methodology uses images of whole-tissue sections stained for dystrophin and spectrin to measure the intensity of the dystrophin signal and the proportion of dystrophin-positive coverage at the sarcolemma of each muscle fiber. To ensure objectivity, the thresholds for dystrophin and spectrin are derived empirically from non-sarcolemmal signal intensity within each tissue section. In addition to metrics of dystrophin expression, this method is readily adaptable for measuring muscle fiber morphology, central nucleation based on a nuclear stain, and expression of many other tissue markers that localize at the sarcolemma or within the sarcoplasm.
In the present study, we demonstrate the sensitivity and reproducibility of this quantification approach with a variety of Duchenne and Becker muscular dystrophy biopsy samples, and quantify the wide range of dystrophin expression in the context of different dystrophin mutations in comparison to healthy muscle tissue. As efforts to restore dystrophin expression in dystrophic muscle bring more potential therapies into clinical trials, this methodology represents a valuable and accessible tool for efficient and precise analysis of muscle markers that reflect the efficacy of these treatments.