Abstract
Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.
Highlights
Duchenne muscular dystrophy (DMD) is a lethal inherited muscle wasting disease caused by mutations in the dystrophin gene that disrupt the open reading frame, preventing production of a functional dystrophin protein [1]
DMD patient biopsies have significantly higher utrophin levels compared to Becker muscular dystrophy (BMD) patients Intensity quantification methods were used to quantify utrophin on mature muscle fibre sarcolemma in sections of quadriceps muscles (Table 1)
The tools that we have developed to quantify sarcolemmal utrophin, dystrophin-associated protein complex (DAPC) proteins and muscle regeneration in muscle biopsies have led to interesting observations correlating levels of utrophin and members of the DAPC and percentage of regenerating fibres to muscle function
Summary
Duchenne muscular dystrophy (DMD) is a lethal inherited muscle wasting disease caused by mutations in the dystrophin gene that disrupt the open reading frame, preventing production of a functional dystrophin protein [1]. Possible treatment strategies in late stage clinical development include the use of antisense oligonucleotides (AONs) to skip mutated dystrophin exons, allowing the production of a BMD-like dystrophin [4,5,6]. One drawback of this approach is that the AONs are exon-specific, so one AON is applicable only to a subset of patients carrying specific mutations. There is considerable interest in utrophin up-regulation as a possible therapeutic option for DMD, as this strategy may have potential for all DMD patients, regardless of their dystrophin mutation
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