Background
Duchenne muscular dystrophy (DMD) is a progressive, X-linked disease, caused by pathogenic variants in the dystrophin gene leading to loss of functional dystrophin protein. Muscle pathology is characterized by myonecrosis and impaired regeneration leading to progressive endomysial fibrosis and fatty replacement. Because the regenerative process is closely linked to the magnitude of muscle damage, accurately measuring the capacity for regeneration remains a significant challenge.
Methods
We developed a method by which regeneration and myonecrosis in human DMD muscle biopsies can be accurately measured. In addition to standard morphometric analyses, regenerating myofibers were identified by immunostaining for the expression of embryonic myosin heavy chain (eMHC) and necrotic fibers were enumerated by immunofluorescent detection of myofibers positive for immunoglobulin G (IgG). The Regenerative Index (RI) was calculated as regeneration (eMHC+ fibers) divided by myonecrosis (IgG+ fibers). Muscle biopsies obtained at ages 7-11 years from 16 boys with DMD and 8 no diagnostic abnormality controls were analyzed and the RI was calculated.
Results
A trend toward an increasing minimal Feret’s diameter, cross-sectional area and circularity was observed with increasing age in DMD boys, with circularity showing the strongest trend. Furthermore, compared to DMD boys 9-11years, those aged 7-8years had significantly reduced muscle fiber circularity. Pax7+ cells (satellite cells) were significantly elevated in DMD boys compared to healthy boys of similar ages, without any observation of age-related changes in Pax7+ cell counts. Notably, the Regenerative Index in DMD boys exhibited a pronounced decline between 7-11 years of age; a significant inverse correlation between RI and age was observed.
Conclusions
The use of eMHC and IgG immunostaining to calculate the RI is a method by which muscle fiber regeneration can be quantified in muscle biopsies. Our studies show significant negative correlation between RI and age in boys from 7-11 years of age.