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Regenerative Index reveals declining muscle regeneration in paediatric patients with Duchenne muscular dystrophy
Preprint   Open access

Regenerative Index reveals declining muscle regeneration in paediatric patients with Duchenne muscular dystrophy

Johnathan K Smid, Charis A McPherson, Jacob G Monast, Shanti S S Rayagiri, Steven A Moore and Michael A Rudnicki
bioRxiv
Cold Spring Harbor Laboratory
01/05/2026
DOI: 10.64898/2026.01.05.697715
PMCID: PMC12803257
PMID: 41542555
url
https://doi.org/10.64898/2026.01.05.697715View
Preprint (Author's original)This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

Duchenne muscular dystrophy (DMD) is a devastating disease manifested in skeletal muscle by repetitious myonecrosis and regeneration. Because the regenerative process is closely linked to the cumulative severity of muscle damage, which is variably distributed within and between muscle groups, accurately quantifying muscle regeneration has remained a significant challenge. Myofibers are delineated by immunostaining for laminin, and subsequent image analysis employed to generate a masked outline precisely within each myofiber boundary. Morphometric parameters including minimal Feret's diameter, cross-sectional area, and circularity were measured for each myofiber. In addition, the number of Pax7-expressing satellite cells were quantified. To evaluate regenerative activity, newly formed myofibers were identified by immunostaining for expression of embryonic myosin heavy chain (eMHC). Necrotic myofibers were enumerated by immunofluorescent detection of immunoglobulin G (IgG) infiltration. The Regenerative Index (RI) was calculated as the number of regenerating (eMHC ) myofibers divided by the number of necrotic (IgG ) myofibers. Determination of RI was performed on muscle biopsies from 10 boys with DMD and 3 non-DMD controls of similar age. 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 7- to 8-years old, the boys 9- to 11-years old had significantly increased myofiber circularity. Pax7-expressing cells were significantly elevated in DMD boys compared to control boys of similar ages, without any observation of age-related changes. Notably, the Regenerative Index in DMD boys exhibited a pronounced decline between 7-11 years of age, and a significant inverse correlation between RI and age was observed. Using eMHC and IgG immunostaining to calculate RI accurately assesses regeneration despite the variation in histopathologic severity between biopsies. This methodology demonstrated a significant negative correlation between RI and age of DMD boys from 7 to 11 years of age.

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