Altered muscle niche contributes to myogenic deficit in the D2-mdx model of severe DMD

Abstract

Lack of dystrophin expression is the underlying genetic basis for Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2-mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2-mdx muscles is associated with an enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports the excessive accumulation of fibroadipogenic progenitors (FAPs), leading to increased fibrosis. Unexpectedly, the extent of damage and degeneration in juvenile D2-mdx muscle is significantly reduced in adults, and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance regenerative myogenesis in the adult D2-mdx muscle, reaching levels comparable to the milder B10-mdx model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with juvenile D2-mdx FAPs reduces their fusion efficacy. Wild-type juvenile D2 mice also manifest regenerative myogenic deficit and glucocorticoid treatment improves their muscle regeneration. Our findings indicate that aberrant stromal cell responses contribute to poor regenerative myogenesis and greater muscle degeneration in juvenile D2-mdx muscles and reversal of this reduces pathology in adult D2-mdx muscle, identifying these responses as a potential therapeutic target for the treatment of DMD.

Fig. 1. Histopathological assessment of disease in D2-mdx and B10-mdx models.

A, B Masson’s trichrome staining and quantification of percent fibrotic tissue area performed on triceps harvested from juvenile (5.5 ± 1.5 wk) and adult (8.5 ± 1.5 mo) D2-mdx and B10-mdx mice. C, D. H&E staining and quantification of percent damaged muscle tissue area performed on triceps harvested from juvenile and adult D2-mdx and B10-mdx mice; damaged areas were characterized by the presence of interstitial mononuclear cells, damaged myofibers, and appearance of small-diameter centrally nucleated fibers (CNFs). E, F Alizarin red staining and quantification of percent calcified fiber area performed on triceps harvested from juvenile and adult D2-mdx and B10-mdx mice. Data represent mean ± SD from n = 6 mice per cohort. *p < 0.05, **p < 0.01 by Mann–Whitney test. Refer to Supplementary Fig. 1.

Fig. 2. Assessment of muscle regeneration in juvenile and adult mdx and WT mice.

A IF images from juvenile (5.5 ± 1.5 wk) and adult (6.5 ± 1 mo) triceps muscle sections from dystrophic mdx mice stained to identify muscle fibers (Laminin-α2) and CNFs (DAPI). Yellow arrowheads show CNFs. B Quantification of CNFs from dystrophic triceps expressed as a percentage of total muscle fibers. C Schematic showing the BrdU ‘myofiber birth dating’ strategy to label proliferating SCs in NTX-injured TA muscles in juvenile (5.5 ± 1.5 wk) and adult (10.5 ± 0.5 mo) WT mice by BrdU administration from 24 to 72 h post injury (green line). Mice were euthanized and tissues were harvested 6 d post-injury. D IF images and quantification of muscle sections from B10-WT and D2-WT TA muscles stained to identify muscle fibers BrdU⁺ CNFs and total CNFs; sections co-stained with Laminin-α2 and DAPI. White arrowheads show BrdU⁺ CNFs while yellow arrowheads show CNFs. E Quantification of CNFs (%) from NTX-injured TA muscles harvested from juvenile and adult B10-WT and D2-WT mice harvested 6 dpi. F Quantification of BrdU⁺ CNFs (%) from juvenile and adult B10-WT and D2-WT NTX-injured TA muscles. Data represent mean ± SD from n = 7–9 mice per cohort (B) or n = 6–12 NTX-injured TA muscles per cohort (E, F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Mann–Whitney test.

Fig. 3. Expression of genes indicative of myogenesis, ECM, and inflammation in juvenile and adult mouse muscles.

A–C Gene expression analysis of myogenic markers, MyoD, MyoG, and Pax7 in juvenile (5.5 ± 1.5 wk) and adult (8.5 ± 1.5 mo) D2-mdx and B10-mdx triceps. D Levels of active TGF-β protein assessed by ELISA from juvenile and adult D2-mdx and B10-mdx triceps. E, F Gene expression analysis of ECM remodeling markers, Postn and Spp1, in juvenile and adult D2-mdx and B10-mdx triceps. G–L Gene expression analysis of inflammatory genes associated with pro-inflammatory (Tnf-α, Il-1b, Il-6) and pro-regenerative (Arg1, Il-10, Cd163) macrophage phenotypes in juvenile and adult D2-mdx and B10-mdx triceps. Data represent mean ± SD from n = 5–6 mice per cohort. *p < 0.05, **p < 0.01 by Mann–Whitney test.

Fig. 4. Investigation of macrophage response to spontaneous injury in mdx muscles.

A Images showing juvenile (5.5 ± 1.5 wk) and adult (8.5 ± 1.5 mo) D2-mdx and B10-mdx triceps muscle cross-sections stained to mark F4/80, iNOS, and CD206 expressing macrophages. B, C. Quantification of F4/80⁺ area (%) per total cross-sectional area (B) and only in damaged areas (areas with abundant F4/80⁺ macrophage infiltration) within cross-sections (C) in juvenile and adult D2-mdx and B10-mdx triceps. D Total macrophages per unit area (mm²) within damaged regions of juvenile and adult D2-mdx and B10-mdx triceps cross-sections. E–G Quantification of the distribution of pro-inflammatory (iNOS⁺/F4/80⁺) (E), and pro-regenerative (CD206⁺/F4/80⁺) (F) macrophages, and the ratio of these macrophages (G) within damaged areas of triceps muscles from juvenile and adult D2-mdx and B10-mdx. Data represent mean ± SD from n = 6 mice per cohort. *p < 0.05, **p < 0.01 by Mann–Whitney test.

Fig. 5. Investigating dynamics of macrophage inflammatory response after acute injury of healthy muscle.

A, B F4/80 expression assessed 5 d or 8 d post-injury (dpi) in TA muscles of juvenile (5.5 ± 1.5 wk) and adult (10.5 mo) D2-WT and B10-WT mice. IF images of muscle sections stained to show the distribution of macrophages (F4/80) in adult B10-WT and D2-WT NTX-injured TA muscles after 5 d and 8 d post-injury; muscles co-stained with DAPI. C, D Quantification of F4/80⁺ area after NTX-injury assessed 5 dpi (C) and 8 dpi (D) in juvenile B10-WT and D2-WT. E, F Quantification of the distribution of pro-inflammatory (iNOS⁺, F4/80⁺) (E) and pro-regenerative (CD206⁺, F4/80⁺) (F) macrophages per damaged area (mm²) in B10-WT and D2-WT NTX-injured TA muscles. Data represent mean ± SD from n = 4 mice per cohort. *p < 0.05, **p < 0.01 by Mann–Whitney test.

Fig. 6. Investigating regenerative response after acute injury of healthy muscle.

A eMHC expression assessed 5 d post-injury (dpi) in TA muscles of juvenile (5.5 ± 1.5 wk) and adult (10.5 mo) D2-WT and B10-WT mice. IF images show the distribution and size of regenerated, eMHC⁺ myofibers (green), co-stained with Laminin-α2 (red) and DAPI. B Quantification of eMHC⁺ myofibers per mm² of damaged tissue present 5 dpi in juvenile and adult D2-WT and B10-WT TA muscles. Data represent mean ± SD from n = 4 mice per cohort. *p < 0.05 by Mann–Whitney test. C Relative frequency plot of eMHC⁺ myofiber area (reported in µm²) at 5 dpi in juvenile and adult D2-WT and B10-WT TA muscles, where fiber area was quantified for 1119 fibers (B10-WT juvenile), 496 fibers (D2-WT juvenile), 829 fibers (B10-WT adult), and 866 fibers (D2-WT adult).

Fig. 7. Analysis of juvenile D2-mdx FAPs on satellite cell function.

A, B IF images and quantification of PDGFRα⁺ FAPs in juvenile and adult D2-mdx and B10-mdx triceps immunostained for (Laminin-α2), PDGFRα (green), and interstitial nuclei (DAPI). C Schematic illustrating the approach for co-culture experiments to evaluate the effect of FAPs on functional properties of healthy SCs. SCs from uninjured B6-WT mice were co-cultured with FAPs isolated from B6-WT mice at 4 d post cardiotoxin (CTX) injury (WT CTX, 7 wk), juvenile D2-mdx (7 wk) mice, or adult D2-mdx (5.5 mo) mice. D Quantification of the SC proliferation monitored by EdU incorporation. E Quantification of the SC differentiation monitored by Myogenin expression. F, G Quantification of the SC fusion index (F) monitored by staining for Desmin (DES) expression with corresponding IF images (G) (Desmin– red, Hoechst—blue). Data represent mean ± SD from n = 6–12 mice per cohort (A, B) or n = 4–6 individual replicates carried out for each functional measure (C–G). *p < 0.05, **p < 0.01, ***p < 0.001, by Mann–Whitney test.

Fig. 8. Analysis of regenerative capacity following acute injury and glucocorticoid treatment.

A Schematic showing details for deflazacort treatment regimen performed in juvenile (4–5 wk) D2-WT mice following acute NTX injury. For additional details refer to methods. B–E Gene expression analysis of inflammatory genes associated with pro-inflammatory (Nos2, Il-1b, Il-6) and pro-regenerative (CD163) macrophage phenotypes in juvenile and adult D2-WT and B10-WT TA muscles after acute injury and deflazacort treatment compared to saline controls. F, G Gene expression analysis of ECM markers, Fn1 and Col1a1, in juvenile and adult D2-WT and B10-WT TA muscles after acute injury and deflazacort treatment compared to saline controls. H IF images showing BrdU⁺ CNFs (Red) as indicated by white arrowheads after acute injury and deflazacort treatment compared to saline controls; sections co-stained with Laminin-α2 (green) and DAPI (blue). I Quantification of BrdU⁺ CNFs (per damaged area) after acute injury and deflazacort treatment compared to saline controls. J, K % F4/80 (J) and PDGFRα (K) area reported within damaged muscle regions after acute injury and deflazacort treatment compared to saline controls. Data represent mean ± SD from n = 5–6 mice per cohort. **p < 0.01, ***p < 0.001 by Mann–Whitney test.