Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study

Abstract

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.

Keywords: collagen turnover; fibrosis; matrix metalloproteinases; muscular dystrophy.

Figure 1

Representative Sirius red stained sections showing collagen content in quadriceps (QD), tibialis anterior (TA), gastrocnemius (GC), and diaphragm (DF) of young (1-month-old) and adult (5 months-old) mice. Original magnification: 20×.

Figure 2

Bar graphs showing the effect of age on collagen content in sections of QD, TA, GC, DF muscles of wt and mdx mice at different age stained with Sirius red. Collagen content was assessed in the endomysium and is expressed as %. Data are mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.

Figure 3

Correlation between muscle (DF) collagen content and physical performances of 5-month-old mdx mice. Collagen percentage was correlated to: WBT 5 and WBT 10 (A,B); distance ran and time to exhaustion in the treadmill exhaustion test (C,D). Pearson correlation coefficients are displayed.

Figure 4

Representative sections of DF muscles stained with Herovici’s staining. The more mature and cross-linked collagen stains purple, whilst the less mature and young collagen (including reticular collagen fibres) stains blue. Original magnification: 20×.

Figure 5

(A–D) Bar graphs showing the percentage of centronucleated muscle fibres in the considered muscles from young and adult wt and mdx mice. Data are mean ± SD. (E–H) Correlations showing the relationship between the percentage of centronucleated skeletal muscle fibres and the collagen content in the same muscles. ** p < 0.01; *** p < 0.005.

Figure 6

Representative micrographs of skeletal muscle sections stained with Alcian blue containing different MgCl₂ concentrations to specifically stain and differentiate total (0.025 M), sulphated (0.3 M), or highly sulphated (0.65 M) GAG/PG content. Original magnification: 20×.

Figure 7

Representative bar graphs showing the score indicating GAG/PG abundance in the different muscles of young and adult wt and mdx mice after staining with Alcian blue containing 0.025 MgCl₂ (A), 0.3 MgCl₂ (B), and 0.65 MgCl₂ (C). Data are mean ± SD. * p < 0.05. (D) Representative micrograph showing BYG immunoreactivity localisation close and around skeletal myofibres. Original magnification: 40×.

Figure 8

Representative bar graphs showing COL-I (A) and COL-III (B) levels in the cell culture supernatants of cultured QD muscle fibroblasts. Gene expression for LH2b (C) and LOX (D) were quantified by real-time PCR. Data are mean ± SD. * p < 0.05; ^# p < 0.05 for t-test.

Figure 9

Representative bar graphs showing MMP-1 (A) and MMP-2 (B) levels in cell culture supernatants of cultured QD muscle fibroblasts analysed by Slot Blot and SDS-zymography, respectively. A representative zymogram is shown in panel (C). Gene expression for TIMP-1 (D) and TIMP-2 (E) were quantified by real-time PCR. Data are mean ± SD. * p < 0.05.