Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, leading to lack of dystrophin. Chronic muscle damage eventually leads to histological alterations in skeletal muscles. The identification of genes and cell types driving tissue remodeling is a key step to develop effective therapies. Here we use spatial transcriptomics in two DMD mouse models differing in disease severity to identify gene expression signatures underlying skeletal muscle pathologies and directly link this to the muscle histology. Deconvolution analysis allowed the identification of cell types contributing to histological alterations. We show how the expression of specific genes is enriched in areas of muscle regeneration (Myl4, Sparc, Hspg2), fibrosis (Vim, Fn1, Thbs4) and calcification (Bgn, Ctsk, Spp1). Finally, our analysis of differentiation dynamics in the severely affected D2-mdx muscle shows a subset of the muscle fibers are predicted to become affected in its future state. Genes associated with tissue remodeling could enable to design new diagnostic and therapeutic strategies for DMD.
Duchenne muscular dystrophy is caused by mutations in the DMD gene, leading to lack of dystrophin. Chronic muscle damage eventually leads to histological alterations in skeletal muscles. The identification of genes and cell types driving tissue remodeling is a key step to developing effective therapies. Here we use spatial transcriptomics in two Duchenne muscular dystrophy mouse models differing in disease severity to identify gene expression signatures underlying skeletal muscle pathology and to directly link gene expression to muscle histology. We perform deconvolution analysis to identify cell types contributing to histological alterations. We show increased expression of specific genes in areas of muscle regeneration (Myl4, Sparc, Hspg2), fibrosis (Vim, Fn1, Thbs4) and calcification (Bgn, Ctsk, Spp1). These findings are confirmed by smFISH. Finally, we use differentiation dynamic analysis in the D2-mdx muscle to identify muscle fibers in the present state that are predicted to become affected in the future state.
Background Becker muscular dystrophy (BMD) is an X‐linked disorder characterized by slow, progressive muscle damage and muscle weakness. Hallmarks include fibre‐size variation and replacement of skeletal muscle with fibrous and adipose tissues, after repeated cycles of regeneration. Muscle histology can detect these features, but the required biopsies are invasive, are difficult to repeat and capture only small muscle volumes. Diffusion‐tensor magnetic resonance imaging (DT‐MRI) is a potential non‐invasive alternative that can calculate muscle fibre diameters when applied with the novel random permeable barrier model (RPBM). In this study, we assessed muscle fibre diameters using DT‐MRI in BMD patients and healthy controls and compared these with histology. Methods We included 13 BMD patients and 9 age‐matched controls, who underwent water‐fat MRI and DT‐MRI at multiple diffusion times, allowing RPBM parameter estimation in the lower leg muscles. Tibialis anterior muscle biopsies were taken from the contralateral leg in 6 BMD patients who underwent DT‐MRI and from an additional 32 BMD patients and 15 healthy controls. Laminin and Sirius‐red stainings were performed to evaluate muscle fibre morphology and fibrosis. Twelve ambulant patients from the MRI cohort underwent the North Star ambulatory assessment, and 6‐min walk, rise‐from‐floor and 10‐m run/walk functional tests. Results RPBM fibre diameter was significantly larger in BMD patients (P = 0.015): mean (SD) = 68.0 (25.3) μm versus 59.4 (19.2) μm in controls. Inter‐muscle differences were also observed (P ≤ 0.002). Both inter‐ and intra‐individual RPBM fibre diameter variability were similar between groups. Laminin staining agreed with the RPBM, showing larger median fibre diameters in patients than in controls: 72.5 (7.9) versus 63.2 (6.9) μm, P = 0.006. However, despite showing similar inter‐individual variation, patients showed more intra‐individual fibre diameter variability than controls—mean variance (SD) = 34.2 (7.9) versus 21.4 (6.9) μm, P < 0.001—and larger fibrosis areas: median (interquartile range) = 21.7 (5.6)% versus 14.9 (3.4)%, P < 0.001. Despite good overall agreement of RPBM and laminin fibre diameters, they were not associated in patients who underwent DT‐MRI and muscle biopsy, perhaps due to lack of colocalization of DT‐MRI with biopsy samples. Conclusions DT‐MRI RPBM metrics agree with histology and can quantify changes in muscle fibre size that are associated with regeneration without the need for biopsies. They therefore show promise as imaging biomarkers for muscular dystrophies.
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