Passive stiffness of fibrotic skeletal muscle in mdx mice relates to collagen architecture

Abstract

Key points The amount of fibrotic material in dystrophic mouse muscles relates to contractile function, but not passive function. Collagen fibres in skeletal muscle are associated with increased passive muscle stiffness in fibrotic muscles. The alignment of collagen is independently associated with passive stiffness in dystrophic skeletal muscles. These outcomes demonstrate that collagen architecture rather than collagen content should be a target of anti‐fibrotic therapies to treat muscle stiffness. Abstract Fibrosis is prominent in many skeletal muscle pathologies including dystrophies, neurological disorders, cachexia, chronic kidney disease, sarcopenia and metabolic disorders. Fibrosis in muscle is associated with decreased contractile forces and increased passive stiffness that limits joint mobility leading to contractures. However, the assumption that more fibrotic material is directly related to decreased function has not held true. Here we utilize novel measurement of extracellular matrix (ECM) and collagen architecture to relate ECM form to muscle function. We used mdx mice, a model for Duchenne muscular dystrophy that becomes fibrotic, and wildtype mice. In this model, extensor digitorum longus (EDL) muscle was significantly stiffer, but with similar total collagen, while the soleus muscle did not change stiffness, but increased collagen. The stiffness of the EDL was associated with increased collagen crosslinking as determined by collagen solubility. Measurement of ECM alignment using polarized light microscopy showed a robust relationship between stiffness and alignment for wildtype muscle that broke down in mdx muscles. Direct visualization of large collagen fibres with second harmonic generation imaging revealed their relative abundance in stiff muscles. Collagen fibre alignment was linked to stiffness across all muscles investigated and the most significant factor in a multiple linear regression‐based model of muscle stiffness from ECM parameters. This work establishes novel characteristics of skeletal muscle ECM architecture and provides evidence for a mechanical function of collagen fibres in muscle. This finding suggests that anti‐fibrotic strategies to enhance muscle function and excessive stiffness should target large collagen fibres and their alignment rather than total collagen.

Skeletal Effects of Bone-Targeted TGFbeta Inhibition in a Mouse Model of Duchenne Muscular Dystrophy

“…It is also possible that serum CTX levels are influenced by the breakdown of muscle collagen. Muscle collagen is increased and is structurally abnormal in mdx mice [ 27 ]; therefore, it might make a bigger contribution to the circulating pool of CTX in mdx than in WT mice.…”

Diaphragm muscle fibrosis involves changes in collagen organization with mechanical implications in Duchenne Muscular Dystrophy

“…In prior studies, collagen organization is related to passive properties of skeletal muscle measured by mechanical testing of intact muscle. 14,15 The contribution of the ECM to the passive properties of skeletal muscle is shown by indirect measurements, comparing properties of single muscle fibers and muscle fiber bundles with intact ECM, [24][25][26] and direct measurements of properties of decellularized muscle fiber bundles. 27 While it is difficult to isolate the influence of organizational parameters on ECM properties with traditional mechanical testing, finite-element (FE) models allow us to isolate the impact of specific structural variations on mechanical properties.…”

“…14 Mdx lower limb muscle shows an increase in collagen fiber alignment. 15 While collagen fiber amount does not predict tissue stiffness, collagen fiber alignment is reported to be a significant predictor of passive lower limb muscle stiffness. 15 However, the mdx lower limb muscle does not mimic the severity of the human phenotype nearly as well as the diaphragm does.…”

“…14 Mdx lower limb muscle shows an increase in collagen fiber alignment. 15 While collagen fiber amount does not predict tissue stiffness, collagen fiber alignment is reported to be a significant predictor of passive . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.…”

“…The copyright holder for this preprint (which this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.438870 doi: bioRxiv preprint 4 lower limb muscle stiffness. 15 However, the mdx lower limb muscle does not mimic the severity of the human phenotype nearly as well as the diaphragm does. Similar to the human condition, mdx mice exhibit impairment in respiratory function 16,17 , decrease in diaphragm muscle fiber cross-sectional area, and increased diaphragm muscle fibrosis.…”