PASSIVE STIFFNESS OF FIBROTIC SKELETAL MUSCLE IN MDX MICE RELATES TO COLLAGEN ARCHITECTURE

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 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
cross-linking 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 fibers with second harmonic
generation imaging revealed their relative abundance in stiff muscles. Collagen
fiber 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 mechanical function
of collagen fibers in muscle. This finding suggests that anti-fibrotic
strategies to enhance muscle function and excessive stiffness should target
large collagen fibers and their alignment rather than total collagen.