Myofibrillar Lattice Remodeling Is a Structural Cytoskeletal Predictor of Diaphragm Muscle Weakness in a Fibrotic mdx (mdx Cmah-/-) Model

Abstract

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of 'structural weakness', intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah-/- diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah-/- diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah-/- single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah-/- genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.

Keywords: cosine angle sum; multiphoton microscopy; muscular dystrophy; skeletal muscle; verniers density.

Figure 1

Extensive fibrosis in H&E muscle cross-sections of EDL and diaphragm muscle from mdx Cmah${}^{-/-}$ mice. Increased fibrosis and scar tissue is observed in mdx Cmah${}^{-/-}$ diaphragm, compared to mdx Cmah${}^{-/-}$ EDL muscle (Scale bar 50 μm).

Figure 2

Marked fibrosis in mdx Cmah${}^{-/-}$ diaphragm and EDL muscle sections assessed through label-free SHG morphometry. (A–C) Imaged and quantified collagen area per muscle tissue area. SHG signals are collected in forward scattered direction and collect both residuals of myosin-II and collagen (A1–A4). The myosin area was extracted via the measured autofluorescence in backward scattered direction and subtracted from the SHG signal resulting in quantifiable collagen areas (B1–B4). mdx Cmah${}^{-/-}$ diaphragms tend to be highly fibrotic (C) more so than mdx Cmah${}^{-/-}$ EDL (D). Combined results demonstrate a highly significant difference in collagen content per muscle area (E,F). Note the different ordinate scales in (C–F) (scale bar: 20 $\mathsf{\mu }$m, *** p < 0.001, (m/n)) relate to animals/cryosections.

Figure 3

Passive visco-elastic stiffness is markedly increased in mdx Cmah${}^{-/-}$ EDL but not in diaphragm muscles. Passive stresses (i.e., specific forces) of whole EDL (top panels, A,B) and diaphragm strips (central panels, C,D) from age-matched wt (n = 3) and mdx Cmah${}^{-/-}$ mice (n = 6). EDL muscles from mdx Cmah${}^{-/-}$ mice present with significantly higher stress values for the same strain. This results in increased dynamic and elastic stiffness in the EDL muscles but not the diaphragm muscles (E,F). Data are means ± SD. *, p < 0.1, **, p < 0.01, ***, p < 0.001 for comparisons between wt and mdx Cmah${}^{-/-}$ muscles. ns, not significant.

Figure 4

Specific active tension is markedly diminished in mdx Cmah${}^{-/-}$ diaphragm but not in EDL muscles. Isometric contractile specific tension of EDL muscle (top panels, A,B) and diaphragm (central panels, C,D) from age-matched wt (n = 3) and mdx Cmah${}^{-/-}$ mice (n = 6). Examples of specific tension traces are shown for a single twitch and for tetanic stimulation. Bottom panel (E) shows specific tension for tetanic stimulation (mean ± SD) (***: p < 0.001).

Figure 5

An improved algorithm allows to quantify structural remodeling in enhanced fibrosis-dystrophic mdx Cmah${}^{-/-}$ EDL and diaphragm muscle. (A) CAS is defined by the integrated local sarcomere orientation with respect to the main fiber axis, pointing towards angular deviations from the perfectly parallel aligned pattern for values below 1. VD provides the number of lattice shifts between neighboring myofibrils (‘Y’ patterns) normalized to the imaged fiber area. (B) Due to extensive fibrosis in mdx Cmah${}^{-/-}$ muscle, an improved algorithm allows further preprocessing steps to detect and extract Verniers and CAS. (C,E) High CAS and low VD as an indicator for ordered myofibrillar ultrastructure, as seen in a healthy myofiber taken from wt diaphragm and EDL, respectively. (D) Example of a highly fibrotic and remodeled mdx Cmah${}^{-/-}$ diaphragm single muscle fiber showing numerous myofibrillar lattice shifts and a high variation in sarcomere orientation, leading to a low CAS and a high VD (as indicated). A substantial amount of extracellular collagen streaks can be seen around the fiber. (F) Example of an mdx Cmah${}^{-/-}$ EDL myofiber with orderly orientation despite a marked myofibrillar lattice shift, reflected by a high CAS and a high VD value. Units: CAS (-), VD ($\frac{\#}{100\mathsf{\mu }{\mathrm{m}}^2}$).

Figure 6

Myofibrillar cytoarchitecture is severely compromised and disordered in single fibers from EDL and diaphragm from adult mdx Cmah${}^{-/-}$ over wt mice. Diaphragm and EDL muscle fibers show a highly significant shift toward decreased CAS (A,B) and increased VD values in mdx Cmah${}^{-/-}$ mice (C,D). Combined results are shown in (E,F). Abscissa numbers correspond to the individual label of each experiment (***: p < 0.001). (m/n) relates to animals/muscle single fibers.