Gene-mediated restoration of normal myofiber elasticity in dystrophic muscles

Abstract

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.

Figure 1

Principles of AFM measurements of muscle resistance to deformation. (a) Scheme of the experimental AFM setup. The tendons of muscle sections glued to a glass coverslip, immersed in DMEM medium, and placed in the “liquid cell” setup of the AFM scanner. (b) Principles of the quantification of muscle elastic properties. Only curves recorded during the approach of the AFM probe to the muscle surface are shown. The subtraction of two curves obtained from one recorded on the glass substrate (showing negligible deformation) from that recorded on the muscle explant, results in the force-versus-indentation dependence used for the determination of the Young's modulus value describing in a quantitative manner the muscle's ability to deform elastically. AFM, atomic force microscopy.

Figure 2

Effect of cytochalasin D on muscle stiffness. Normalized Young's modulus value distributions performed on thick longitudinal sections of (a) wild-type (N_TOT = 126) or (b) dystrophic (N_TOT = 150) mice muscle (black bars) are shown in comparison with those obtained from an adjacent section of the same muscle treated with cytochalasin D (gray bars, N_TOT = 192 and N_TOT = 168, respectively). The distributions were fitted with Gaussian curves, where the centers of distribution denote the Young's modulus mean value.

Figure 3

Localization of muscle fibers expressing utrophin and effects on muscle function. Fluorescence microscopy analysis of mouse TA muscle after electrotransfer of utrophin and GFP expression vector plasmids was performed on (a) a whole muscle or on (b) a transversal muscle section. Muscle regions showing the brightest area were sectioned longitudinally and assayed by AFM as described in the Materials and Methods section. The Young's modulus value distribution of control male mdx muscles are shown in c (N_TOT = 252, black columns), whereas gray columns illustrate the distribution of utrophin-expressing muscle sections (N_TOT = 186). The latter distribution shows two contributions: unaffected values corresponding to those of dystrophic muscle fibers (31% of recordings) and values elevated after electrotransfer (69%). Phasic responses were recorded under isometric conditions from electrically stimulated TA muscle of dystrophic mice after in vivo electroporation of plasmids encoding GFP alone (MDX-GFP) or GFP and utrophin (MDX-Utro), as displayed in d. TA muscles from normal mice after electroporation of the GFP plasmid alone (C57-GFP) were used for comparison. Traces represent average from six to nine muscles per group after normalization to the muscle cross-sectional surface. For clarity, only one error bar every 10 ms is shown. Note that the specific force of GFP-expressing dystrophic TA is significantly lower than that of its normal counterpart, and that the introduction of the utrophin plasmid into the dystrophic TA muscle fully restored force. AFM, atomic force microscopy.

Figure 4

Time course of utrophin-mediated mdx muscle improved stiffness. The TA muscles of male mdx mice were explanted and assayed by AFM 1 week after the electrotransfer of either (a) the GFP expression vector alone (N_TOT = 480), owr of both the GFP and (b) the utrophin-expression vectors (N_TOT = 705). Muscles treated as for the a and b were assayed 1 month (c, N_TOT = 443 and d, N_TOT = 680) and 2 months (e, N_TOT = 402, and f, N_TOT = 690) after vector electrotransfer. AFM, atomic force microscopy.

Figure 5

Effect of utrophin and dystrophin expression on muscle function as probed by AFM. The TA muscles of male mdx mice were explanted and assayed by AFM either (a, c) 1 month after electrotransfer of the utrophin-expression plasmid alone (c, N_TOT = 364) or after mock electrotransfer performed in the absence of expression vector (a, N_TOT = 504), or 3 months after transduction with an small nuclear RNA (snRNA)-AAV viral vector–mediating dystrophin RNA exon skipping (d, N_TOT = 374) or with the nontransduced contralateral controls (b, N_TOT = 290). AFM, atomic force microscopy.