Skeletal Muscle Fibrosis in the mdx/utrn+/- Mouse Validates Its Suitability as a Murine Model of Duchenne Muscular Dystrophy

Abstract

Various therapeutic approaches have been studied for the treatment of Duchenne muscular dystrophy (DMD), but none of these approaches have led to significant long-term effects in patients. One reason for this observed inefficacy may be the use of inappropriate animal models for the testing of therapeutic agents. The mdx mouse is the most widely used murine model of DMD, yet it does not model the fibrotic progression observed in patients. Other murine models of DMD are available that lack one or both alleles of utrophin, a functional analog of dystrophin. The aim of this study was to compare fibrosis and myofiber damage in the mdx, mdx/utrn+/- and double knockout (dko) mouse models. We used Masson’s trichrome stain and percentage of centrally-nucleated myofibers as indicators of fibrosis and myofiber regeneration, respectively, to assess disease progression in diaphragm and gastrocnemius muscles harvested from young and aged wild-type, mdx, mdx/utrn+/- and dko mice. Our results indicated that eight week-old gastrocnemius muscles of both mdx/utrn+/- and dko hind limb developed fibrosis whereas age-matched mdx gastrocnemius muscle did not (p = 0.002). The amount of collagen found in the mdx/utrn+/- diaphragm was significantly higher than that found in the corresponding diaphragm muscles of wild-type animals, but not of mdx animals (p = 0.0003). Aged mdx/utrn+/- mice developed fibrosis in both diaphragm and gastrocnemius muscles compared to wild-type controls (p = 0.003). Mdx diaphragm was fibrotic in aged mice as well (p = 0.0235), whereas the gastrocnemius muscle in these animals was not fibrotic. We did not measure a significant difference in collagen staining between wild-type and mdx gastrocnemius muscles. The results of this study support previous reports that the moderately-affected mdx/utrn+/- mouse is a better model of DMD, and we show here that this difference is apparent by 2 months of age.

MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin

“…We compared multiple paradigms to suppress skeletal muscle fibrosis in an animal model suitable to test and compare modes of treatment. The animal model used for proof of principle was the mdx/utrn +/-mouse, known for the extent of fibrosis found in the GAS muscle as early as 3 months of age (19). We constructed a potentially promising vector for translational studies using AAV containing miR-29c expressed from a miR-30 backbone in an EF1α intron driven by a CMV promoter.…”

“…An important part of the work to be presented in this report is the study of antifibrotic therapies in the haploinsufficient, mdx/utrn +/-mice (18,19). In addition to a complete absence of dystrophin, this model lacks one copy of utrophin (utrn), which is known to partially compensate for dystrophin loss (20).…”

“…In addition to a complete absence of dystrophin, this model lacks one copy of utrophin (utrn), which is known to partially compensate for dystrophin loss (20). Gutpell and colleagues showed that as early as 8 weeks of age, the gastrocnemius (GAS) muscle of mdx/ utrn +/-mice show significantly more fibrosis than mdx mice (19). In the current study, we validated these findings in GAS and found that fibrosis was accompanied by a reduction of miR-29c transcripts.…”

Exercise mitigates the effects of hyperhomocysteinemia on adverse skeletal muscle remodeling.

“…It has been well established that mdx mice, a model that represents muscular dystrophy in humans, display a decrease in skeletal muscle mass, myocyte cross sectional area and muscular strength, as well as elevated macrophage infiltration and muscle fibrosis (Bogdanovich et al, 2002;Desguerre et al, 2012;Gutpell, Hrinivich, & Hoffman, 2015;Nakatani et al, 2008). Gutpell et al demonstrate that 8 week old mdx/utrn +/-mice, which lack dystrophin and utrophin (another crucial protein in skeletal muscle structure), display extensive fibrosis in the gastrocnemius and diaphragm; while age matched mdx mice only displayed fibrosis in the diaphragm (Gutpell et al, 2015).…”

“…Gutpell et al demonstrate that 8 week old mdx/utrn +/-mice, which lack dystrophin and utrophin (another crucial protein in skeletal muscle structure), display extensive fibrosis in the gastrocnemius and diaphragm; while age matched mdx mice only displayed fibrosis in the diaphragm (Gutpell et al, 2015). Other studies suggest that myostatin inhibition significantly ameliorates muscular function in mdx mice, producing elevated muscle weight, contractile force and cross sectional area, as well as attenuating macrophage infiltration into the skeletal muscle (Bogdanovich et al, 2002;Nakatani et al, 2008).…”