Inhibition of muscle fibrosis and improvement of muscle histopathology in dysferlin knock-out mice treated with halofuginone

Summary. Absence of, or loss-of-function mutations in the dysferlin gene (dysf) result in dysferlinopathy, characterized by increased muscle inflammation, collagen deposition and deterioration in muscle function. We evaluated halofuginone efficacy in improving muscle histopathology in mice with deleted dysf transmembrane domain. Quadriceps sublumbar and longissimus muscles of 9-month-old dysf-/- mice treated with halofuginone for 4 months exhibited a reduction in centrally-nucleated myofibers, inflammatory infiltrates and collagen content. Late onset of dysferlinopathy makes it ideal for evaluating the efficacy of early treatments on late outcome. The dysf-/- mice were treated with halofuginone for 3 to 4 months starting at 1, 5 or 9 months of age, and quadricep muscle histopathology was evaluated at 12 months. Collagen content and number of centrally nucleated myofibers decreased after early halofuginone treatment, administered when myofibers with central nuclei and inflammatory infiltrates are evident, but there was almost no fibrosis. When administered at the beginning of fibrosis it resulted in a further decrease in the number of centrally-nucleated myofibers with no additional decrease in collagen levels. Cardiac fibrosis was almost completely abolished following early halofuginone treatment. Halofuginone inhibited Smad3 phosphorylation and its translocation to the nucleus and increased the activity of matrix metalloproteinases 9 and 2 responsible for resolution of pre-existing collagen. Macrophage and myofibroblast invasion into the dystrophic muscle at the site of myofibers with central nuclei was inhibited by halofuginone. These results suggest that early halofuginone treatment can prevent the late outcome of dysferlinopathy and can cause resolution of the established fibrosis when administered at later stages.