piperidines and Muscular-Dystrophies--Limb-Girdle

Dysferlinopathies are a non-lethal group of late-onset muscular dystrophies. Here, we evaluated the fusion ability of primary myoblasts from young dysf

Topics: Animals; Disease Models, Animal; Dysferlin; Fibrosis; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Piperidines; Protein Synthesis Inhibitors; Quinazolinones

In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.

Topics: Animals; Disease Models, Animal; Dysferlin; Humans; Mice; Mice, Knockout; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Myoblasts; Phosphatidylinositol 3-Kinases; Piperidines; Quinazolinones; Synaptotagmins

Halofuginone is a leading agent in preventing fibrosis and inflammation in various muscular dystrophies. We hypothesized that in addition to these actions, halofuginone directly promotes the cell-cycle events of satellite cells in the mdx and dysf(-/-) mouse models of early-onset Duchenne muscular dystrophy and late-onset dysferlinopathy, respectively. In both models, addition of halofuginone to freshly prepared single gastrocnemius myofibers derived from 6-week-old mice increased BrdU incorporation at as early as 18h of incubation, as well as phospho-histone H3 (PHH3) and MyoD protein expression in the attached satellite cells, while having no apparent effect on myofibers derived from wild-type mice. BrdU incorporation was abolished by an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated protein kinase, suggesting involvement of this pathway in mediating halofuginone's effects on cell-cycle events. In cultures of myofibers and myoblasts isolated from dysf(-/-) mice, halofuginone reduced Bax and induced Bcl2 expression levels and induced Akt phosphorylation in a time-dependent manner. Addition of an inhibitor of the phosphinositide-3-kinase/Akt pathway reversed the halofuginone-induced cell survival, suggesting this pathway's involvement in mediating halofuginone's effects on survival. Thus, in addition to its known role in inhibiting fibrosis and inflammation, halofuginone plays a direct role in satellite cell activity and survival in muscular dystrophies, regardless of the mutation. These actions are of the utmost importance for improving muscle pathology and function in muscular dystrophies.

Topics: Animals; Apoptosis; Cell Cycle; Cell Survival; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Muscle Fibers, Skeletal; Muscular Dystrophies, Limb-Girdle; Muscular Dystrophy, Duchenne; Phosphatidylinositol 3-Kinases; Piperidines; Proto-Oncogene Proteins c-akt; Quinazolinones; Satellite Cells, Skeletal Muscle; Signal Transduction

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.

Topics: Animals; Collagen; Disease Models, Animal; Dysferlin; Fibrosis; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Phosphorylation; Piperidines; Protein Synthesis Inhibitors; Quinazolinones; Smad3 Protein; Treatment Outcome