Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which resultsin dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmd mdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmd mdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmd mdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.

In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophindeficient zebrafish, mdx 5cv mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.

BackgroundDuchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the ANKYRIN1(ANK1) gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types.ResultsInhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor β, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers in vivo, as these mice had impaired muscle regeneration.ConclusionsThese studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology.

Skeletal muscle regeneration is a highly complex and regulated process that involves muscle precursor proliferation and differentiation and probably requires the participation of heparin binding growth factors such as FGFs, HGF and TGFβ. Heparan sulfate proteoglycans, key components of cell-surfaces and ECM, modulate growth factor activities and influence cell growth and differentiation. Their expression in forming muscle masses during development and in cell culture, suggest their participation in the regulation of myogenesis. In the present study, heparan sulfate proteoglycan expression in skeletal muscle regeneration induced by barium chloride injection was evaluated. Expression of muscle differentiation markers and neuromuscular junction (NMJ) components was characterized. Immunoblots with anti-Δ-heparan sulfate antibody showed that four major species - perlecan, glypican, syndecan-3 and syndecan-4 - were transiently up-regulated. The first three were detected at the surface or basement membranes of newly formed myotubes by specific indirect immunofluorescence. Syndecan-3, a satellite cell marker, showed the earliest and most significant increase. Experiments involving myoblast grafting into regenerating muscle showed that C2C12 cell clones, with inhibited syndecan-3 expression resulting from antisense transfection, presented a normal proliferation rate but an impaired capacity to fuse and form skeletal muscle fibers. These data constitute the first in vivo evidence suggesting the requirement of a specific heparan sulfate proteoglycan for successful skeletal muscle regeneration.

The onset and progression of skeletal muscle regeneration are controlled by a complex set of interactions between muscle precursor cells and their environment. Decorin is the main proteoglycan present in the extracellular matrix (ECM) of adult muscle while biglycan expression is lower, but both are increased in mdx mice dystrophic muscle. Both of these small leucine-rich proteoglycans (SLRPs) can bind other matrix proteins and to the three TGF-beta isoforms, acting as modulators of their biological activity. We evaluated biglycan and decorin expression in skeletal muscle during barium chloride-induced skeletal muscle regeneration in mice. A transient and dramatic up-regulation of biglycan was associated with newly formed myotubes, whereas decorin presented only minor variations. Studies both in vitro and in intact developing newborn mice showed that biglycan expression is initially high and then decreases during skeletal muscle differentiation and maturation. To further evaluate the role of biglycan during the regenerative process, skeletal muscle regeneration was studied in biglycan-null mice. Skeletal muscle maintains its regenerative capacity in the absence of biglycan, but a delay in regenerated fiber growth and a decreased expression of embryonic myosin were observed despite to normal expression of MyoD and myogenin. Transient up-regulation of decorin during muscle regeneration in these mice may possibly obscure further roles of SLRPs in this process.

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BackgroundDuchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-β) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy.Methodsmdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-β signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice.Resultsmdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-β signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells.ConclusionsThese results suggest that andrographolide could be used to improve quality of life in individuals with DMD.