cobra-cardiotoxin-proteins and Muscular-Dystrophies

Muscular dystrophy (MD) is a disease characterized by skeletal muscle necrosis and the progressive accumulation of fibrotic tissue. While transforming growth factor (TGF)-β has emerged as central effector of MD and fibrotic disease, the cell types in diseased muscle that underlie TGFβ-dependent pathology have not been segregated. Here, we generated transgenic mice with myofiber-specific inhibition of TGFβ signaling owing to expression of a TGFβ type II receptor dominant-negative (dnTGFβRII) truncation mutant. Expression of dnTGFβRII in myofibers mitigated the dystrophic phenotype observed in δ-sarcoglycan-null (Sgcd(-/-)) mice through a mechanism involving reduced myofiber membrane fragility. The dnTGFβRII transgene also reduced muscle injury and improved muscle regeneration after cardiotoxin injury, as well as increased satellite cell numbers and activity. An unbiased global expression analysis revealed a number of potential mechanisms for dnTGFβRII-mediated protection, one of which was induction of the antioxidant protein metallothionein (Mt). Indeed, TGFβ directly inhibited Mt gene expression in vitro, the dnTGFβRII transgene conferred protection against reactive oxygen species accumulation in dystrophic muscle and treatment with Mt mimetics protected skeletal muscle upon injury in vivo and improved the membrane stability of dystrophic myofibers. Hence, our results show that the myofibers are central mediators of the deleterious effects associated with TGFβ signaling in MD.

Topics: Animals; Cell Membrane; Cobra Cardiotoxin Proteins; Crotoxin; Disease Models, Animal; Drug Combinations; Gene Expression Profiling; Gene Expression Regulation; Humans; Metallothionein; Mice; Mice, Transgenic; Muscular Dystrophies; Mutation; Myofibrils; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sarcoglycans; Satellite Cells, Skeletal Muscle; Signal Transduction; Transforming Growth Factor beta; Transgenes

Muscle progenitor cells (satellite cells) function in the maintenance and repair of adult skeletal muscle. Side population (SP) cells are enriched in repopulating activity and also reside in adult skeletal muscle. In this study, we observed that Abcg2 is a determinant of the SP cell phenotype. Using reverse transcription polymerase chain reaction and immunohistochemical techniques, we localized Abcg2-expressing cells in the interstitium and in close approximation to the vasculature of adult skeletal muscle. Muscle SP cells are able to differentiate into myotubes and increase in number after cardiotoxin-induced muscle injury. Similar to myogenic progenitor cells, muscle SP cells express Foxk1 and are decreased in number in Foxk1 mutant skeletal muscle. Using emerging technologies, we examine the molecular signature of muscle SP cells from normal, injured, and Foxk1 mutant skeletal muscle to define common and distinct molecular programs. We propose that muscle SP cells are progenitor cells that participate in repair and regeneration of adult skeletal muscle.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Differentiation; Cell Separation; Cobra Cardiotoxin Proteins; Flow Cytometry; Forkhead Transcription Factors; Green Fluorescent Proteins; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Muscles; Muscular Dystrophies; Neoplasm Proteins; Nuclear Proteins; Nucleic Acid Hybridization; Phenotype; Regeneration; Reverse Transcriptase Polymerase Chain Reaction; RNA; Satellite Cells, Skeletal Muscle; Stem Cells; Time Factors; Transcription Factors; Transcription, Genetic

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.

Topics: Adenoviridae; Animals; Antigens; beta-Galactosidase; CD4 Antigens; Cobra Cardiotoxin Proteins; Cytoskeletal Proteins; Dystrophin; Genetic Vectors; Green Fluorescent Proteins; Immunosuppression Therapy; Luminescent Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscle, Skeletal; Muscular Dystrophies; Regeneration; Sarcolemma; Tacrolimus; Up-Regulation; Utrophin