ABSTRACT
Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.
Subject(s)
Cellular Microenvironment , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Animals , Cell Proliferation , Cells, Cultured , Dystrophin/metabolism , Extracellular Matrix/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Hypertrophy , Immunohistochemistry , Male , Mice, Inbred C57BL , Mice, Transgenic , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Myosin Heavy Chains/metabolism , Oligonucleotide Array Sequence Analysis , PAX7 Transcription Factor/genetics , PAX7 Transcription Factor/metabolism , Satellite Cells, Skeletal Muscle/cytology , Stress, Mechanical , Tamoxifen/pharmacology , Time Factors , Transcriptome/drug effects , Transcriptome/genetics , Weight-BearingABSTRACT
TNF promotes skeletal muscle weakness, in part, by depressing specific force of muscle fibers. This is a rapid, receptor-mediated response, in which TNF stimulates cellular oxidant production, causing myofilament dysfunction. The oxidants appear to include nitric oxide (NO); otherwise, the redox mechanisms that underlie this response remain undefined. The current study tested the hypotheses that 1) TNF signals via neuronal-type NO synthase (nNOS) to depress specific force, and 2) muscle-derived reactive oxygen species (ROS) are essential co-mediators of this response. Mouse diaphragm fiber bundles were studied using live cell assays. TNF exposure increased general oxidant activity (P < 0.05; 2',7'-dichlorodihydrofluorescein diacetate assay) and NO activity (P < 0.05; 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate assay) and depressed specific force across the full range of stimulus frequencies (1-300 Hz; P < 0.05). These responses were abolished by pretreatment with N(ω)-nitro-L-arginine methyl ester (L-NAME; a nonspecific inhibitor of NOS activity), confirming NO involvement. Genetic nNOS deficiency replicated L-NAME effects on TNF-treated muscle, diminishing NO activity (-80%; P < 0.05) and preventing the decrement in specific force (P < 0.05). Comparable protection was achieved by selective depletion of muscle-derived ROS. Pretreatment with either SOD (degrades superoxide anion) or catalase (degrades hydrogen peroxide) depressed oxidant activity in TNF-treated muscle and abolished the decrement in specific force. These findings indicate that TNF signals via nNOS to depress contractile function, a response that requires ROS and NO as obligate co-mediators.
Subject(s)
Diaphragm/physiology , Muscle Strength/physiology , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide/metabolism , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Cells, Cultured , Diaphragm/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/physiologyABSTRACT
Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2',6'-dimethyl-Tyr-Lys-Phe-NH(2), decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m(2): control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus.
