SUMMARYAn important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca 2+ sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.

MyoD, a master regulator of myogenesis, exhibits a circadian rhythm in its mRNA and protein levels, suggesting a possible role in the daily maintenance of muscle phenotype and function. We report that MyoD is a direct target of the circadian transcriptional activators CLOCK and BMAL1, which bind in a rhythmic manner to the core enhancer of the MyoD promoter. Skeletal muscle of Clock Δ19 and Bmal1 −/− mutant mice exhibited ∼30% reductions in normalized maximal force. A similar reduction in force was observed at the single-fiber level. Electron microscopy (EM) showed that the myofilament architecture was disrupted in skeletal muscle of Clock Δ19 , Bmal1 −/− , and MyoD −/− mice. The alteration in myofilament organization was associated with decreased expression of actin, myosins, titin, and several MyoD target genes. EM analysis also demonstrated that muscle from both Clock Δ19 and Bmal1 −/− mice had a 40% reduction in mitochondrial volume. The remaining mitochondria in these mutant mice displayed aberrant morphology and increased uncoupling of respiration. This mitochondrial pathology was not seen in muscle of MyoD −/− mice. We suggest that altered expression of both Pgc-1α and Pgc-1β in Clock Δ19 and Bmal1 −/− mice may underlie this pathology. Taken together, our results demonstrate that disruption of CLOCK or BMAL1 leads to structural and functional alterations at the cellular level in skeletal muscle. The identification of MyoD as a clock-controlled gene provides a mechanism by which the circadian clock may generate a muscle-specific circadian transcriptome in an adaptive role for the daily maintenance of adult skeletal muscle.circadian clock | myofilaments | mitochondria A fundamental, evolutionarily conserved property of most organisms, from cyanobacteria to plants and animals, is the daily cycling of their internal physiology as well as certain behaviors in animals, such as sleep and feeding (1). The timing of these circadian rhythms is synchronized to the environment by external cues, with light and nutrient availability being two of the most salient entrainment cues (2). The synchronization of endogenous circadian rhythms with the daily cycles in the environment provides an adaptive mechanism for organisms to anticipate cyclic demands on cellular physiology and behavior (3, 4). At the molecular level, the circadian clock represents a well-defined gene regulatory network composed of transcriptional-translational feedback loops (5). The positive arm of the loop is composed of the transcription factors CLOCK and BMAL1, which heterodimerize and bind to E-box elements on target genes such as Per1 to drive the negative part of the feedback loop (5). More recently, the same molecular clock factors that have been identified in the central clock in the suprachiasmatic nucleus have been found to exist in most peripheral tissues (reviewed in ref. 6).We recently characterized the circadian transcriptome of adult skeletal muscle. These mRNAs exhibit significant oscillation in gene expression with a repeating period len...

Circadian rhythms are approximate 24-h behavioral and physiological cycles that function to prepare an organism for daily environmental changes. The basic clock mechanism is a network of transcriptional-translational feedback loops that drive rhythmic expression of genes over a 24-h period. The objectives of this study were to identify transcripts with a circadian pattern of expression in adult skeletal muscle and to determine the effect of the Clock mutation on gene expression. Expression profiling on muscle samples collected every 4 h for 48 h was performed. Using COSOPT, we identified a total of 215 transcripts as having a circadian pattern of expression. Real-time PCR results verified the circadian expression of the core clock genes, Bmal1, Per2, and Cry2. Annotation revealed cycling genes were involved in a range of biological processes including transcription, lipid metabolism, protein degradation, ion transport, and vesicular trafficking. The tissue specificity of the skeletal muscle circadian transcriptome was highlighted by the presence of known muscle-specific genes such as Myod1, Ucp3, Atrogin1 (Fbxo32), and Myh1 (myosin heavy chain IIX). Expression profiling was also performed on muscle from the Clock mutant mouse and sarcomeric genes such as actin and titin, and many mitochondrial genes were significantly downregulated in the muscle of Clock mutant mice. Defining the circadian transcriptome in adult skeletal muscle and identifying the significant alterations in gene expression that occur in muscle of the Clock mutant mouse provide the basis for understanding the role of circadian rhythms in the daily maintenance of skeletal muscle.

Analysis of skeletal muscle cross sections is an important experimental technique in muscle biology. Many aspects of immunohistochemistry and fluorescence microscopy can now be automated, but most image quantification techniques still require extensive human input, slowing progress and introducing the possibility of user bias. MyoVision is a new software package that was developed to overcome these limitations. The software improves upon previously reported automatic techniques and analyzes images without requiring significant human input and correction. When compared with data derived by manual quantification, MyoVision achieves an accuracy of ≥94% for basic measurements such as fiber number, fiber type distribution, fiber cross-sectional area, and myonuclear number. Scientists can download the software free from www.MyoVision.org and use it to automate the analysis of their own experimental data. This will improve the efficiency and consistency of the analysis of muscle cross sections and help to reduce the burden of routine image quantification in muscle biology. NEW & NOTEWORTHY Scientists currently analyze images of immunofluorescently labeled skeletal muscle using time-consuming techniques that require sustained human supervision. As well as being inefficient, these techniques can increase variability in studies that quantify morphological adaptations of skeletal muscle at the cellular level. MyoVision is new software that overcomes these limitations by performing high-content analysis of muscle cross sections with minimal manual input. It is open source and freely available.

Skeletal muscle development, nutrient uptake, and nutrient utilization is largely coordinated by growth hormone (GH) and its downstream effectors, in particular, IGF-1. However, it is not clear which effects of GH on skeletal muscle are direct and which are secondary to GH-induced IGF-1 expression. Thus, we generated mice lacking either GH receptor (GHR) or IGF-1 receptor (IGF-1R) specifically in skeletal muscle. Both exhibited impaired skeletal muscle development characterized by reductions in myofiber number and area as well as accompanying deficiencies in functional performance. Defective skeletal muscle development, in both GHR and IGF-1R mutants, was attributable to diminished myoblast fusion and associated with compromised nuclear factor of activated T cells import and activity. Strikingly, mice lacking GHR developed metabolic features that were not observed in the IGF-1R mutants, including marked peripheral adiposity, insulin resistance, and glucose intolerance. Insulin resistance in GHR-deficient myotubes derived from reduced IR protein abundance and increased inhibitory phosphorylation of IRS-1 on Ser 1101. These results identify distinct signaling pathways through which GHR regulates skeletal muscle development and modulates nutrient metabolism. IntroductionMammalian skeletal muscle has evolved to perform a diverse set of functions, including locomotion, breathing, protecting internal organs, and coordinating global energy expenditure. Skeletal muscle is formed and regenerated through a highly regulated process characterized by myoblast differentiation and fusion into multinucleated syncytia. During embryonic development, specification of mesodermal precursor cells into the myogenic lineage is controlled by signals from surrounding tissues and requires upregulation of several factors, including paired-box transcription factor 7 (pax-7) and basic helix-loop-helix transcriptional activators of the myogenic regulatory factor family, MyoD and Myf-5 (1). The proliferating precursor cells/myoblasts withdraw from the cell cycle and initiate muscle-specific gene expression (2, 3). Myoblasts then initially fuse to form nascent myotubes, with relatively few nuclei, through a highly ordered set of cellular events, including recognition, adhesion, alignment, and membrane union. Subsequent recruitment and fusion of additional myoblasts gives rise to multinucleated myotubes that ultimately mature to give rise to skeletal muscle fibers. The fusion process is controlled, in part, by the actions of calcium-sensitive transcription factors of the nuclear factor of activated T cells (NFAT) family (4, 5). During myoblast fusion,