Skeletal muscle is a heterogeneous tissue comprised of muscle fiber and mononuclear cell types that, in addition to movement, influences immunity, metabolism and cognition. We investigated the gene expression patterns of skeletal muscle cells using RNA-seq of subtype-pooled single human muscle fibers and single cell RNA-seq of mononuclear cells from human vastus lateralis, mouse quadriceps, and mouse diaphragm. We identified 11 human skeletal muscle mononuclear cell types, including two fibro-adipogenic progenitor (FAP) cell subtypes. The human FBN1+ FAP cell subtype is novel and a corresponding FBN1+ FAP cell type was also found in single cell RNA-seq analysis in mouse. Transcriptome exercise studies using bulk tissue analysis do not resolve changes in individual cell-type proportion or gene expression. The cell-type gene signatures provide the means to use computational methods to identify cell-type level changes in bulk studies. As an example, we analyzed public transcriptome data from an exercise training study and revealed significant changes in specific mononuclear cell-type proportions related to age, sex, acute exercise and training. Our single-cell expression map of skeletal muscle cell types will further the understanding of the diverse effects of exercise and the pathophysiology of muscle disease.Skeletal muscle is a complex heterogeneous tissue consisting of multinucleated muscle fibers, immune cells, endothelial cells, muscle stem cells (satellite cells), non-myogenic mesenchymal progenitors (e.g., fibro-adipogenic progenitors, or FAPs), and other mononuclear cells 1 . To improve the understanding of skeletal muscle cell types and their transcriptional signatures, we studied human and mouse skeletal muscle mononuclear cells by single-cell RNA-sequencing and single human muscle fiber subtypes by RNA-seq.The majority of skeletal muscle is composed of the multinucleated fibers that facilitate movement. These muscle fibers include several fiber types of differing metabolic and functional properties 2-4 . While slow-twitch (or Type I) muscle fibers possess high oxidative capacity, fast-twitch (or Type II) muscle fibers have a high glycolytic capacity and are capable of supplying more power than Type I fibers 2-4 . Fiber-type composition differs across individuals and can change by as much as 10-30% during exercise training regimens 5-7 . Furthermore, the transcriptomic response to physical activity is different in each fiber-type as each fiber-type responds differently to different modes of exercise 8,9 . Crucially, muscle fibers secrete myokines, which both act locally within muscle tissue as well as influence other organs and tissues via hormone-like signaling 10 . Myokines may be responsible for the immune-, metabolism-, and cognition-related benefits of physical activity, as well as the chronic diseases that are caused by lack of physical activity (insulin resistance, cardiovascular disease, etc.) 10 .Besides multinucleated fibers, skeletal muscle contains many mononuclear cells, such as immune cells,...

Ferry A, Bonnieu A, Ollendorff V, Favier FB. REDD1 deletion prevents dexamethasone-induced skeletal muscle atrophy. Am J Physiol Endocrinol Metab 307: E983-E993, 2014. First published October 14, 2014 doi:10.1152/ajpendo.00234.2014.-REDD1 (regulated in development and DNA damage response 1) has been proposed to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) during in vitro hypoxia. REDD1 expression is low under basal conditions but is highly increased in response to several catabolic stresses, like hypoxia and glucocorticoids. However, REDD1 function seems to be tissue and stress dependent, and its role in skeletal muscle in vivo has been poorly characterized. Here, we investigated the effect of REDD1 deletion on skeletal muscle mass, protein synthesis, proteolysis, and mTORC1 signaling pathway under basal conditions and after glucocorticoid administration. Whereas skeletal muscle mass and typology were unchanged between wildtype (WT) and REDD1-null mice, oral gavage with dexamethasone (DEX) for 7 days reduced tibialis anterior and gastrocnemius muscle weights as well as tibialis anterior fiber size only in WT. Similarly, REDD1 deletion prevented the inhibition of protein synthesis and mTORC1 activity (assessed by S6, 4E-BP1, and ULK1 phosphorylation) observed in gastrocnemius muscle of WT mice following single DEX administration for 5 h. However, our results suggest that REDD1-mediated inhibition of mTORC1 in skeletal muscle is not related to the modulation of the binding between TSC2 and 14-3-3. In contrast, our data highlight a new mechanism involved in mTORC1 inhibition linking REDD1, Akt, and PRAS40. Altogether, these results demonstrated in vivo that REDD1 is required for glucocorticoidinduced inhibition of protein synthesis via mTORC1 downregulation. Inhibition of REDD1 may thus be a strategy to limit muscle loss in glucocorticoid-mediated atrophy. regulated in development and DNA damage response 1; protein synthesis; mechanistic target of rapamycin; autophagy; glucocorticoids; proline-rich Akt substrate of 40 kDa

Cytokine interleukin-6 (IL-6) is an essential regulator of satellite cell-mediated hypertrophic muscle growth through the transcription factor signal transducer and activator of transcription 3 (STAT3). The importance of this pathway linked to the modulation of myogenic regulatory factors expression in rat skeletal muscle undergoing hypertrophy following resistance exercise, has not been investigated. In this study, the phosphorylation and nuclear localization of STAT3, together with IL-6/STAT3-responsive gene expression, were measured after both a single bout of resistance exercise and 10 weeks of training. Flexor Digitorum Profundus muscle samples from Wistar rats were obtained 2 and 6 hours after a single bout of resistance exercise and 72 h after the last bout of either 2, 4, or 10 weeks of resistance training. We observed an increase in IL-6 and SOCS3 mRNAs concomitant with phosphorylation of STAT1 and STAT3 after 2 and 6 hours of a single bout of exercise (p<0.05). STAT3-dependent early responsive genes such as CyclinD1 and cMyc were also upregulated whereas MyoD and Myf5 mRNAs were downregulated (p<0.05). BrdU-positive satellite cells increased at 2 and 6 hours after exercise (p<0.05). Muscle fiber hypertrophy reached up to 100% after 10 weeks of training and the mRNA expression of Myf5, c-Myc and Cyclin-D1 decreased, whereas IL-6 mRNA remained upregulated. We conclude that the IL-6/STAT1/STAT3 signaling pathway and its responsive genes after a single bout of resistance exercise are an important event regulating the SC pool and behavior involved in muscle hypertrophy after ten weeks of training in rat skeletal muscle.

support-information-section). Key pointsr A hallmark trait of ageing skeletal muscle health is a reduction in size and function, which is most pronounced in the fast muscle fibres.r We studied older men (74 ± 4 years) with a history of lifelong (>50 years) endurance exercise to examine potential benefits for slow and fast muscle fibre size and contractile function.r Lifelong endurance exercisers had slow muscle fibres that were larger, stronger, faster and more powerful than young exercisers (25 ± 1 years) and age-matched non-exercisers (75 ± 2 years).r Limited benefits with lifelong endurance exercise were noted in the fast muscle fibres. r These findings suggest that additional exercise modalities (e.g. resistance exercise) or other therapeutic interventions are needed to target fast muscle fibres with age.

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research. This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men.