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Abstract Background Skeletal muscle (SkM) is a large, secretory organ that produces and releases myokines that can have autocrine, paracrine, and endocrine effects. Whether extracellular vesicles (EVs) also play a role in the SkM adaptive response and ability to communicate with other tissues is not well understood. The purpose of this study was to investigate EV biogenesis factors, marker expression, and localization across cell types in the skeletal muscle. We also aimed to investigate whether EV concentrations are altered by disuse atrophy. Methods To identify the potential markers of SkM-derived EVs, EVs were isolated from rat serum using density gradient ultracentrifugation, followed by fluorescence correlation spectroscopy measurements or qPCR. Single-cell RNA sequencing (scRNA-seq) data from rat SkM were analyzed to assess the EV biogenesis factor expression, and cellular localization of tetraspanins was investigated by immunohistochemistry. Finally, to assess the effects of mechanical unloading on EV expression in vivo, EV concentrations were measured in the serum by nanoparticle tracking analysis in both a rat and human model of disuse. Results In this study, we show that the widely used markers of SkM-derived EVs, α-sarcoglycan and miR-1, are undetectable in serum EVs. We also found that EV biogenesis factors, including the tetraspanins CD63, CD9, and CD81, are expressed by a variety of cell types in SkM. SkM sections showed very low detection of CD63, CD9, and CD81 in myofibers and instead accumulation within the interstitial space. Furthermore, although there were no differences in serum EV concentrations following hindlimb suspension in rats, serum EV concentrations were elevated in human subjects after bed rest. Conclusions Our findings provide insight into the distribution and localization of EVs in SkM and demonstrate the importance of methodological guidelines in SkM EV research.

Abstract Background Translational capacity (i.e. ribosomal mass) is a key determinant of protein synthesis and has been associated with skeletal muscle hypertrophy. The role of translational capacity in muscle atrophy and regrowth from disuse is largely unknown. Therefore, we investigated the effect of muscle disuse and reloading on translational capacity in middle‐aged men (Study 1) and in rats (Study 2). Methods In Study 1, 28 male participants (age 50.03 ± 3.54 years) underwent 2 weeks of knee immobilization followed by 2 weeks of ambulatory recovery and a further 2 weeks of resistance training. Muscle biopsies were obtained for measurement of total RNA and pre‐ribosomal (r)RNA expression, and vastus lateralis cross‐sectional area (CSA) was determined via peripheral quantitative computed tomography. In Study 2, male rats underwent hindlimb suspension (HS) for either 24 h (HS 24 h, n = 4) or 7 days (HS 7d, n = 5), HS for 7 days followed by 7 days of reloading (Rel, n = 5) or remained as ambulatory weight bearing (WB, n = 5) controls. Rats received deuterium oxide throughout the study to determine RNA synthesis and degradation, and mTORC1 signalling pathway was assessed. Results Two weeks of immobilization reduced total RNA concentration (20%) and CSA (4%) in men (both P ≤ 0.05). Ambulatory recovery restored total RNA concentration to baseline levels and partially restored muscle CSA. Total RNA concentration and 47S pre‐rRNA expression increased above basal levels after resistance training (P ≤ 0.05). In rats, RNA synthesis was 30% lower while degradation was ~400% higher in HS 7d in soleus and plantaris muscles compared with WB (P ≤ 0.05). mTORC1 signalling was lower in HS compared with WB as was 47S pre‐rRNA (P ≤ 0.05). With reloading, the aforementioned parameters were restored to WB levels while RNA degradation was suppressed (P ≤ 0.05). Conclusions Changes in RNA concentration following muscle disuse and reloading were associated with changes in ribosome biogenesis and degradation, indicating that both processes are important determinants of translational capacity. The pre‐clinical data help explain the reduced translational capacity after muscle immobilization in humans and demonstrate that ribosome biogenesis and degradation might be valuable therapeutic targets to maintain muscle mass during disuse.

Summary: Using in vivo muscle stem cell (satellite cell)-specific extracellular vesicle (EV) tracking, satellite cell depletion, in vitro cell culture, and single-cell RNA sequencing, we show satellite cells communicate with other cells in skeletal muscle during mechanical overload. Early satellite cell EV communication primes the muscle milieu for proper long-term extracellular matrix (ECM) deposition and is sufficient to support sustained hypertrophy in adult mice, even in the absence of fusion to muscle fibers. Satellite cells modulate chemokine gene expression across cell types within the first few days of loading, and EV delivery of miR-206 to fibrogenic cells represses Wisp1 expression required for appropriate ECM remodeling. Late-stage communication from myogenic cells during loading is widespread but may be targeted toward endothelial cells. Satellite cells coordinate adaptation by influencing the phenotype of recipient cells, which extends our understanding of their role in muscle adaptation beyond regeneration and myonuclear donation.

In vitro examinations of the effect of saturated fatty acids on skeletal muscle insulin action often use only one or two different fatty acid species, which does not resemble the human plasma fatty acid profile. We compared graded concentrations (0.1-0.8 mM) of 3 different lipid mixtures: 1) a physiologic fatty acid mixture (NORM; 40% saturated fatty acids), 2) a physiologic mixture high in saturated fatty acids (HSFA; 60% saturated fatty acids), and 3) 100% palmitate (PALM) on insulin signaling and fatty acid partitioning into triacylglycerol (TAG) and diacylglycerol (DAG) in cultured muscle cells. As expected, PALM readily impaired insulin-stimulated pAktThr308/Akt and markedly increased intracellular DAG content. In contrast, the fatty acid mixtures only modestly impaired insulin-stimulated pAktThr308M/Akt, and we found no differences between NORM and HSFA. Importantly, NORM and HSFA did not increase DAG content, but instead dose-dependently increased TAG accumulation. Therefore, the robust impairment in insulin signaling found with palmitate exposure was attenuated with physiologic mixtures of fatty acids, even with a very high proportion of saturated fatty acids. This may be explained in part by selective partitioning of fatty acids into neutral lipid (i.e., TAG) when muscle cells were exposed to physiologic lipid mixtures.