| Popis: |
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. |
