Extracellular vesicle distribution and localization in skeletal muscle at rest and following disuse atrophy

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.

Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training

“…This was consistent with the nanosight sizing data showing peaks in the small EV size range (110 and 160 nms.). Roughly 0.5%-2% of circulating EVs were muscle-derived (SGCA + ), which is consistent with a growing number of studies (Annibalini et al, 2019;Brahmer et al, 2019;Estrada et al, 2021;Guescini et al, 2015;Ismaeel et al, 2023) demonstrating that only a small fraction of circulating EVs at rest or following exercise originate from muscle. Since SGCA may not be the most accurate marker for muscle-derived EVs (Estrada et al, 2021), we also checked for presence of musclederived miRNAs (myomiRs; miRNA-1, -133a and b, -206, -486), which were present at only very low amounts, confirming low abundance of circulating muscle-derived EVs.…”

Predicting cell type-specific extracellular vesicle biology using an organism-wide single cell transcriptomic atlas – insights from theTabula Muris

“…What is known is that SkM myofibers secrete far more EVs than the same mass of adipose tissue [2], these EVs can be found within SkM tissues [62] as well as the circulation [2] and they accumulate in distant recipient tissues like white adipose during exercise where they enhance lipolysis [63]. Since SkM tissue is also the largest tissue (by mass) in non-obese humans, this evidence places SkM as one of the most abundant sources of EVs in the entire body.…”

“…An acute bout of exercise increases the circulating abundance of tetraspanin-expressing EVs [44, 45] and while SkM is a logical source for some of these EVs, their cellular origin(s) have not been well defined experimentally. What is known is that SkM myofibers secrete far more EVs than the same mass of adipose tissue [2], these EVs can be found within SkM tissues [62] as well as the circulation [2] and they accumulate in distant recipient tissues like white adipose during exercise where they enhance lipolysis [63]. Since SkM tissue is also the largest tissue (by mass) in non-obese humans, this evidence places SkM as one of the most abundant sources of EVs in the entire body.…”

From Theory to Therapy: The Advancements of Extracellular Vesicles in Immunotherapy

“…Proteins and carbohydrates on the EV surface can target them to specific cells [30][31][32][33] and their biophysical characteristics, such as charge or size, regulate their systemic and tissue distribution. [34][35][36][37] And third, they are multifactorial in nature, integrating multiple regulatory and effector molecules, such as receptor ligands on the surface or RNA-based regulatory elements in their lumen. [38][39][40][41][42][43] With this, EVs have a distinct advantage over many other immunotherapeutic approaches.…”