Skeletal muscle releases extracellular vesicles with distinct protein and microRNA signatures that function in the muscle microenvironment

Abstract

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish skeletal muscle-derived EVs (SkM-EVs) from others, whether the skeletal muscle releases EVs in vivo and how much SkM-EVs account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings confirm previous studies showing that skeletal muscle cells release exosome-like EVs with specific protein and miRNA profiles in vivo and show that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

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

“…This finding prompted immunohistochemical staining of the tetraspanins in the skeletal muscle cross-sections, which demonstrated hardly any detection within myofibers and instead accumulation within the interstitium. This is consistent with the recent findings of Watanabe et al, showing EVs concentrated in the muscle interstitium, attached to extracellular matrix (ECM)-like structures by transmission electron microscopy [46]. Within muscle, satellite cellderived EVs have been previously shown to influence early phases of myofiber growth in response to overload by regulating extracellular matrix (ECM)-related factors in both the myofiber and FAPs [67,68].…”

“…Beyond the lack of detection in pure serum or plasma-derived EV preparations, it is critical to note that α-sarcoglycan is not exclusively expressed in skeletal muscle, but is also expressed in cardiac muscle and in the lung, further questioning the designation of α-sarcoglycan-positive EVs as skeletal muscle EVs [45]. Similarly, APT2A1, β-enolase, and desmin have been suggested as marker proteins to identify skeletal muscle-derived EVs in vivo [46]; however, these proteins can be even more abundant in cardiac muscle tissue than skeletal muscle [47][48][49]. To address issues of tissue specificity, Estrada et al recently developed a skeletal muscle myofiber-specific fluorescent reporter mouse [50].…”

Circulating exosome‐like vesicle and skeletal muscle microRNAs are altered with age and resistance training

“…However, Estrada et al (2022) utilized a skeletal muscle myofibre-specific dual fluorescent reporter mouse to determine that only a small portion of ELVs secreted from myofibres reach the circulation (Estrada et al, 2022), making the likelihood of detecting these subpopulations challenging. In line with this, work by Watanabe et al (2022) suggests that while there are specific protein and miRNA profiles within SkM-derived ELVs, they are predominately concentrated in skeletal muscle interstitium, and are likely to play a more significant role within the muscle microenvironment. Herein, our findings confirm previous reports from Guescini et al (2015) who observed the presence of TSG101 and muscle-specific miRNAs in α-SGCA + captured ELVs.…”

“…In line with this, work by Watanabe et al. (2022) suggests that while there are specific protein and miRNA profiles within SkM‐derived ELVs, they are predominately concentrated in skeletal muscle interstitium, and are likely to play a more significant role within the muscle microenvironment. Herein, our findings confirm previous reports from Guescini et al.…”

Isolation of small extracellular vesicles from regenerating muscle tissue using Tangential Flow Filtration and Size Exclusion Chromatography

“…Particularly, there is an overwhelming evidence for the therapeutic use of EVs in muscle related pathologies [14][15][16][17][18][19][20][21]. In line with this, Wnt proteins, a conserved family of secreted glycoproteins that govern essential developmental, growth and regenerative stem cell processes, are a representative example of long-distance signaling and myoregenerative potential [22].…”