Identifying protein markers of skeletal muscle small extracellular vesicle secretion during exercise

Sale, Benedict ORCID: 0000-0002-1877-6910 (2022). Identifying protein markers of skeletal muscle small extracellular vesicle secretion during exercise. University of Birmingham. M.Sc.

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Abstract

Extracellular Vesicles (EVs) are membranous particles which act as a method of cellular communication between cells and tissues through the transfer of lipids, proteins, and RNA cargo. Exosomes are a subpopulation of EV which are derived from the cell cytosol, meaning the packing of proteins into exosomes is pre-determine intracellularly based on the origin (i.e., adipose vs muscle) and condition (i.e., exercise vs sedentary) from which they are influenced, making them interesting candidates for biomarker discovery. During exercise, there is an acute rapid release of small EVs (sEVs), consisting of both exosomes and small microvesicles, into the circulation which has been proposed to provide a method of tissues crosstalk during exercise (Whitham et al., 2018). Due to the lack of a methodological approach that allows for the clear separation of various tissue derived sEVs, the contribution of skeletal muscle to the increase in systemic sEVs during exercise is unclear. In this regard, whether skeletal muscle sEVs play a role in tissue crosstalk during exercise is not known. With the aim of identifying novel candidate protein markers of skeletal muscle sEV secretion, the present study curated, re-analyzed and overlayed currently available proteomic datasets regarding muscle derived EV bound proteins that are also exercise responsive in vitro and in vivo; recognizing ANXA6 and CD44 as candidate protein markers of muscle sEV secretion. Immunoblotting of ANXA6, CD44 and alpha sarcoglycan (SGCA), a previously proposed muscle specific sEV marker, amongst several different cell lines revealed that ANXA6 and CD44 were enriched (P < 0.05) but not specifically expressed in C2C12 myotube sEVs. SGCA however, was highly unique to myotube sEVs compared to other cell lines (P < 0.05). Noticeably, in vitro stimulation by ionomycin (IM) caused a ~4-fold increase in the secretion of sEVs from C2C12 myotubes detected by nanoparticle tracking analysis (NTA), of which these affects were reflected by increases in ANXA6+ (P < 0.05, 158.29%) and CD44+ (P < 0.05, 180.69%) sEVs but not SGCA (P = 0.6244, 6.58%). This suggests that the secretion of sEVs from muscle might be somewhat calcium regulated. Mechanical contraction, induced by electrical pulse stimulation (EPS), did not produce similar results. Seemingly EPS as a model is limited in its ability to generate sufficient calcium transients in C2C12s (Olsson et al., 2015), which might explain this response. Finally, acute high intensity interval exercise (HIIE) recapitulated the increase in systemic sEVs previously reported by others (Fruhbeis et al., 2015; Whitham et al., 2018; Brahmer et al., 2019; Vanderboom et al., 2021) as indicated by the increase in CD9+ (P <0.05, 28.28%), CD63+ (P < 0.05, 68.64%) and CD81+ (P < 0.05, 13.16%) sEVs immediately post exercise. However, these changes were only observed in CD44+ sEVs isolated by immunomagnetic precipitation (IMP) (P < 0.05, 879.84%) and not ultracentrifugation (UC) (P = 0.1002, 33.82%) suggesting that the purity of isolation forms part of the observed changes in specific protein markers. Overall, it is unclear as to whether skeletal muscle sEVs are liberated into the circulation with acute exercise and more research is needed to conclude the confidence of protein markers including ANXA6 and CD44 as sufficient markers of skeletal muscle sEV release in the circulation. Similarly, calcium influx into the sarcolemma with contraction might present a method of sEV secretion in vivo and as such warrant further investigation into the mechanisms regulating EV biogenesis and secretion from skeletal muscle. Finally, the present study highlights how the use of currently available proteomic data can be adopted to inform targeted methods in novel protein marker discovery and validation.

Type of Work: Thesis (Masters by Research > M.Sc.)
Award Type: Masters by Research > M.Sc.
Supervisor(s):
Supervisor(s)EmailORCID
Whitham, MartinUNSPECIFIEDUNSPECIFIED
Wadley, AlexanderUNSPECIFIEDUNSPECIFIED
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department: School of Sport, Exercise and Rehabilitation Sciences
Funders: None/not applicable
Subjects: Q Science > Q Science (General)
Q Science > QP Physiology
Q Science > QR Microbiology
URI: http://etheses.bham.ac.uk/id/eprint/12501

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