Zackary Valenti (Colorado State University), Andrea Estrada (Colorado State University), Nicholas Williams (Colorado State University, Ursinus College), Gabriella Hehn (Colorado State University), Nicole Kruh-Garcia (Colorado State University), Dan Lark (Colorado State University)
Skeletal muscle (SkM) is the largest tissue in the human body and dynamic in its metabolic activity. Cells within the SkM niche communicate with other cells, at least in part, via extracellular vesicles (EVs). EVs reach target cells locally or after traveling through the circulation, but there is very limited evidence that SkM myofiber EVs reach the circulation in vivo. Furthermore, we do not know whether EV secretion differs across metabolically active tissues (i.e. SkM vs white adipose tissue (WAT)). These knowledge gaps limit our ability to understand the endogenous function of EVs and thus limit our ability to use EVs as biomarkers and/or therapies. Here we hypothesized that metabolic activity predicts EV secretion between tissues, and that SkM myofibers contribute to the circulating EV pool in vivo. To address these hypotheses, we studied tissue-specific EV secretion ex vivo and generated a SkM-specific version of the popular mT/mG mouse to track SkM EVs circulating in vivo.
METHODS
Studies were approved by the Colorado State University IACUC. All mice were on a C57BL/6J background and studied at ~21 weeks old. Mice were anesthetized with isoflurane. SkM (vastus medialis (VM), soleus (SOL) or plantaris (PLA)) and WAT (epididymal) were cut into ~5 mg pieces and placed in serum-free DMEM for 24 hours at 37°C in a CO2 incubator (Fig. 1A). Conditioned media was collected and EVs were isolated using Exoquick-TC or size exclusion chromatography. EV abundance was determined via nanoparticle tracking analysis on a Horiba ViewSizer 3000. EV secretion was normalized to culture media volume and tissue mass and thus expressed as particles/ml/mg tissue.
SkM-mG/mT mice were generated by crossing mT/mG and ACTA-Cre expressing mice to reach homozygous floxed alleles and heterozygous expression of Cre recombinase. In this mouse, SkM fibers express membrane-targeted enhanced green fluorescent protein (mG) while non-SkM cells and EVs express a membrane-targeted tdTomato (mT) (Fig. 1B). Whole blood was obtained via cardiac puncture into EDTA coated tubes and centrifuged at 10k x g for 20 minutes at 4°C. 50 µl of plasma was incubated on a microfluidics chip coated with anti-CD9 or anti-CD81 antibody. Chips were washed and spots imaged for mG, mT and CD63 as blue, yellow and red fluorescence, respectively (Fig. 1C).
RESULTS
VM secretes about 100x more EVs than WAT independent of EV isolation method (Fig. 2A amp; 2B). SOL secretes more EVs than PLA (Fig. 2C). Circulating EVs express CD9 more frequently than CD81 (Fig. 2D). EVs from SkM-mG/mT mice express more mG relative to WT and mT mice (Fig. 2E). 4-5% of circulating CD9+ and CD81+ EVs from SkM-mG/mT mice express mG (Fig. 2F), representing SkM myofiber EVs. 10-20% of circulating EVs express tdTomato (Fig. 2G), representing non-SkM myofiber EVs.
CONCLUSIONS
Here we show that 1) SkM secretes more EVs than WAT, 2) oxidative SkM (SOL) secretes more EVs than glycolytic SkM (PLA), and 3) circulating SkM myofiber-derived EVs express tetraspanins and account for ~5% of circulating EVs. These data advance our understanding of EV secretion from SkM tissue and provide direct evidence that SkM myofiber-derived EVs reach the circulation in vivo. These findings demonstrate the relevance of SkM-myofiber derived EVs in physiological systems and provide an approach to begin understanding the in vivo function of SkM EVs.
AHA Innovative Project Award (IPA1834110052 to DSL) NSF REU (1757514 supported NW)
Outline of EV methods used. A) Assay workflow for measuring ex vivo EV secretion in mouse vastus medialis (VM), soleus (SOL) or plantaris (PLA) and epididymal white adipose tissue (WAT) and isolation via Exoquick-TC or size exclusion chromatography (SEC). B) Design of the SkM-mG/mT mouse and the secretion of mG (SkM myofiber-derived) or mT (non-SkM myofiber-derived) EVs. C) Immunocapture and single particle fluorescence detection of mG, mT and CD63 in SkM-mG/mT mouse plasma.; Ex vivo EV secretion and circulating SkM EVs in vivo. A) Exoquick-TC (EQ-TC) VM and WAT EVs in 9 male mice. B) SEC SkM and WAT EVs in 6 male mice. C) EQ-TC EVs from SOL and PLA in 6 female mice. [EV] was adjusted to tissue mass (mg). D) CD9 or CD81 EVs in plasma. E) Single EV mG (eGFP) from WT, mT or mG mouse plasma. F) % of CD9+ or CD81+ EVs expressing mG. G) % of plasma CD9+ or CD81+ EVs expressing mT. * p < 0.05 in paired Student’s two-way t-test. 8 male SkM-mG/mT mice studied in D, F, G.
