Cell culture

Monolayer cultures of C2C12 and L6 myoblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, Waltham, MA) supplemented with 10% (L6) or 20% (C2C12) fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT) and 1X Penicillin/Streptomycin (Thermo Fisher) at 37 °C in a humidified 10% CO2-90% air atmosphere incubator, as previously described [23, 24].

Animals and experimental procedures

All procedures were approved by the University of Kentucky’s Institutional Animal Care and Use Committee. Male Brown Norway/F344 rats at 10 months of age (National Institute on Aging, Bethesda, MD) were used in this study. Rats were randomly assigned into one of four groups: weight-bearing control conditions (WB), hindlimb suspension (HS) for 4 h (4h HS), HS for 24 h (24h HS), and HS for 7 days (7d HS). Rats were allowed free access to food and water at all times and were housed on a 12:12-h light-dark cycle. Hindlimb suspension was performed as previously described [22]. Briefly, a tail device containing a hook was attached with gauze and cyanoacrylate glue while the animals were anesthetized with isoflurane (2% by inhalation). The tail device was connected via a thin cable to a pulley sliding on a vertically adjustable stainless steel bar running longitudinally above a high-sided cage. The system was designed in such a way that the rats could not rest their hindlimbs against any side of the cage but could move around the cage on their front limbs and could reach water and food easily. Cages were randomly placed in the room, and the room temperature was 27 °C.

Blood and tissue collection

At the end of the experimental period, rats were anesthetized with pentobarbital sodium, and blood was immediately collected through cardiac puncture. Rats were euthanized, and the soleus muscles were excised, weighed, and used for ex vivo collection of muscle EVs (right) or dissected, weighed, frozen in liquid nitrogen, and stored at − 80 °C for later biochemical analyses (left). For immunohistochemistry (IHC) analyses, the soleus muscles were covered in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen-cooled isopentane, and stored at − 80 °C. The soleus muscles were used in these experiments because in rats, the soleus, which is almost exclusively type I, is especially susceptible to hindlimb suspension-induced muscle atrophy [25, 26]. Furthermore, the smaller size of the soleus limits issues of oxygen diffusion during ex vivo muscle assessments [27]. The gastrocnemius muscles were used for single-cell RNA sequencing (scRNA-seq) as described in [28]. The serum was isolated by allowing blood to clot at room temperature for 30 min before centrifugation for 10 min at 2000g at 4 °C. The serum supernatant was collected and stored at − 80 °C until analysis. The number of animals used in each experiment is listed in the figure legends.

Human participants

Serum and muscle biopsies obtained from the vastus lateralis from a previously published bed rest study (young group, age: 23 ± 3 years) were used for EV and RNA isolation, respectively [29]. Human participants were recruited at the University of Utah under an approved Institutional Review Board protocol, and the study conformed to the Declaration of Helsinki. Bed rest (5 days; Monday–Friday) took place according to the protocol and safety guidelines described in detail in the original publication [29]. For total RNA isolation from the muscle, samples were homogenized in TRIzol Reagent (Invitrogen, Waltham, MA), and 1-bromo-3-chloropropane was added for phase separation. Finally, 2-propanol was used to precipitate the RNA, and RNA was pelleted by centrifugation (12,000g for 10 min). The Bio-Rad iScript Reverse Transcription Supermix (1708841, Bio-Rad Laboratories, Hercules, CA) was used for cDNA synthesis from 1 μg of total RNA. Real-time PCR was used to determine the relative mRNA expression of the tetraspanins, CD63, CD9, and CD81. PCR reactions used primer sets and Applied Biosystems PowerUp SYBR Green Master Mix (A25742, Applied Biosystems, Waltham, MA).

Ex vivo collection of muscle EVs

For ex vivo experiments, the soleus muscle was excised intact, rinsed with Krebs-Henseleit buffer (KHB) (118.5mM NaCl, 1.2mM MgSO4, 4.7mM KCl, 1.2mM KH2PO4, 25mM NaHCO3, 2.5mM CaCl2; pH 7.4), and then suspended prior to incubation in continuously gassed (95% O2/5% CO2) KHB supplemented with 5 mM glucose at 37 °C for 1 h. EV abundance in the KHB was measured using nanoparticle tracking analysis (NTA) (Zetaview®, Particle Metrix, Meerbusch, Germany) immediately after the 1-h incubation period. The Zetaview® instrument uses a laser scattering video microscope to track individual nanoparticle movement under Brownian motion, measuring the size and concentration [30].

Serum EV isolation

EVs isolated from rat and human serum for miRNA analysis were isolated from 500 μL of serum with ExoQuick Exosome Precipitation Solution (System Biosciences (SBI), Palo Alto, CA). The serum was first centrifuged at 3000g for 15 min to remove debris, and the supernatant was collected and filtered through a 0.22-μm low-binding PVDF filter (Millex-GV; Millipore, Tullagreen, Ireland). Approximately 240 μL of ExoQuick was added to the sample and incubated at 4 °C overnight. The ExoQuick-serum mixture was centrifuged at 1500g for 30 min to pellet the EVs. The supernatant was removed, and the EV pellet was reconstituted in 300 μL of PBS.

To isolate a purer sample of EVs and better assess the contribution of EVs to the overall particle population in rat serum, we used a slightly modified density gradient ultracentrifugation (DGUC) protocol from Onodi et al. [31]. First, rat serum was centrifuged 2500g for 15 min at 4 °C to remove the debris, and the supernatant was collected. The supernatant was filtered through a 0.22-μm low-binding PVDF filter (Millex-GV; Millipore, Tullagreen, Ireland). The sample was layered on top of an iodixanol (OptiPrep™, BioVision Inc., Milpitas, CA) density gradient. The iodixanol was diluted to 50, 30, and 10% in 0.25M sucrose/10mM Tris buffer, and a discontinuous gradient was formed by layering 3.66 mL of the 50, 30, and 10% iodixanol solutions in a 13-mL ultracentrifuge tube (Beckman Coulter, Pasadena, CA). The volume of the filtered serum sample was brought up to 1 mL with PBS if necessary and then layered onto the top of the discontinuous gradient. The samples were centrifuged in a SW41 Ti rotor for 24 h at 120,000g at 4 °C. Twelve 1-mL fractions of the density gradient layers were collected (F1–F12).

EV miRNA isolation and expression

Total RNA was isolated from EVs as previously described [22] using the commercial miRCURY RNA Isolation Kit (Exiqon, Woburn, MA). miRNA concentrations were quantified with a small RNA kit on an Agilent Bioanalyzer (Agilent, Santa Clara, CA), followed by reverse transcription of miRNA performed with 10 ng of total RNA using the miRCURY LNA RT kit (Qiagen, Hilden, Germany). RT-qPCR reactions used the miRCURY LNA SYBR Green PCR kit (Qiagen) and the appropriate miRCURY LNA primer sets for the miRNAs of interest (Qiagen). miRNA expression was normalized to the expression of UniSp6, an exogenous spike-in that resembles miRNAs, using the −ΔCT method [32].

EV protein isolation and protein expression

Total protein was isolated from EVs using Pierce RIPA lysis buffer with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA), and protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher). For Western blotting, samples were prepared in Laemmli buffer, boiled at 95 °C for 5 min, and 5 μg protein was loaded. Proteins were separated by SDS-PAGE using 4–15% TGX Gels (Criterion, Bio-Rad, Hercules, CA) by running at 200 V at room temperature. Proteins were transferred for 60 min at 100 V on ice onto a nitrocellulose membrane in 20% methanol Tris-glycine buffer. The Revert Total Protein Stain Kit (Li-Cor Biosciences, Lincoln, NE) or Ponceau S solution (Thermo Fisher) was used to stain total protein, and the membranes were imaged to verify transfer efficiency and loading. The membranes were subsequently blocked in 5% nonfat dry milk in Tris-buffered saline-Tween (TBS-T, 0.1% Tween-20) for 1 h at room temperature, then incubated overnight at 4 °C in primary antibody (anti-CD63, EXOAB-CD63A-1; System Biosciences, Palo Alto, CA and anti-Apolipoprotein A1 (ApoA1, 701239; Thermo Fisher) at a 1:1000 dilution in 5% nonfat dry milk in TBS-T. The membranes were then washed before incubation in goat anti-rabbit secondary antibodies (EXOAB-CD63A-1; System Biosciences) (1:10,000 dilution) for 1 h at room temperature. Blots were developed with enhanced chemiluminescence (Clarity Western ECL Substrate, Bio-Rad), imaged, and quantified with ImageJ (National Institutes of Health).

Fluorescence correlation spectroscopy (FCS) of EVs

To assess α-sarcoglycan protein levels, Western blotting was performed as described above for CD63, and the membranes were incubated with anti-α-sarcoglycan antibody (Santa Cruz, SC-271321) (1:1000 dilution). To further determine the number of EVs that are positive for α-sarcoglycan, we used fluorescence correlation spectroscopy (FCS). FCS is a powerful technique that can quantitatively evaluate picomolar concentrations, with sensitivity that can be up to a single-molecule level [33, 34]. Specifically, an anti-α-sarcoglycan antibody was used (Santa Cruz, SC-271321). The antibody was first labeled with CF488 dye using antibody labeling kits (Mix-n-Stain, Biotium) following the manufacturer’s antibody labeling protocol. Fifty ng/mL CF488 labeled antibody was added to each EV sample and allowed to incubate for 60 min at room temperature. The vesicles were purified from free dye using a 5000-molecular weight cutoff size exclusion column (PD Minitrap G25, GE Healthcare) as described previously [35]. Briefly, the binding of fluorescently labeled anti-α-sarcoglycan antibody to EVs was confirmed via FCS based on their diffusion times. All FCS measurements were done as reported previously by Fu et al. [36]. Briefly, 40 μL of fluorescently labeled EVs were placed onto a coverslip mounted on an Olympus IX83 microscope equipped with a PicoQuant PicoHarp 300 time-correlated single photon counting (TCSPC) system. We employed a 488-nm laser (50 μW) to excite the fluorescent labels, and a 60× water immersion objective was used to focus this laser beam into the sample solution. Two avalanche photodiodes (APDs) were used for photon detection, and the signal was directed to a PicoHarp 300 TCSPC module controller. All measurements were performed 30 μm above the glass surface in the sample solution. For the unconjugated fluorophore, the fitted autocorrelation functions (ACF) yield a diffusion time (τD) of 0.21 ± 0.02 ms. A longer diffusion time of 2.5 ± 0.2 ms was observed for the CF-488-labeled anti-α-sarcoglycan antibody. The immunolabeled (anti-α-sarcoglycan-CF488 antibody) EVs exhibited a diffusion time of 32 ± 5 ms. In order to calculate the average number of immunolabeled (anti-α-sarcoglycan-CF488 antibody) EVs within the focal volume, the FCS focal volume was first calibrated using commercially available 0.1-μm tetra speck beads with a known diffusion constant and concentration. The number of vesicles per mL of solution was determined using NTA, and the number of labeled vesicles per mL was determined using FCS and the calibrated size of the focal volume.

Mononuclear cell isolation and scRNA-seq

Cell isolations were performed as previously described in mice [37] and modified slightly for rats [28]. Briefly, the gastrocnemius muscles from WB and HS male rats were excised and placed in muscle dissociation media (MDM) (Hams F-10 (Gibco, USA), 10% Horse Serum (Thermo Fisher), 1% penicillin/streptomycin (Gibco), 800 U/ml Collagenase II (Gibco)), and minced using sterilized surgical equipment. The muscle homogenate was then incubated in MDM for 1 h at 37 °C with gentle agitation. Following incubation, samples underwent further incubation in 1000 U/ml Collagenase II (Gibco) and 11 U/ml dispase (Gibco) for 30 min at 37 °C. The single-cell suspension was passed through an 18-gauge needle approximately 10 times prior to 0.2-μm filtration. Single cells were incubated in propidium iodide to identify dying/dead cells for removal via fluorescence-activated cell sorting (Sony Biotechnology, USA). Single-cell suspensions from each group were added to a Chromium Controller (10X Genomics, USA) using the Single Cell 3’ Reagent Kit per manufacturer’s instructions and sequenced on an Illumina HiSeq platform (Novogene, USA), yielding 200 million reads/sample.

Data processing and cell population annotation

scRNA-seq data were processed using the Partek Genomics Suite (Partek, USA) as previously described [28]. Briefly, following data quality control, samples were aligned to the rn6 genome and low-quality cells and/or reads were excluded based on the following criteria: mitochondrial reads exceeding 20%, an indication of doublets via read counts/cell, lowly expressed genes in only 0.01% of total cells, and high expression of myofiber-related RNA resulting from muscle mincing. Following dimensionality reduction, graph-based clustering was used in combination with known muscle mononuclear cell-related gene markers for population annotation [11, 28, 38].

Mononuclear cell EV-related gene expression

Bubble plots were generated using the Extracellular Vesicle Biogenesis GO term (http://www.informatics.jax.org/vocab/gene_ontology/GO:0140112) in combination with the identified mononuclear cell populations. Following the filtration of genes represented by the selected GO term (GO: 0140112), a bubble plot was made with the average expression of the gene of interest represented by heatmap, and the percent of cells expressing each gene represented by the size of the bubble. Cell populations are grouped by sample for population-specific comparison.

Immunohistochemistry (IHC)

The muscles were cut on a cryostat at − 23 °C (7 μm), air-dried, and stored at − 20 °C. Slides were air-dried, rehydrated, and fixed in 4% paraformaldehyde (PFA) for 20 min at the time of staining. For CD63/DAPI/laminin staining, sections were incubated with mouse anti-CD63 IgG1 antibody (1:100 dilution, ab108950, Abcam, Cambridge, UK) and rabbit anti-laminin IgG antibody (1:100 dilution, L9393, Sigma-Aldrich, St. Louis, MO) overnight at 4 °C. Slides were washed in PBS, then incubated with Alexa Fluor 488 goat anti-mouse IgG1 (1:250 dilution, A11001, Invitrogen, Waltham, MA) and Alexa Fluor 594 goat anti-rabbit IgG (1:250 dilution, A11012, Invitrogen) secondary antibodies for 1 h at room temperature. Slides were washed in PBS and mounted with VectaShield fluorescent mounting media with DAPI (H-1200-10, Vector Laboratories, Newark, CA). For CD9/DAPI/dystrophin staining, sections were incubated with rabbit anti-CD9 IgG (1:100 dilution, SA35-08, Invitrogen) and mouse anti-dystrophin IgG2b (1:250 dilution, 08168, Sigma-Aldrich) overnight, followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG (1:250 dilution, A11012, Invitrogen) and Alexa Fluor 647 goat anti-mouse IgG2b (1:250 dilution, A32728, Invitrogen) for 1 h at room temperature. For CD81/DAPI/dystrophin staining, sections were incubated with rabbit anti-CD81(1:100 dilution, SN206-01, Novus Biologicals, Centennial, CO) and mouse anti-dystrophin IgG2b (1:250 dilution, 08168, Sigma-Aldrich) overnight, followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG (1:250 dilution, A11012, Invitrogen) and Alexa Fluor 647 goat anti-mouse IgG2b (1:250 dilution, A32728, Invitrogen) for 1 h at room temperature. For Pax7/CD9/DAPI/WGA staining, sections were subjected to epitope retrieval using sodium citrate (10 mM, pH 6.5) at 92 °C, followed by blocking of endogenous peroxidase activity with 3% hydrogen peroxide in PBS. Sections were incubated overnight in mouse anti-Pax7 IgG1 (1:100 dilution, Developmental Studies Hybridoma Bank, Iowa City, IA) and rabbit anti-CD9 IgG (1:100 dilution, SA35-08, Invitrogen), followed by incubation in goat anti-mouse biotin-conjugated secondary antibody (dilution 1:1,000, 115-065-205; Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor 647 goat anti-rabbit IgG (1:250 dilution, A32733, Invitrogen) for 1 h at room temperature. Next, sections were incubated with streptavidin-HRP (1:500 dilution, S-911, Invitrogen) and Texas Red-conjugated Wheat Germ Agglutinin (WGA) (1:50 dilution, W21405, Invitrogen) at room temperature for 1 h, before incubation in Tyramide Signal Amplification (TSA) Alexa Fluor 488 (1:500 dilution, B40953, Invitrogen). Sections were mounted with VectaShield fluorescent mounting media with DAPI (H-1200-10, Vector Laboratories).

Images were captured with a Zeiss upright microscope (AxioImager M1, Oberkochen, Germany). To quantify the percentage of nuclei (DAPI+) expressing CD63, MyoVision software was used for automated analysis of nuclear density in cross-sections [39], and nuclei-expressing CD63 (identified as DAPI+/CD63+ events) were counted manually in a blinded manner by the same assessor for all sections using the Zen Blue software.

Statistical analysis

Differences between the two groups (HS vs WB) were analyzed by unpaired Student’s t-tests. When comparing 4 groups, a one-way ANOVA was used, with Tukey’s multiple comparisons test for post hoc analysis. A two-way ANOVA was used to assess the differences in particle concentrations between WB and HS across fractions of the density gradient. Paired t-tests were used to examine the changes in measures from pre- to post-immobilization in human samples. All statistical analyses were performed in GraphPad Prism (v7.00, GraphPad Software, La Jolla, CA), and statistical significance was set at an α < 0.05.