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

Abstract

Concurrent resistance and endurance exercise training (CET) has well-studied benefits; however, inherent hormonal and genetic differences alter adaptive responses to exercise between sexes. Extracellular vesicles (EVs) are factors that contribute to adaptive signaling. Our purpose was to test if EV characteristics differ between men and women following CET. 18 young healthy participants underwent 12-weeks of CET. Prior to and following CET, subjects performed an acute bout of heavy resistance exercise (AHRET) consisting of 6 × 10 back squats at 75% 1RM. At rest and following AHRET, EVs were isolated from plasma and characteristics and miRNA contents were analyzed. AHRET elevated EV abundance in trained men only (+51%) and AHRET-induced changes were observed for muscle-derived EVs and microvesicles. There were considerable sex-specific effects of CET on EV miRNAs, highlighted by larger variation following the 12-week program in men compared to women at rest. Pathway analysis based on differentially expressed EV miRNAs predicted that AHRET and 12 weeks of CET in men positively regulates hypertrophy and growth pathways more so than in women. This report highlights sex-based differences in the EV response to resistance and concurrent exercise training and suggests that EVs may be important adaptive signaling factors altered by exercise training.

Keywords: exercise; extracellular vesicles; sex differences.

FIGURE 1

Schematic overview of study methodology. Prior to the chronic training program subjects had blood draws taken before and after an acute bout of heavy resistance exercise training (AHRET, acute bout of heavy resistance exercise; 6 × 10 at 75% 1 RM on the back squat). Subjects then underwent 12 weeks of resistance exercise training, following which they repeated the AHRET bout. Extracellular vesicles (EVs) were isolated from plasma samples via a combined size exclusion chromatography and ultrafiltration technique (SEC‐UF). EV characteristics and contents were analyzed via Nanoparticle Tracking Analysis, Imaging Flow Cytometry, and small RNA sequencing. Figure generated using BioRender.

FIGURE 2

Acute exercise elevates EV release in trained men but not women. EV particle concentration (particles/mL plasma) and mean size (nm) as measured by Nanoparticle Tracking Analysis (NTA; a and b). Representative image of EV particle size distribution as measured by NTA (c). Protein concentration of isolated EVs as measured by BCA assay (d). Concentration data are displayed per mL of plasma used for EV isolation. N = 9. Statistical testing was done via three‐way ANOVA.

FIGURE 3

EV surface marker expression is altered by AHRET in men and women. The proportion of EVs expressing SGCA (a), CD9 (b), VAMP3 (c), and THSD (d) were measured via Imaging Flow Cytometry and are shown as percentage of total gated EVs. Representative images of EV particles with bright‐field (BF) image of each particle and subsequent fluorescent channel images showing presence or absence of muscle‐derived EVs (SGCA+), microvesicles (VAMP3+), exosomes (CD9+) and apoptotic bodies (THSD+) vesicles are shown (e). N = 9. Statistical testing was done via three‐way ANOVA.

FIGURE 4

Sex‐based differences in EV miRNA contents in response to chronic exercise training. EV miRNA contents at each timepoint as measured via Bioanalyzer (a). Venn diagram depicting the 25 most enriched miRNAs contained within plasma‐EVs, before and after training in resting men and women (b). Volcano plots of statistical significance (p‐value) versus magnitude of change (fold change of POST vs PRE training modality) for chronic training comparisons in men and women. Black dots represent miRNAs that were significantly higher following the specific training and red dots represent miRNAs that were significantly lower following training (p ≤ 0.05). The number of up‐ and downregulated miRNAs after training are depicted in each graph (c). Total EV miRNA statistical analysis was performed with a three‐way ANOVA. Differentially expressed miRNAs were identified using edgeR software. N = 5. Statistical significance set at p = 0.05.

FIGURE 5

General target pathways of differentially expressed EV miRNAs. The top 10 most significant KEGG pathways containing target genes of the differentially expressed miRNAs for each of the four chronic exercise comparisons in men and women, determined using DIANA miRPath (a–d). Pathways are grouped into general KEGG designations and then sorted by p‐value. A Venn diagram depicts the overlapping target pathways between each of the comparisons (e).

FIGURE 6

Predicted regulation of exercise‐related pathways by EV miRNAs. Heatmaps were generated in Ingenuity Pathways Analysis depicting the predicted impact (z‐score) of EV miRNA changes (post vs pre) on Exercise‐relevant physiological functions (a) and intracellular signaling pathways (b). Red is predicted to be upregulated following chronic training and green is predicted to be downregulated. Depicted comparisons include effects of an acute bout of exercise, 12‐weeks of chronic training, and an acute bout of exercise following chronic training in men and women. A table depicting the average changes of prominent exercise‐related signaling pathways (hypertrophy and myokine) and exercise‐related physiological functions (muscle, bone, and metabolism) were compiled and averaged from the IPA generated heatmaps (c). Arrows pointing up denote significant upregulation, arrows pointing down denote significant downregulation, and sideways arrows depict no significant differences. Functions with multiple arrows had varied responses.

FIGURE 7

Predicted and measured Akt pathway regulation at rest following chronic resistance training in men and women. IPA generated schematic of the Akt pathway (a) in men (top) and women (bottom). Color coded symbols represent direction of predicted mRNA regulation by differentially expressed EV miRNAs from pre‐ to post‐chronic resistance exercise training and are described in the prediction legend. Total amount of Akt from biopsied skeletal muscle, normalized to total protein, before and after chronic resistance exercise training in resting men and women, respectively, measured via western blotting (b). Representative blot shown, blot single lane images have been vertically spliced to show resting conditions and male–female images side‐by‐side. Complete blot images can be found in supplementary materials. N = 5. Akt was analyzed via two‐way ANOVA.