Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training.
Author(s): Kargl CK, Sterczala AJ, Santucci D, Conkright WR, Krajewski KT, Martin BJ, Greeves JP, O'Leary TJ, Wardle SL, Sahu A, Ambrosio F, Nindl BC
Publication: Physiol Rep, 2024, Vol. 12, Page e16016
PubMed ID: 38697940 PubMed Review Paper? No
Purpose of Paper
This paper compared extracellular vesicle (EV) abundance, size, protein content, subtype populations, and miRNA expression among EVs obtained from plasma collected from healthy male and female volunteers before and after an acute heavy resistance exercise training (AHRET) workout that occurred before and after a 12-week training program. The authors also compared Protein kinase B (AKT) expression in muscle biopsies collected before and after the 12-week training program.
Conclusion of Paper
As expected, female volunteers in the study were on average shorter, with lower body mass, a higher percentage body of fat, a lower VO2peak, and a lower repetition maximum (RM) than male volunteers both before and after the 12-week training program. Both male and female volunteers gained body mass and increased squat RM and total weight during AHRET while maintaining percentage body fat after the 12-week training program. There was a significant interaction of chronic training and acute exercise on EV abundance and protein content. EV abundance and protein concentration were higher in plasma specimens obtained from men post-AHRET relative to pre-AHRET after completing the 12-week training program but did not differ pre-and post-AHRET when measured before the training program was initiated. In contrast, pre- and post-AHRET measurements of EV abundance and protein concentration did not differ in females either before initiation of the training program or after its conclusion. The mean size of EVs was unaffected by AHRET or the training program and there was no difference in mean size between EVs from male and female volunteers. AHRET affected the proportion of SGCA+, CD9+ and VAMP3+ EVs but the effect on CD9+ EVs was sex and training dependent.
The concentration of miRNA isolated from EVs was higher in specimens from women than men and increased after training. Eighteen of the 25 most abundant miRNAs were consistent across sexes and all timepoints (pre-/post-AHRET and before and after completion of a training program). The authors identified miRNAs differentially expressed after training in men and women and found overrepresentation of miRNAs involved in signal transduction, metabolism, endocrine signaling, cell growth and protein turnover, but only the “Adherens junction pathway” was affected by training in all four groups (men and women, pre- and post-AHRET). Further investigation found that miRNAs in pathways involved with muscle, bone, connective tissue, inflammation, and metabolism displayed changes with AHRET and training. There was an increase in AKT protein post-training relative to pre-training in muscle biopsies from men but not women.
Studies
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Study Purpose
This study compared extracellular vesicle (EV) abundance, size, protein content, subtype populations, and miRNA expression among EVs obtained from plasma collected from healthy male and female volunteers before and after an acute heavy resistance exercise training (AHRET) workout that occurred before and after a 12-week training program. The authors also compared AKT expression in muscle biopsies collected before and after the 12-week training program. The study included specimens from 18 healthy volunteers (9 men and 9 women; aged 18-36 years) who had performed 30 min physical exercise ≥3 times a week but were not training for a competitive event and had not had a weight fluctuation ≥10 lbs in 2 months. Blood and a muscle biopsy specimen were collected before, and blood specimens were also collected after, an AHRET workout consisting of six sets of ten back squats at 75% repetition maximum (RM) both at the start of the study and after 12 weeks of a concurrent resistance and endurance exercise training program. Blood was collected using 21- or 23-gauge needles into EDTA Vacutainer tubes, and plasma was separated by centrifugation at 1500 × g for 15 min at 4°C and stored at -80°C. After thawing on ice, EVs were isolated from plasma by size exclusion chromatography using Generation 2 qEV1 70 nm columns and concentrated using Protein concentrator tubes with 50 kDa filters before storage at -80°C. EV yield and size profile were analyzed by nanoparticle tracking analysis. EV protein content was analyzed by BCA assay. EV population proportions were analyzed by flow cytometry using antibodies against CD9, VAMP3, and thrombospondin (THSD-1). RNA was extracted from EVs using the Plasma/Serum Exosome Purification and RNA Isolation Mini Kit and stored at −80°C. RNA was evaluated using a 2100 Bioanalyzer, sequencing libraries were prepared using the QIAseq miRNA library kit and then sequenced on a NextSeq500 instrument. Reads were mapped to the miRNAs using the Qiagen GeneGlobe RNA-seq analysis portal and differential expression analysis was performed using edgeR. Pathway analysis was conducted using DIANA mirpath v.3 and the Ingenuity Pathway Analysis software. Muscle biopsies were collected from the vastus lateralis using a modified Bergstrom needle technique prior to AHRET. Connective tissue and fat were removed from all biopsies before flash freezing in liquid nitrogen and storage at -80°C. Tissue specimens were homogenized at 4°C in T-PER lysis buffer containing HALT protease and a phosphatase inhibitor cocktail using a Bullet Blender Tissue Homogenizer. Lysates were centrifuged at 14,000 × g for 5 min at 4°C, and supernatant protein was quantified by BCA and AKT was quantified by Western Blot.
Summary of Findings:
As expected, female volunteers in this study were on average shorter, with a lower body mass, a higher percentage of body fat, a lower VO2peak, and a lower RM than male volunteers both before and after the training program; however, both male and female volunteers gained body mass and increased squat RM and total weight during AHRET while maintaining the same percentage body fat after the 12-week program. There was a significant interaction between chronic training and acute exercise on EV abundance and protein content (P=0.048 and P=0.0133, respectively). EV abundance and protein concentration were higher in specimens obtained from men post-AHRET relative to pre-AHRET after completing the 12 week training program (P=0.042 and P=0.019), but did not differ pre-and post-AHRET when measured before the training program was initiated. In contrast, pre- and post-AHRET measurements of EV abundance and protein concentration did not differ in females either before initiation of the training program or after its conclusion. The mean size of EVs was unaffected by AHRET or training and the mean size of EVs did not differ when male and female volunteers were compared.
After AHRET, there was a 33% decrease in the proportion of SGCA+ (muscle-derived) EVs relative to pre-AHRET specimens (P=0.023), and this decrease was independent of training and volunteer sex. A significant interaction between volunteer sex, training program, and AHRET on the proportion of CD9+ EVs was identified (P=0.019). In specimens from men, CD9+ EVs increased post-AHRET prior to the initiation of the training program but decreased post-AHRET after the 12 weeks of training. Interestingly, a trend toward the reverse was observed in females, as CD9+ EVs decreased post-AHRET prior to the initiation of the training program but increased post-AHRET after 12 weeks of training. Following AHRET, there was a 27% decrease in the proportion of VAMP3+ microvesicles (P=0.022) before the training program. The proportion of THSD-1+ apoptotic bodies was unaffected by AHRET or the training program.
The concentration of miRNA from EVs was 184% higher in specimens from women than men (P=0.0242) and increased after the 12-week training program (P=0.0309). Eighteen of the 25 most abundant miRNAs were consistent across sexes and all timepoints (pre-/post-AHRET and pre-/post- the 12 week training program). The authors identified miRNAs differentially expressed after training in men and women and found overrepresentation of miRNAs involved in signal transduction, metabolism, endocrine signaling, cell growth, and protein turnover, but only the “Adherens junction pathway” was affected by the 12-week training in all four groups (men and women, pre- and post-AHRET). Further investigation found that miRNAs in pathways involved with muscle, bone, connective tissue, inflammation, and metabolism displayed changes with AHRET and the training program. In muscle biopsies there was an increase in AKT protein post-training relative to pre-training in men but not women.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Light scattering RNA Next generation sequencing Protein Western blot Protein Colorimetric assay Protein Flow cytometry Cell count/volume Flow cytometry RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Pre-AHRET before training program
Post-AHRET before training program
Pre-AHRET after 12 week training program
Post-AHRET after 12 week training program
Preaquisition Patient gender Female
Male