MicroRNAs in Extracellular Vesicles in Sweat Change in Response to Endurance Exercise

Abstract

Background: To date, microRNAs (miRs) carried in extracellular vesicles (EVs) in response to exercise have been studied in blood but not in non-invasively collectable body fluids. In the present study, we examined whether six exercise-responsive miRs, miRs-21, -26, -126, -146, -221, and -222, respond to acute endurance exercise stimuli of different intensities in sweat.

Methods: We investigated the response of miRs isolated from sweat and serum EVs to three endurance exercise protocols: (1) maximal aerobic capacity (VO_{2 max} ), (2) anaerobic threshold (AnaT), and (3) aerobic threshold (AerT) tests. Sauna bathing was used as a control test to induce sweating through increased body temperature in the absence of exercise. All protocols were performed by the same subjects (n = 8, three males and five females). The occurrence of different miR carriers in sweat and serum was investigated via EV markers (CD9, CD63, and TSG101), an miR-carrier protein (AGO2), and an HDL-particle marker (APOA1) with Western blot. Correlations between miRs in sweat and serum (post-sample) were examined.

Results: Of the studied miR carrier markers, sweat EV fractions expressed CD63 and, very weakly, APOA1, while the serum EV fraction expressed all the studied markers. In sweat EVs, miR-21 level increased after AerT and miR-26 after all the endurance exercise tests compared with the Sauna (p < 0.050). miR-146 after AnaT correlated to sweat and serum EV samples (r = 0.881, p = 0.004).

Conclusion: Our preliminary study is the first to show that, in addition to serum, sweat EVs carry miRs. Interestingly, we observed that miRs-21 and -26 in sweat EVs respond to endurance exercise of different intensities. Our data further confirmed that miR responses to endurance exercise in sweat and serum were triggered by exercise and not by increased body temperature. Our results highlight that sweat possesses a unique miR carrier content that should be taken into account when planning analyses from sweat as a substitute for serum.

Keywords: acute exercise response; circulating microRNA; leukocyte; sauna; serum.

FIGURE 1

Schematic illustration of the study setup. After eligibility screening subjects (n = 8) went through a familiarization period followed by following tests in the same order: (1) VO_2max test, (2) Sauna, (3) anaerobic threshold endurance test (AnaT), and (4) aerobic threshold endurance test (AerT). Same sample set was harvested in each of the tests.

FIGURE 2

Western blot (WB) images from pooled (n = 4/group) EVs isolated from sweat and serum (post-samples). EV marker CD63 was detected both from sweat (A) and serum (B) samples, whereas EV markers CD9 and TGS101 were detected only from serum samples (D,F vs. C,E). Similarly, AGO2 was only present in serum samples (G vs H). HDL marker APOA1 was clearly present in serum samples (J) and showed weak positive signal also from some of the sweat samples (I).

FIGURE 3

miR expression in sweat EVs in control (Sauna) or endurance exercise tests (n = 6–8). The expression of miR-21 was increased after AerT (A), whereas miR-26 was increased after VO_2max, AnaT and AerT tests compared with sauna (B). There were no differences in miRs -126 (C), -146 (D), -221 (E) or -221 (F). *p ≤ 0.050. Figure shows mean + SEM.

FIGURE 4

miR expression in serum EVs before and after control (Sauna) or endurance exercise tests (n = 7–8). The expression of miR-21 and miR-222 were increased after AnaT (A,F). There were no changes in the rest of the studied miRs in any of the exercise tests (B–E). *p ≤ 0.050. Figure shows mean + SEM.