Study of Extracellular Vesicles from Accessible Biological Fluids to Characterize Individual Responses to Physical Activity

Background: Regularly performed exercise promotes skeletal muscle health through the modulation of muscle regeneration, repair and remodelling. Unfortunately, not all individuals respond the same to an exercise protocol; some are highly trainable while others respond poorly or only marginally. To investigate the molecular mechanisms involved in interpersonal training variability, extracellular vesicles isolated from accessible biological fluids would represent a complex and dynamic diagnostic tool. Aim: The present project aims to develop a protocol for the isolation of EVs from athletes’ plasma, serum and saliva to characterize individual responses to physical activity. Methods: To isolate EVs, plasma and serum samples were collected from marathoners pre- and post-race and analysed by SEC, while saliva samples from football players pre- and post-training were processed by serial ultracentrifugation. NTA was utilized to measure EV size distribution and concentration, and BCA assay was performed for total protein content. Finally, Dot Blot analysis was employed for the antibody-based detection of different EV markers. Preliminary Results: For all athletes’ samples, NTA analysis showed typical EV size and distribution. Concerning marathoners’ samples, Dot Blot showed that most analysed targets (CD63, CD9, HSP60, CD171, CD56) increased in post-race. Regarding football players’ salivary EVs, Dot Blot revealed higher CD63 and HSP60 positivity levels in EXOs compared to MVs, suggesting that the protocol was able to properly isolate EV subpopulations. Moreover, CD63 and HSP60 increased in most post-exercise samples. Conclusions: The obtained results highlight an increase in EVs in response to physical activity, which could reveal metabolic changes and muscle adaptation to exercise. EVs could therefore represent an innovative source of exercise biomarkers that would allow the development of standardized exercise protocols both in a clinical and elite athlete setting.