Plasma-derived extracellular vesicles released after endurance exercise exert cardioprotective activity through the activation of antioxidant pathways

Abstract

Cardiovascular diseases (CVD) can cause various conditions, including an increase in reactive oxygen species (ROS) levels that can decrease nitric oxide (NO) availability and promote vasoconstriction, leading to arterial hypertension. Physical exercise (PE) has been found to be protective against CVD by helping to maintain redox homeostasis through a decrease in ROS levels, achieved by increased expression of antioxidant enzymes (AOEs) and modulation of heat shock proteins (HSPs). Extracellular vesicles (EVs) circulating in the body are a major source of regulatory signals, including proteins and nucleic acids. Interestingly, the cardioprotective role of EVs released after PE has not been fully described. The aim of this study was to investigate the role of circulating EVs, obtained through Size Exclusion Chromatography (SEC) of plasma samples from healthy young males (age: 26.95 ± 3.07; estimated maximum oxygen consumption rate (VO_2max): 51.22 ± 4.85 (mL/kg/min)) at basal level (Pre_EVs) and immediately after a single bout of endurance exercise (30' treadmill, 70% heart rate (HR) -Post_EVs). Gene ontology (GO) analysis of proteomic data from isolated EVs, revealed enrichment in proteins endowed with catalytic activity in Post_EVs, compare to Pre_EVs, with MAP2K1 being the most significantly upregulated protein. Enzymatic assays on EVs derived from Pre and Post samples showed increment in Glutathione Reductase (GR) and Catalase (CAT) activity in Post_EVs. At functional level, Post_EVs, but not Pre_EVs, enhanced the activity of antioxidant enzymes (AOEs) and reduced oxidative damage accumulation in treated human iPS-derived cardiomyocytes (hCM) at basal level and under stress conditions (Hydrogen Peroxide (H₂O₂) treatment), resulting in a global cardioprotective effect. In conclusion, our data demonstrated, for the first time, that a single 30-min endurance exercise is able to alter the cargo of circulating EVs, resulting in cardioprotective effect through antioxidant activity.

Keywords: Antioxidant activity; Cardioprotection; Catalytic activity; Extracellular vesicles; Physical exercise; Redox capacity.

Fig. 1

A) Schematic representation of the methods B) Particles total number and diameter (nm) in plasma before isolation. Statistical significances were determined using unpaired t-test (*p < 0.05 and ****p < 0.0001); C) Representative Particle Metrix concentration and size distribution of isolated EVs; D) TEM images of Pre_EVs and Post_EVs; E) Immunoblot analysis of EVs markers and plasma contaminant; F) SPRi report.

Fig. 2

A) PCA of Pre_EVs and Post_EVs; B) Vulcano plot of differential expressed protein from Post_EVs vs Pre_EVs; C) Enrichment analysis of Post_EVs upregulated proteins.

Fig. 3

A) Analysis of GR and CAT activity on Pre_EVs and Post_EVs; B) ROS concentration (measured as DCF fluorescent signal) on Pre_EVs and Post_EVs; Statistical significances were determined using unpaired t-test (***p < 0.001, ****p < 0.0001). C) analysis of GR, CAT, GPx and G6PD activity in hCM + PBS, hCM + Pre_EVs and hCM + Post_EVs; D) TBARS levels in hCM + PBS, hCM + Pre_EVs and hCM + Post_EVs; E) ROS concentrations (measured as DHE fluorescent signal) in hCM + PBS, hCM + Pre_EVs and hCM + Post_EVs; Statistical significance were determined using one-way anova (*p < 0.05,****p < 0.0001).

Fig. 4

A) pHSP27 immunoblot analysis on hCM + PBS, hCM + Pre_EVs and hCM + Post_EVs. B) Representative images of hCM + PBS, hCM + Pre_EVs, hCM + Post_EVs and Ctrl+ (hCM treated with H2O2) immunostained for NRF2 (red), cardiac-troponin T (cTnT-green) and DAPI; On the right panel, quantification of NRF2 nuclear translocation. Statistical significance were determined using one-way anova (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

A) ROS concentrations (measured as DHE fluorescent signal) and Viability (measured as CCK8 activity) analysis on hCM + PBS vs hCM + H₂O₂; B) ROS concentrations (measured as DHE fluorescent signal) in hCM + H₂O₂vs hCM + H₂O₂+Post_EVs; C) Vitality (measured as CCK8 activity) analysis in hCM + H₂O₂vs hCM + H₂O₂+Post_EVs; D) GR activity in hCM + H₂O₂vs hCM + H₂O₂+Post_EVs; Statistical significances were determined using unpaired t-test (*p < 0.05, **p < 0.01,****p < 0.0001).

Supplementary Fig. 1

Schematic overview of blood sampling, exercise protocol and subject characteristics

Supplementary Fig. 2

Schematic representation of ROS detection (measured as DHE fluorescent signal) in hCM

Supplementary Fig. 3

RT-RealTime-PCR for SOD1; HMOX1; CAT and NQO1 genes on hCM + PBS, hCM + Pre_EVs and hCM + Post_EVs. Statistical significances were determined using unpaired t-test (*p < 0.05)