Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream

Abstract

In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and alpha-sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1-5% of the total and that 60-65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.

Fig 1. Density gradient separation of plasma EVs.

Plasma EVs were purified using the serial ultracentrifugation protocol. The obtained pellet was then further separated using the Optiprep iodixanol density gradient. Fractions containing total EVs were identified by Western blot analysis with antibodies against Tsg101 (a) while EVs originating from muscle tissue were identified by the anti-SGCA (b). MiR-206 expression levels were quantified from each fraction using a specific TaqMan MicroRNA probe, expression levels were normalized versus the spike-in cel-miR-39 exogenously added and reported as fold change compared to the fraction 6 (c).

Fig 2. Contour plot of plasma EVs.

EVs were evaluated (P3) using size beads, identified by °(1 μm), *(2 μm) and (5.2 μm) (a). This gate strategy was performed to define the proper gate for events smaller than 1μm (P3), including MVs. Selected events are shown in contour plots (c) and (d), from samples labelled by goat anti-mouse (GaM) FITC (FL1-A) and anti-SGCA+ GaM FITC. P4 represents the area of FL1 positivity, drawn taking into account cluster distribution into the “negative” sample (MVs labelled with GaM only). Percentages are related to the following subpopulations: Purple: SGCA+ events; Blue: events less than 1 μm; Green: CD81⁺/SGCA⁺ events, shown in Fig 2A and 2B.

Fig 3. Contour plots of size-selected EVs.

Briefly, green clusters are those gated on the basis of their smaller size and SGCA positivity (previous contour plots: green cluster) and visualized for: a) for CD81 (FL4-A) and FSC localization; b) CD81 (FL4-A) and SGCA positivity (FL1-A), contour plot emphasizing co-expression of both markers.

Fig 4. Isolation of alpha-sarcoglycan⁺ EVs from plasma using immunoaffinity capturing.

Anti-alpha-sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA⁺ sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = Cq_miR-206-Cq_miR-16) (b). Moreover, the quantification of miR-16 ratio in the SGCA⁺ sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = Cq_miR-16-Cq_cel-miR-39) (c). Asterisks denote significant changes (p<0.05).

Fig 5. Expression levels in specific EV miRNAs correlate with aerobic fitness.

For each volunteer (n = 18), baseline miRNA levels from plasma EVs under resting condition were assigned to the corresponding aerobic fitness level estimated as VO_2max. MiRNA expression levels were reported as -ΔCq (where ΔCq = Cq_miR-206-Cq_miR-16). A direct significant correlation (r = correlation coefficient) was observed between levels of miR-1, miR-133b, miR-206, miR-499 and miR-181a-5p (baseline) and VO_2max. MiR-24 expression; which is stably expressed in blood, is the negative control.

Fig 6. Modulation in circulating EV miRNAs in response to an aerobic exercise bout.

The expression levels of miR-1, -133a, -133b, -146a, -181a-5p, -206, and -499 were evaluated in the circulating EVs of healthy volunteers (n = 7) at rest (Rest) and 1-hour after a 45-min aerobic exercise bout at 65% VO_2max (Post-Exercise). MiRNA expression levels were reported as -ΔΔCt obtained subtracting the ΔCq at rest (where ΔCq = Cq_miR-206-Cq_miR-16) from the ΔCq Post-Exercise. MiR-24 is the negative control. Asterisks denote significant changes (p<0.05).