Effects of high‐intensity ultrasound irradiation on exosomes release from cultured myotubes

Abstract

Purpose Exosomes from skeletal muscle have endocrine actions and exert therapeutic effects on other organs. Intracellular calcium induces the exosomes release from cells, and ultrasound irradiation is also known to increase the intracellular calcium in cells. The aim of this study was to investigate the effects of ultrasound irradiation on the release of exosomes from myotubes. Methods C2C12 myotubes were exposed to ultrasound intensity of 0, 1, 2, or 3 W/cm2 (frequency; 1 MHz, and duty cycle; 20%) for 5 minutes. Exosomes were isolated from the culture medium by ultracentrifugation. The concentration of exosomes and positive rate of CD63, an exosome marker, were analyzed by Nanoparticles Tracking Analysis and flow cytometry, respectively. P < 0.05 was considered statistically significant (Tukey-Kramer test). Results The concentration of exosomes in the ultrasound intensity at 3 W/cm2 was 2.6 times higher than that in control without ultrasound irradiation and the concentration of Ca2+ at 6 hours after stimulation compared to control. In addition, the concentration of exosomes in the ultrasound intensity at 2 W/cm2 was 1.9 times higher than control and did not significantly increase the concentration of calcium. The concentration level of exosomes in ultrasound at 1 W/cm2 was equivalent to that of control. The positive rate of CD63 was more than 80% in all groups. Cell viability did not decrease among all groups. Discussion This study found the facilitatory effects of high-intensity ultrasound on the release of exosomes from myotubes. Intracellular calcium level has been reported as a factor to promote exosomes release, and high-intensity ultrasound promoted the intracellular calcium in the present study. These results suggested the high-intensity ultrasound could facilitate calcium influx in myotubes, resulting in the enhanced release of exosomes. Conclusion High-intensity ultrasound irradiation could increase intracellular calcium level and promote the release of exosomes from cultured myotubes.