Activation of mechanosensitive transcription factors in murine C2C12 myoblasts by focused low-intensity pulsed Ultrasound (FLIPUS)

Quantitative Ultrasound (QUS) is a promising technique for bone tissue evaluation. Focused transducers used for QUS also have a capability to be applied for tissue-regenerative purposes and can provide spatially limited deposition of acoustic energy. We describe a focused LIPUS (FLIPUS) system, which has been developed for the stimulation of cell monolayers in the defocused far field of the transducer through the bottom of the well plate. Tissue culture well plates, carrying the cells, were incubated in a special chamber, immersed in a temperature controlled water tank. The ultrasound was introduced at 3.6 MHz, pulsed at 100 Hz repetition frequency with 27.8% duty cycle. The calibrated output intensity corresponded to ISATA = 44.5 ± 7.1 mW/cm 2 . In this study we evaluated mechanical response of murine C2C12 myoblastic cells to ultrasound. FLIPUS induced 1.5 fold increase (p<0.05) in AP-1 promoter activity already after 5-minute of FLIPUS exposure. The Sp1 binding was enhanced by about 20% (p<0.05) after 5-min FLIPUS stimulation and the trend persisted for 30 min (p<0.01) and 1 hour (p<0.01) stimulation times. Increase of 70% in activation of TEAD-binding sequences (p<0.01) was observed after the 5 min FLIPUS treatment. The proliferation of the C2C12 cells was up-regulated after the 5-minlong FLIPUS exposure. These results imply the pro-proliferative nature of the selected FLIPUS-intensity in C2C12s, with potential functional role of the described mechanosensitive transcription factors. The use of FLIPUS as a visualization and regenerative technique can be further applied in clinical settings as a device providing monitoring and healing of the damaged tissue.