Activation of Mechanosensitive Transcription Factors in Murine C2C12 Mesenchymal Precursors by Focused Low-Intensity Pulsed Ultrasound (FLIPUS).

In this study we investigated the mechanoresponse of C2C12 mesenchymal precursor cells to focused low-intensity pulsed ultrasound (FLIPUS). The set-up has been developed for in-vitro stimulation of adherent cells in the defocused far field of the ultrasound propagating through the bottom of the well plate. Twenty-four-well tissue culture plates, carrying the cell monolayers, were incubated in a temperature controlled water tank. The ultrasound was applied at 3.6 MHz frequency, pulsed at 100 Hz repetition frequency with a 27.8% duty cycle, and calibrated output intensity of ISATA = 44.5 ± 7.1 mW/cm2. Numerical sound propagation simulations showed no generation of standing waves in the well plate. The response of murine C2C12 cells to FLIPUS was evaluated by measuring activation of mechanosensitive transcription factors, i.e., activator protein 1 (AP-1), specificity protein 1 (Sp1) and transcriptional enhancer factor (TEF or TEAD), and expression of mechanosensitive genes, i.e., c-fos, c-jun, HB-GAM, and Cyr-61. FLIPUS induced 50% (p ≤ 0.05) and 70% (p ≤ 0.05) increases in AP-1 and TEAD promoter activities, respectively, when stimulated for 5 minutes. The Sp1 activity was enhanced by about 20% (p ≤ 0.05) after 5-min FLIPUS exposure and the trend persisted for 30 min (p ≤ 0.05) and 1 hour (p≤0.05) stimulation times. Expressions of mechanosensitive genes c-fos (p ≤ 0.05), c-jun (p ≤ 0.05), HB-GAM (p ≤ 0.05) and Cyr61 (p ≤ 0.05) was enhanced in response to 5-min FLIPUS stimulation. The increase in proliferation of C2C12s occurred after the FLIPUS stimulation (p ≤ 0.05), with AP-1, SP1 and TEAD possibly regulating the observed cellular activities.

PMID: 27392348 DOI: 10.1109/TUFFC.2016.2586972