Low-Intensity Ultrasound-Exposed Microbubbles Provoke Local Hyperpolarization of the Cell Membrane Via Activation of BKCa Channels

Ultrasound (US) contrast agents have gained wide interest in gene therapy as many researchers reported increased membrane permeability and transfection efficiency by sonoporation in the presence of US contrast agents. We recently demonstrated an increase in cell membrane permeability for Ca2+ in rat cardiomyoblast (H9c2) cells insonified in the presence of microbubbles. In the present study, we specifically investigated whether US-exposed microbubbles have an effect on the cell membrane potential and whether Ca2+-dependent potassium (BKCa) channels are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane. H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts with a frequency of 1 MHz with a peak-to-peak pressure of 0.1 or 0.5 MPa. Pulse repetition frequency was set to 20 Hz, with a duty cycle of 0.2%. Cells were insonified during 30 s in the presence of Sonovue™ microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-aminonaphtylethenylpyridinium (di-4-ANEPPS). The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BKCa channels. Surprisingly, despite the previously reported Ca2+ influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean intensity of fluorescence (MIF) to 118.6 ± 2.5% (p < 0.001, n = 267) at 0.1 MPa and 125.7 ± 5.9% (p < 0.001, n = 161) at 0.5 MPa at t = 74 s, respectively, compared with “no US” (100.3 ± 3.4%, n = 52). This hyperpolarization was caused by the activation of BKCa channels, as iberiotoxin completely prevented hyperpolarization. (MIFt74 = 100.6 ± 1.4%; p < 0.001, n = 267) and 0.5 MPa (MIFt74 = 88.8 ± 2.0%; p< 0.001, n = 193), compared with 0.1 and 0.5 MPa microbubbles without iberiotoxin. In conclusion, US-exposed microbubbles elicit a Ca2+ influx, which leads to activation of BKCa channels and a subsequent, local hyperpolarization of the cell membrane. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis. (E-mail: ljm.juffermans@vumc.nl)