Evaluating muscular membrane perturbation upon pulsed electric field exposure

Nanosecond pulsed electric fields (nsPEF) are high voltage (1-15 kV/cm) nanosecond energy waveforms that can impact cellular activity. On a physical level, a nsPEF generates transient membrane perturbations in the form of nanopores to allow cation influx resulting in localized membrane depolarization. On a physiological level, a nsPEF exposure can activate receptors and channels on the membrane as well as second messenger cascades, both of which results in subcellular modulation that lasts beyond the nsPEF duration. An ongoing challenge is to characterize the extent/sequence of physiological events induced by nsPEF exposure, and potential to interplay with physical effects induced by the pulse. In our laboratory, C2C12 mouse myoblast cells have been demonstrated to be a useful in vitro model, as it is feasible to differentiate these immortalized progenitors into terminally transformed myotubes. From previous efforts, we quantified YO-PRO -1 (YO-PRO-1) uptake as a measurement of membrane perturbation, and concluded that membrane damage is proportional to applied pulsed electric field voltage. To expand upon these findings, we evaluated to what extent YOPRO-1 uptake at the membrane is physical or physiological in nature. Interestingly, the P2X7 receptor complex has been extensively studied utilizing YO-PRO-1 uptake as marker of apoptotic activity. For this reason, we tested the role of P2X7 receptor complex activation to mediate YO-PRO-1 uptake during pulsed electric field exposure. By blocking the P2X7 receptor, we reduced nsPEF-induced YO-PRO-1 uptake by 31.57%. Our results demonstrate that the P2X7 receptor complex is a subcellular candidate responsible for YO-PRO-1 uptake upon nsPEF exposure in myotubes.