Extracellular Ca2+ drives cytoskeletal remodeling and cell swelling following nanosecond pulsed electric field exposure

Cell swelling and cytoskeletal disruption are known to be secondary effects of cell membrane permeabilization induced by nanosecond pulsed electric fields (nsPEFs). In this study, we used healthy and cancer urothelial cells to investigate the role of Ca²⁺ influx on cytoskeleton remodeling and morphological changes of cells following exposure. A train of 200 nsPEFs (300 ns pulse duration, 10 Hz), delivered via contact electrodes, effectively permeabilized the cell membrane in an isosmotic physiological solution. Subsequent shrinkage of the actin cortex and a reduction in actin fluorescence were observed only in the presence of extracellular Ca²⁺. In its absence, no significant changes in the phalloidin-stained actin cortex were detected. Time-lapse imaging using scanning ion conductance microscopy (SICM) revealed that a significantly greater and more immediate increase in projected cell area and cell volume occurred after nsPEFs exposure in a solution containing Ca²⁺ compared to a solution without Ca²⁺. These findings demonstrate that Ca²⁺ is a key driver of actin cytoskeleton disintegration and morphological changes following membrane permeabilization with nsPEFs.