Diffuse, non-polar electropermeabilization and reduced propidium uptake distinguish the effect of nanosecond electric pulses

Abstract

Ca2+ activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca2+ transients was expectedly higher for nsEP (36 kV/cm) than for msEP (0.09 kV/cm) but the respective doses were similar (190 and 460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca2+ level by >10 nM in cells with blocked voltage-gated Ca2+ channels and depleted Ca2+ depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These electric field values were at 150-230% of stimulation thresholds for both msEP and nsEP, notwithstanding a 400,000-fold difference in pulse duration. For comparable levels of electroporative Ca2+ uptake, msEP caused at least 10-fold greater uptake of propidium than nsEP, suggesting increased yield of larger pores. Electroporation by msEP started Ca2+ entry abruptly and locally at the electrode-facing poles of cell, followed by a slow diffusion to the center. In a stark contrast, nsEP evoked a "supra-electroporation" pattern of slower but spatially uniform Ca2+ entry. Thus nsEP and msEP had comparable dose efficiency, but differed profoundly in the size and localization of electropores.

Keywords: Calcium activation; Cardiomyocytes; Electropermeabilization; Electroporation; Nanosecond electric pulses.

Fig. 1

Spontaneous, nsEP-induced, and msEP-induced Ca²⁺ transients in rat embryonic cardiomyocytes. A–C: different individual cells subjected to subthreshold (A) and suprathreshold stimuli (B, C). Stimulation parameters are given in the legends. Vertical dotted lines show when the stimuli were applied. Insets show typical shapes of nsEP and msEP.

Fig. 2

Intense msEP and nsEP cause sustained elevation of cytosolic Ca²⁺ and trigger asynchronous firing in previously quiescent cells. Stimulation parameters are given in the legends; vertical dotted lines show when the stimuli were applied. Horizontal dashed lines show the basal level of Ca²⁺ (about 100 nM). In three different cells, msEP (A) and nsEP (B, C) caused a lasting but reversible Ca²⁺elevation. A: A recovered cell generates regular Ca²⁺ transients in response to non-electroporative stimuli. B and C: Asynchronous Ca²⁺ oscillations depend on the entry of extracellular Ca²⁺ through L-type Ca²⁺ channels. They are reversibly blocked by nifedipine or perfusion by a Ca²⁺-free solution (horizontal bars).

Fig. 3

A cocktail of caffeine, CPA, and verapamil fully blocks Ca²⁺ transients in response to previously effective stimuli and enables the observation of electroporative Ca²⁺ entry after more intense stimuli. A and B: representative experiments with msEP and nsEP, respectively. The perfusion with the cocktail (horizontal bar) transiently increases the cytosolic Ca²⁺, followed by its drop below the basal level. An inset zoom (B) shows a small Ca²⁺ response with 10x magnification. See Fig. 2 for other details.

Fig. 4

The amplitude of electroporative Ca²⁺ entry as a function of stimulus intensity for msEP and nsEP. A: 10-ns pulses require much higher electric field to cause electroporation than 4-ms pulses. Measurements were performed in cells blocked with 10 mM caffeine, 10 μM CPA, and 10 mM verapamil. The horizontal dotted line at 0.01 μM delimits the lowest detectable Ca²⁺ increase, so the datapoints falling below this line are considered subthreshold for electroporation. Mean values +/− s.e. for 5–8 experiments; the error bars may be not visible when they are smaller than the central symbol. Ca²⁺ entry for datapoints above the dotted line was statistically significant at p<0.05 or better. The labels next to the datapoints are the respective dose values, in J/g. The dashed lines are the best power function fits of data above 0.01 μM. Two open symbols at 0 Ca²⁺ uptake are the values subthreshold for stimulation (in cells not blocked with the drugs). B: same data expressed in % to the minimum electric field known to be effective for stimulation of unblocked cells, namely 0.09 and 36 kV/cm for msEP and nsEP, respectively. The dashed area is the common power function fit and the shaded corridor sets the limits of a 95% confidence interval. See text for more details.

Fig. 5

Propidium uptake triggered by nsEP and msEP at intensities equipotent for electroporative Ca²⁺ uptake. A: propidium uptake was studied at 3 different msEP and nsEP intensities, which were chosen to cause the same Ca²⁺ entry by electroporation (see text and Fig. 4). The uptake of propidium (as measured 9 min after the stimulus) from nsEP was reduced significantly (p<0.01) while Ca²⁺ uptake was the same. Mean values +/− s.e for 5–8 experiments. Labels indicate the electric field applied, kV/cm. B: sample traces of propidium uptake caused by nsEP and msEP at the indicated E-field. Vertical dotted line marks the application of the pulse.

Fig. 6

The difference between conventional electroporation (top) and supra-electroporation (bottom) is visualized by different patterns of Ca²⁺ entry. Cardiomyocytes were incubated with the same inhibitors as in Fig. 4. Cells were permeabilized to Ca²⁺ by a single 4-ms pulse (top) or a 10-ns pulse (bottom). Shown are representative images at indicated time points after the electric shock. Ca²⁺ concentration is coded by intensity-modulated pseudocolor. The positions of anode and cathode stimulating electrodes are designated in the first panel by “+” and “−”. Note Ca²⁺entry from the cell poles after 4-ms pulse, and entry without apparent localization after the 10-ns pulse. See text for more details. Drawings above and below the cell images show a vertical cross-section of a cell in the plane between two electrodes; arrows point to sites of Ca²⁺ entry after msEP and nsEP, respectively.

Fig. 7

Different patterns of cytosolic Ca²⁺ increase after electroporation by msEP (left panels) and nsEP (right panels). Each panel shows data for one representative cell stimulated by a single pulse (dotted vertical line, at 0 s) at the indicated electric field. In each cell, Ca²⁺ level was monitored in three regions selected at the anode-facing pole of the cell (a), at the cathode-facing pole (c), or in the middle of the cell (m). With 4-ms pulses, Ca²⁺ entry starts from the poles followed by slow diffusion to the center. With 10-ns pulses, Ca²⁺ increased synchronously in all measured regions.