Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions

Abstract

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca²⁺, Cd²⁺, Zn²⁺, and Ba²⁺ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca²⁺ and Ba²⁺ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5-2 kV/cm, much lower than >7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba²⁺ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca²⁺ entry could elicit calcium-induced calcium release (CICR) followed by Ca²⁺ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca²⁺ and Ba²⁺ proved as sensitive nanoporation markers, with Ba²⁺ being more reliable for monitoring membrane damage and resealing.

Keywords: electropermeabilization; electroporation; membrane integrity; membrane repair; nanopores; nsEP; nsPEF.

Figure 1

Effect of extracellular divalent cations on the fluorescence of CHO cells loaded with Calbryte (A) or Fluo-8 (B,C) dyes. The time-lapse imaging began in a physiological solution with 2 mM Ca²⁺ (considered as a baseline, 100%), which was replaced at 1–2 min by a modified solution with a divalent cation marked in the legend (see Methods for details). Note that 2 mM of Cd²⁺ or Zn²⁺ increased the emission of either dye, while Ca²⁺ and Ba²⁺ did not. Mean +/− s.e., n > 30 cells per group. Panel C shows fluorescence traces of the response to Zn²⁺ from several individual cells loaded with Fluo-8, without averaging.

Figure 2

Traces of Ca²⁺ (A,B) and Ba²⁺ (C,D) fluorescence change in CHO cells in response to a single 300-ns pulse at the indicated electric field strength (kV/cm). Images were taken every 10 s, and the pulse was delivered at 27 s into the experiment. Panels A and B show the same data at different vertical scales, to emphasize the statistically significant response already at 2.3 kV/cm (p < 0.01 compared to 0 kV/cm, two-tailed t-test) and its disproportional enhancement by the calcium-induced calcium release (CICR) once its threshold is exceeded (see text and [33] for more detail). Panel D uses the data from panel C to plot the fluorescence change per 10-s intervals between the sequential readings. The values were fitted with a double-exponential function (see text and [11] for detail). Mean +/− s.e., n = 30–45 cells for most groups.

Figure 3

The effect of the ionic environment on the detection of membrane permeabilization in CHO and BPAE cells (A,B and C,D, respectively). Cells were loaded with Calbryte dye. Images were taken every 10 s (CHO) or every 5 s (BPAE). A single 300-ns pulse at the indicated intensity (kV/cm) was delivered at 27 s. Panels B and D show the same data as A and C, respectively, but on an expanded vertical scale. The labels identify whether the extracellular solution contained 2 mM Ca²⁺; or 2 mM of Ba²⁺; or none of them was added deliberately (“nominal Ca²⁺”); or the free Ca²⁺ level was further reduced with EGTA (“0.5 mM EGTA” and “1 mM EGTA”). Sham exposures were performed for all ionic conditions, with similar results; shown are the data for sham exposure in 2 mM Ca²⁺ only. Mean +/ s.e., 10–20 cells per each condition.

Figure 4

Mobilization of intracellular Ca²⁺ by intense nanosecond pulsed electric field (nsPEF) in CHO (A) and BPAE (B) cells. The extracellular solution did not contain any added Ca²⁺ or Ba²⁺ and was supplemented with 0.5 mM EGTA (CHO) or 1 mM EGTA (BPAE). The electric field strength (kV/cm) for a single 300-ns pulse is indicated in the labels. See Figure 3 and text for more details.

Figure 5

The different time course of fluorescence in Calbryte-loaded CHO cells during 30 min after nsPEF in the medium with 2 mM Ca²⁺ (A,B) or Ba²⁺ (C). Images were taken once a minute. A single 300-ns pulse at 0 (sham exposure), 12.1, or 19 kV/cm was delivered at 122 s. Panels A and B show the same data, with an expanded vertical scale in B. Dashed lines are the polynomial (B) and linear (C) fits through the last 10–15 data points. Mean ± s.e., n = 22–38.

Figure 6

Dose-response and thresholds for the detection of membrane permeabilization by the entry of Ca²⁺ and Ba²⁺ ions or nucleic stains YO-PRO-1, TO-PRO-3, and propidium (Pr). The data were collected in CHO cells (A) and in two different sets of experiments in HEK cells (B and C). For Ca²⁺ and Ba²⁺ detection, cells were loaded with Calbryte dye (A and C) or with Fluo-4 (B). In all experiments, fluorescence images of cells were taken every 2–10 s for up to 4.5 or 9.5 min after nsPEF (traces not shown). Plotted are the values measured in the last image of each time series (except for Ca²⁺ data in panel B, which are the peak response values measured in 20–30 s after nsPEF). Note different units and baseline values for Ca²⁺ and Ba²⁺ detection (100%, Y-axes on the left) and for the detection of the nucleic stains (0 a.u., Y-axes on the right). The data points immediately above the baseline were fit with a linear function (dashed lines); the response thresholds were estimated by the intercept of the linear fit with the baseline. In panel A, the experiments with Ba²⁺ salts of different purity (99.9% and 99.999%) produced similar results and were pooled together for fitting. Asterisks designate the first measurement that was significantly above the baseline (p < 0.05 or better, one sample t-test). Mean +/− s.e., n = 14–40.

Figure 7

Permeabilization of CHO cells by a burst of 10, 300-ns pulses at 100 Hz. The membrane disruption was detected by a change in Calbryte fluorescence when cells were kept in a physiological solution with 2 mM Ba²⁺ (A,B) or by YO-PRO-1 fluorescence (C). Panel B shows sharp fluorescence peaks in individual cells exposed at 4.1 kV/cm and their average (+/− s.e.); the peaks were presumably caused by the mobilization of Ca²⁺ from the intracellular stores. Panel D shows the dose response for nsPEF effect traces in panels A and C, using the last data point in each time series. The electric field thresholds were estimated by linear fitting of the plotted values (see Figure 6 and text for more detail). Mean +/− s.e., n = 26–52.

Figure 8

Polarity effect in the permeabilization of HEK cells by a single 600-ns pulse at 10 kV/cm. Cells were loaded with Calbryte dye and incubated in the physiological solution with 2 mM Ca²⁺ (A,B) or 2 mM Ba²⁺ (C,D). The acquisition rate was 3048 frames/s and the pulse was delivered at zero time point. Panels B and D show the same traces as A and C, respectively, at a higher time resolution. Panel E shows the difference in the fluorescence measured at the anode- and cathode-facing poles of each individual cell, averaged for all cells in the group. The inset illustrates preferential anodic entry of Ca²⁺ in a representative cell. The first image taken in the bright field shows the cell boundaries and the directions to the cathode and anode electrodes relative to the cell. The next images show the Calbryte fluorescence signal at indicated times before and after nsPEF. All images are 17 × 17 µm. Mean +/− s.e., for nine experiments with Ba²⁺ and 13 with Ca²⁺; error bars are plotted in one direction only, except for panel E. The difference between Ca²⁺ readings from the opposite poles (E) is significant at p < 0.01 (one-sample t-test).