Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability

Abstract

It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0-80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.

Figure 1. The electrical pulsing conditions used in this study.

a) The 600 ns pulses with 15 ns (dark line) and 150 ns rise time (gray line). The pulses with 15 ns rise time were obtained under the best impedance-matching condition. The 150 ns rise time was obtained by adding an inductance in series with the cuvette, which caused a slight impedance mismatch and therefore a small negative tail. b) The instantaneous power of the electric pulses for the 15 ns and 150 rise times. The total energy was calculated as 0.15 J and 0.17 J for pulses with 15 ns and 150 ns rise times, respectively.

Figure 2. Effects of EGTA on calcium mobilization and forward light scatter in response to nsPEFs.

Cells were prepared for analysis of calcium with Fluo-4 with and without EGTA as indicated in Materials and Methods EGTA. N1-S1 cells were then treated with a single 600 ns pulse at the indicated electric fields. Ten minutes after treatment, cells were analyzed for Fluo-4 fluorescence as an indicator of changes in intracellular calcium (X-axis) and forward light scatter as an indicator of cell size (Y-axis) by flow cytometry. The figure represents a typical experiment.

Figure 3. Calcium influx is a better indicator than propidium iodide for nsPEF effects on plasma membranes.

Fluo-4 and PI fluorescence were determined in parallel. N1-S1 cells were exposed to single 600 ns pulses at indicated electric fields. For Fluo-4, cells were loaded with the fluorophore as indicated in Experimental Procedures, incubated in the presence or absence of EGTA to chelate extracellular calcium, exposed to nsPEFs and analyzed for green fluorescence by flow cytometry 10 minutes after pulses. For PI, cells were exposed to nsPEFs, PI was added immediately and cells were analyzed for red fluorescence by flow cytometry 10 minutes after pulses. Significant increases in Fluo-4 were observed for all electric fields at and above the 10 kV/cm as indicated by the (+) and for PI above 50 kV/cm as indicated by the (*). There were increases in Fluo-4 florescence in the presence of EGTA. n = 3, p<0.01.

Figure 4. Effect of different pulse shapes on Fluo-4 fluorescence as a marker for plasma membranes and TMRE as a marker for mitochondria membranes.

Cells were labeled with the respective markers as indicated in Material and Methods. N1-S1 cells were exposed to single 600 ns pulses at the indicated electric fields with two different waveforms (15 ns rise time and 150 ns rise time) as shown in Figure 1. Effects on ΔΨm with TMRE are shown in panel A; effects on calcium influx with Fluo-4 are shown in panel B. The results represent three separate experiments carried out in duplicate. (#n = 3, p<0.02 vs. 0 kV/cm). Significant differences on both parameters are indicated in Figures 5 and 6.

Figure 5. Effects of calcium influx, dissipation of ΔΨm and viability in response to nsPEFs with.

Cells were treated and analyzed as indicate in the legend to Figure 4. Effects on increases in calcium influx and loss of ΔΨm were determine 10 minutes after treatment and cell viability was determined 24 hours after treatment (panels A and B) as described in Material and Methods. Panels C and D show corresponding waveforms. Significant differences from control sham treatment are indicated for all electric fields greater than and equal to the symbol for calcium (+n = 3, p<0.001), ΔΨm (*, n = 3, p<0.03) and viability (#, n = 3, p<0.001). (Correction: ANOVA with Student-Newman-Keuls).

Figure 6. Effects of EGTA and time on ΔΨm.

Cells were loaded with TMRE and treated in the presence or absence of EGTA with one 600 ns pulse at the indicated electric fields. Ten and 30 minutes later, TMRE fluorescence was determined by flow cytometry. * = p<0.001 vs. 0 kV/cm at time point; +p = 0.026 vs. 80 kV/cm+EGTA; #p = 0.05 vs 0 kV/cm +EGTA.