Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study.

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns-10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15-20% higher LD50, p < 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30-40% lower LD50 (p < 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation.

Control of the Electroporation Efficiency of Nanosecond Pulses by Swinging the Electric Field Vector Direction.

Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as "bipolar cancellation," enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing the electric field in the pairs (180° direction change) caused 2-fold (1 Hz) or 20-fold (833 kHz) weaker electroporation than the train of single nsEPs. Reducing the angle between pulse directions in the pairs weakened cancellation and replaced it with facilitation at angles <160° (1 Hz) and <130° (833 kHz). Facilitation plateaued at about three-fold stronger electroporation compared to single pulses at 90-100° angle for both nsEP frequencies. The profound dependence of the efficiency on the angle enables novel protocols for highly selective focal electroporation at one electrode in a three-electrode array while avoiding effects at the other electrodes. Nanosecond-resolution imaging of cell membrane potential was used to link the selectivity to charging kinetics by co- and counter-directional nsEPs.

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

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.

Excitation and electroporation by MHz bursts of nanosecond stimuli.

Intense nanosecond pulsed electric field (nsPEF) is a novel modality for cell activation and nanoelectroporation. Applications of nsPEF in research and therapy are hindered by a high electric field requirement, typically from 1 to over 50 kV/cm to elicit any bioeffects. We show how this requirement can be overcome by engaging temporal summation when pulses are compressed into high-rate bursts (up to several MHz). This approach was tested for excitation of ventricular cardiomyocytes and peripheral nerve fibers; for membrane electroporation of cardiomyocytes, CHO, and HEK cells; and for killing EL-4 cells. MHz compression of nsPEF bursts (100-1000 pulses) enables excitation at only 0.01-0.15 kV/cm and electroporation already at 0.4-0.6 kV/cm. Clear separation of excitation and electroporation thresholds allows for multiple excitation cycles without membrane disruption. The efficiency of nsPEF bursts increases with the duty cycle (by increasing either pulse duration or repetition rate) and with increasing the total time "on" (by increasing either pulse duration or number). For some endpoints, the efficiency of nsPEF bursts matches a single "long" pulse whose amplitude and duration equal the time-average amplitude and duration of the bursts. For other endpoints this rule is not valid, presumably because of nsPEF-specific bioeffects and/or possible modification of targets already during the burst. MHz compression of nsPEF bursts is a universal and efficient way to lower excitation thresholds and facilitate electroporation.

Excitation of murine cardiac myocytes by nanosecond pulsed electric field.

INTRODUCTION: Opening of voltage-gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. METHODS AND RESULTS: Fluorescent imaging of optical APs (FluoVolt) and Ca2+ -transients (Fluo-4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200-nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin-sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P < 0.001). Inhibition of l-type calcium and sodium-calcium exchanger currents reduced the SSD amplitude and increased the AP threshold ( P < 0.05). The threshold for Ca 2+ -transients (1.40 ± 0.04 kV/cm) was not significantly affected by a tetrodotoxin-verapamil cocktail, suggesting the activation of a Ca 2+ entry pathway independent from the opening of Na + or Ca 2+ voltage-gated channels. Removal of external Ca 2+ decreased the SSD amplitude ( P = 0.004) and blocked Ca 2+ -transients but not APs. The incidence of transient afterdepolarization waves was decreased by verapamil and by removal of external Ca 2+ ( P = 0.002). CONCLUSIONS: The study established that nsPEF stimulation caused calcium entry into cardiac myocytes (including routes other than voltage-gated calcium channels) and SSD. Tetrodotoxin-sensitive APs were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from l-type calcium and sodium-calcium exchanger currents.

Neuronal excitation and permeabilization by 200-ns pulsed electric field: An optical membrane potential study with FluoVolt dye.

Electric field pulses of nano- and picosecond duration are a novel modality for neurostimulation, activation of Ca2+ signaling, and tissue ablation. However it is not known how such brief pulses activate voltage-gated ion channels. We studied excitation and electroporation of hippocampal neurons by 200-ns pulsed electric field (nsPEF), by means of time-lapse imaging of the optical membrane potential (OMP) with FluoVolt dye. Electroporation abruptly shifted OMP to a more depolarized level, which was reached within <1ms. The OMP recovery started rapidly (τ=8-12ms) but gradually slowed down (to τ>10s), so cells remained above the resting OMP level for at least 20-30s. Activation of voltage-gated sodium channels (VGSC) enhanced the depolarizing effect of electroporation, resulting in an additional tetrodotoxin-sensitive OMP peak in 4-5ms after nsPEF. Omitting Ca2+ in the extracellular solution did not reduce the depolarization, suggesting no contribution of voltage-gated calcium channels (VGCC). In 40% of neurons, nsPEF triggered a single action potential (AP), with the median threshold of 3kV/cm (range: 1.9-4kV/cm); no APs could be evoked by stimuli below the electroporation threshold (1.5-1.9kV/cm). VGSC opening could already be detected in 0.5ms after nsPEF, which is too fast to be mediated by the depolarizing effect of electroporation. The overlap of electroporation and AP thresholds does not necessarily reflect the causal relation, but suggests a low potency of nsPEF, as compared to conventional electrostimulation, for VGSC activation and AP induction.

Frequency spectrum of induced transmembrane potential and permeabilization efficacy of bipolar electric pulses.

In this paper a simple prediction method for the bipolar pulse cancellation effect is proposed, based on the frequency analysis of the TMP spectra of a single cell and the computed relative global spectral content up to a defined frequency threshold. We present a spectral analysis of pulses applied in experiments, and we extract the induced TMP from a microdosimetric model of the cell. The induced TMP computation is carried out on a hemispherical multi-layered cell model in the time domain. The analysis is presented for a variety of unipolar and bipolar input signals in the nanosecond and the microsecond time scales. Our evaluations are in good agreement with experimental results for bipolar pulse cancellation of electropermeabilization-induced Ca2+ influx using 300ns, 750kV/m pulses and with other results reported in recent literature.

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

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.

Multiple nanosecond electric pulses increase the number but not the size of long-lived nanopores in the cell membrane.

Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr) in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at<4µM) and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60ns, 13.2kV/cm, 10Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.

Calcium-mediated pore expansion and cell death following nanoelectroporation.

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca(2+) after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca(2+) level from the nominal 2-5µM to 2mM for the first 60-90min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca(2+), the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2mM Ca(2+) in the medium. With the nominal Ca(2+), most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2mM Ca(2+), the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca(2+)-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.

Picosecond and Terahertz Perturbation of Interfacial Water and Electropermeabilization of Biological Membranes.

Non-thermal probing and stimulation with subnanosecond electric pulses and terahertz electromagnetic radiation may lead to new, minimally invasive diagnostic and therapeutic procedures and to methods for remote monitoring and analysis of biological systems, including plants, animals, and humans. To effectively engineer these still-emerging tools, we need an understanding of the biophysical mechanisms underlying the responses that have been reported to these novel stimuli. We show here that subnanosecond (≤500 ps) electric pulses induce action potentials in neurons and cause calcium transients in neuroblastoma-glioma hybrid cells, and we report complementary molecular dynamics simulations of phospholipid bilayers in electric fields in which membrane permeabilization occurs in less than 1 ns. Water dipoles in the interior of these model membranes respond in less than 1 ps to permeabilizing electric potentials by aligning in the direction of the field, and they re-orient at terahertz frequencies to field reversals. The mechanism for subnanosecond lipid electropore formation is similar to that observed on longer time scales-energy-minimizing intrusions of interfacial water into the membrane interior and subsequent reorganization of the bilayer into hydrophilic, conductive structures.

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity.

Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca(2+) mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1-60), and amplitudes (15-60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-µs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.

Primary pathways of intracellular Ca(2+) mobilization by nanosecond pulsed electric field.

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which determines multifarious downstream effects. By using fast ratiometric Ca(2+) imaging with Fura-2, we quantified the external Ca(2+) uptake, compared it with Ca(2+) release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca(2+) channels, so that the nsPEF-induced [Ca(2+)](i) changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca(2+)](i) increase by Ca(2+) influx from the outside and Ca(2+) efflux from the ER, with the E-field thresholds of about 9 and 19kV/cm, respectively. Above these thresholds, the amplitude of [Ca(2+)](i) response increased linearly by 8-10nM per 1kV/cm until a critical level between 200 and 300nM of [Ca(2+)](i) was reached. If the critical level was reached, the nsPEF-induced Ca(2+) signal was amplified up to 3000nM by engaging the physiological mechanism of Ca(2+)-induced Ca(2+)-release (CICR). The amplification was prevented by depleting Ca(2+) from the ER store with 100nM thapsigargin, as well as by blocking the ER inositol-1,4,5-trisphosphate receptors (IP(3)R) with 50µM of 2-aminoethoxydiphenyl borate (2-APB). Mobilization of [Ca(2+)](i) by nsPEF mimicked native Ca(2+) signaling, but without preceding activation of plasma membrane receptors or channels. NsPEF stimulation may serve as a unique method to mobilize [Ca(2+)](i) and activate downstream cascades while bypassing the plasma membrane receptors.

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses.

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.

Dihydroethidium-derived fluorescence in electrically stressed cells indicates intracellular microenvironment modifications independent of ROS.

Intracellular reactive oxygen species (ROS) generation is widely suggested as a trigger for biological consequences of electric field exposures, such as those in electroporation applications. ROS are linked with membrane barrier function degradation, genetic damage, and complex events like immunological cell death. Dihydroethidium (DHE) is commonly used to monitor ROS in cells. DHE is linked to intracellular ROS by a primary oxidation product, Ethidium (Eth+), that shows increased fluorescence upon binding to polynucleotides. We observed changes in DHE-derived fluorescence in Chinese hamster ovary (CHO) cells post 300-ns electric pulse exposures, comparing them to tert-butyl-hydroperoxide (t-BHP) induced oxidative stress. Immediate intracellular fluorescence changes were noted in both cases, but with distinct localization patterns. After electrical stress, cytosolic DHE-derived fluorescence intensity decreases, and nucleolar intensity increases. Conversely, t-BHP exposure increases DHE-derived fluorescence uniformly across the cell. Surprisingly, fluorescence patterns after electrical stress in Eth+-loaded cells is identical to those in DHE-loaded cells, in kinetics and localization patterns. These findings indicate that DHE-derived fluorescence changes after pulsed electric field stress are not due to intracellular ROS generation leading to DHE oxidation, but rather indicate stress-induced intracellular microenvironment alterations affecting Eth+ fluorescence.

Next generation CANCAN focusing for remote stimulation by nanosecond electric pulses.

Focusing electric pulse effects away from electrodes is a challenge because the electric field weakens with distance. Previously we introduced a remote focusing method based on bipolar cancellation, a phenomenon of low efficiency of bipolar nanosecond electric pulses (nsEP). Superpositioning two bipolar nsEP into a unipolar pulse canceled bipolar cancellation ("CANCAN" effect), enhancing bioeffects at a distance despite the electric field weakening. Here, we introduce the next generation (NG) CANCAN focusing with unipolar nsEP packets designed to produce bipolar waveforms near electrodes (suppressing electroporation) but not at the remote target. NG-CANCAN was tested in CHO cell monolayers using a quadrupole electrode array and labeling electroporated cells with YO-PRO-1 dye. We routinely achieved 1.5-2 times stronger electroporation in the center of the quadrupole than near electrodes, despite a 3-4-fold field attenuation. With the array lifted 1-2 mm above the monolayer (imitating a 3D treatment), the remote effect was enhanced up to 6-fold. We analyzed the role of nsEP number, amplitude, rotation, and inter-pulse delay, and showed how remote focusing is enhanced when re-created bipolar waveforms exhibit stronger cancellation. Advantages of NG-CANCAN include the exceptional versatility of designing pulse packets and easy remote focusing using an off-the-shelf 4-channel nsEP generator.

Action spectra and mechanisms of (in) efficiency of bipolar electric pulses at electroporation.

The reversal of the electric field direction inhibits various biological effects of nanosecond electric pulses (nsEP). This feature, known as "bipolar cancellation," enables interference targeting of nsEP bioeffects remotely from stimulating electrodes, for prospective applications such as precise cancer ablation and non-invasive deep brain stimulation. This study was undertaken to achieve the maximum cancellation of electroporation, by quantifying the impact of the pulse shape, duration, number, and repetition rate across a broad range of electric field strengths. Monolayers of endothelial cells (BPAE) were electroporated in a non-uniform electric field. Cell membrane permeabilization was quantified by YO-PRO-1 (YP) dye uptake and correlated to local electric field strength. For most conditions tested, adding an opposite polarity phase reduced YP uptake by 50-80 %. The strongest cancellation, which reduced YP uptake by 95-97 %, was accomplished by adding a 50 % second phase to 600-ns pulses delivered at a high repetition rate of 833 kHz. Strobe photography of nanosecond kinetics of membrane potential in single CHO cells revealed the temporal summation of polarization by individual unipolar nsEP applied at sub-MHz rate, leading to enhanced electroporation. In contrast, there was no summation for bipolar pulses, and increasing their repetition rate suppressed electroporation. These new findings are discussed in the context of bipolar cancellation mechanisms and remote focusing applications.

BK channel ß1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific ß1 subunit regulate excitation­contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel ß1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK ß1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of ß1 knockout is eliminated by specific M2 receptor antagonism. The role of BK ß1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with ß1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK ß1 knockout or by paxilline block of BK channels. Normalization of ß1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/ß1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation­contraction coupling to more negative voltage ranges.

Interference targeting of bipolar nanosecond electric pulses for spatially focused electroporation, electrostimulation, and tissue ablation.

Stimulation and electroporation by nanosecond electric pulses (nsEP) are distinguished by a phenomenon of bipolar cancellation, which stands for a reduced efficiency of bipolar pulses compared to unipolar ones. When two pairs of stimulating electrodes are arrayed in a quadrupole, bipolar cancellation inhibits nsEP effects near the electrodes, where the electric field is the strongest. Two properly shaped and synchronized bipolar nsEP overlay into a unipolar pulse towards the center of the electrode array, thus canceling the bipolar cancellation (a "CANCAN effect"). High efficiency of the re-created unipolar nsEP outweighs the weakening of the electric field with distance and focuses nsEP effects to the center. In monolayers of CHO, BPAE, and HEK cells, CANCAN effect achieved by the interference of two bipolar nsEP enhanced electroporation up to tenfold, with a peak at the quadrupole center. Introducing a time interval between bipolar nsEP prevented the formation of a unipolar pulse and eliminated the CANCAN effect. Strong electroporation by CANCAN stimuli killed cells over the entire area encompassed by the electrodes, whereas the time-separated pulses caused ablation only in the strongest electric field near the electrodes. The CANCAN approach is promising for uniform tumor ablation and stimulation targeting away from electrodes.

The role of reactive oxygen species in the immunity induced by nano-pulse stimulation.

Reactive oxygen species (ROS) are byproducts of tumor cells treated with Nano-Pulse Stimulation (NPS). Recently, ROS have been suggested as a contributing factor in immunogenic cell death and T cell-mediated immunity. This research further investigated the role of NPS induced ROS in antitumor immunity. ROS production in 4T1-luc breast cancer cells was characterized using three detection reagents, namely, Amplex Red, MitoSox Red, and Dihydroethidium. The efficiency of ROS quenching was evaluated in the presence or absence of ROS scavengers and/or antioxidants. The immunogenicity of NPS treated tumor cells was assessed by ex vivo dendritic cell activation, in vivo vaccination assay and in situ vaccination with NPS tumor ablation. We found that NPS treatment enhanced the immunogenicity of 4T1-luc mouse mammary tumor, resulted in a potent in situ vaccination protection and induced long-term T cell immunity. ROS production derived from NPS treated breast cancer cells was an electric pulse dose-dependent phenomenon. Noticeably, the dynamic pattern of hydrogen peroxide production was different from that of superoxide production. Interestingly, regardless of NPS treatment, different ROS scavengers could either block or promote ROS production and stimulate or inhibit tumor cell growth. The activation of dendritic cells was not influenced by blocking ROS generation. The results from in vivo vaccination with NPS treated cancer cells suggests that ROS generation was not a prerequisite for immune protection.