Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

INTRODUCTION: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCM assay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). METHODS: Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. RESULTS: Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). CONCLUSION: These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Cancellation of nerve excitation by the reversal of nanosecond stimulus polarity and its relevance to the gating time of sodium channels.

The initiation of action potentials (APs) by membrane depolarization occurs after a brief vulnerability period, during which excitation can be abolished by the reversal of the stimulus polarity. This vulnerability period is determined by the time needed for gating of voltage-gated sodium channels (VGSC). We compared nerve excitation by ultra-short uni- and bipolar stimuli to define the time frame of bipolar cancellation and of AP initiation. Propagating APs in isolated frog sciatic nerve were elicited by cathodic pulses (200 ns-300 µs), followed by an anodic (canceling) pulse of the same duration after a 0-200-µs delay. We found that the earliest and the latest boundaries for opening the critical number of VGSC needed to initiate AP are, respectively, between 11 and 20 µs and between 100 and 200 µs after the onset of depolarization. Stronger depolarization accelerated AP initiation, apparently due to faster VGSC opening, but not beyond the 11-µs limit. Bipolar cancellation was augmented by reducing pulse duration, shortening the delay between pulses, decreasing the amplitude of the cathodic pulse, and increasing the amplitude of the anodic one. Some of these characteristics contrasted the bipolar cancellation of cell membrane electroporation (Pakhomov et al. in Bioelectrochemistry 122:123-133, 2018; Gianulis et al. in Bioelectrochemistry 119:10-19, 2017), suggesting different mechanisms. The ratio of nerve excitation thresholds for a unipolar cathodic pulse and a symmetrical bipolar pulse increased as a power function as the pulse duration decreased, in remarkable agreement with the predictions of SENN model of nerve excitation (Reilly and Diamant in Health Phys 83(3):356-365, 2002).

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.

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects.

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.

A molecular insight into the electro-transfer of small molecules through electropores driven by electric fields.

The transport of chemical compounds across the plasma membrane into the cell is relevant for several biological and medical applications. One of the most efficient techniques to enhance this uptake is reversible electroporation. Nevertheless, the detailed molecular mechanism of transport of chemical species (dyes, drugs, genetic materials, …) following the application of electric pulses is not yet fully elucidated. In the past decade, molecular dynamics (MD) simulations have been conducted to model the effect of pulsed electric fields on membranes, describing several aspects of this phenomenon. Here, we first present a comprehensive review of the results obtained so far modeling the electroporation of lipid membranes, then we extend these findings to study the electrotransfer across lipid bilayers subject to microsecond pulsed electric fields of Tat11, a small hydrophilic charged peptide, and of siRNA. We use in particular a MD simulation protocol that allows to characterize the transport of charged species through stable pores. Unexpectedly, our results show that for an electroporated bilayer subject to transmembrane voltages in the order of 500mV, i.e. consistent with experimental conditions, both Tat11 and siRNA can translocate through nanoelectropores within tens of ns. We discuss these results in comparison to experiments in order to rationalize the mechanism of drug uptake by cells. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

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.

Pulsed electric field performance calculator tool based on an in vitro human cardiac model.

Introduction: Pulsed Field Ablation (PFA) is a novel non-thermal method for cardiac ablation, relying on irreversible electroporation induced by high-energy pulsed electric fields (PEFs) to create localized lesions in the heart atria. A significant challenge in optimizing PFA treatments is determining the lethal electric field threshold (EFT), which governs ablation volume and varies with PEF waveform parameters. However, the proprietary nature of device developer's waveform characteristics and the lack of standardized nonclinical testing methods have left optimal EFTs for cardiac ablation uncertain. Methods: To address this gap, we introduced a laboratory protocol employing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in monolayer format to evaluate the impact of a range of clinically relevant biphasic pulse parameters on lethal EFT and adiabatic heating (AH). Cell death areas were assessed using fluorescent dyes and confocal microscopy, while lethal EFTs were quantified through comparison with electric field numerical simulations. Results and conclusion: Our study confirmed a strong correlation between cell death in hiPSC-CMs and the number and duration of pulses in each train, with pulse repetition frequency exerting a comparatively weaker influence. Fitting of these results through machine learning algorithms were used to develop an open-source online calculator. By estimating lethal EFT and associated temperature increases for diverse pulse parameter combinations, this tool, once validated, has the potential to significantly reduce reliance on animal models during early-stage device de-risking and performance assessment. This tool also offers a promising avenue for advancing PFA technology for cardiac ablation medical devices to enhance patient outcomes.

Human in vitro neurocardiac coculture (ivNCC) assay development for evaluating cardiac contractility modulation.

Two of the most prominent organ systems, the nervous and the cardiovascular systems, are intricately connected to maintain homeostasis in mammals. Recent years have shown tremendous efforts toward therapeutic modulation of cardiac contractility and electrophysiology by electrical stimulation. Neuronal innervation and cardiac ganglia regulation are often overlooked when developing in vitro models for cardiac devices, but it is likely that peripheral nervous system plays a role in the clinical effects. We developed an in vitro neurocardiac coculture (ivNCC) model system to study cardiac and neuronal interplay using human induced pluripotent stem cell (hiPSC) technology. We demonstrated significant expression and colocalization of cardiac markers including troponin, α-actinin, and neuronal marker peripherin in neurocardiac coculture. To assess functional coupling between the cardiomyocytes and neurons, we evaluated nicotine-induced ß-adrenergic norepinephrine effect and found beat rate was significantly increased in ivNCC as compared to monoculture alone. The developed platform was used as a nonclinical model for the assessment of cardiac medical devices that deliver nonexcitatory electrical pulses to the heart during the absolute refractory period of the cardiac cycle, that is, cardiac contractility modulation (CCM) therapy. Robust coculture response was observed at 14 V/cm (5 V, 64 mA), monophasic, 2 ms pulse duration for pacing and 20 V/cm (7 V, 90 mA) phase amplitude, biphasic, 5.14 ms pulse duration for CCM. We observed that the CCM effect and kinetics were more pronounced in coculture as compared to cardiac monoculture, supporting a hypothesis that some part of CCM mechanism of action can be attributed to peripheral nervous system stimulation. This study provides novel characterization of CCM effects on hiPSC-derived neurocardiac cocultures. This innervated human heart model can be further extended to investigate arrhythmic mechanisms, neurocardiac safety, and toxicity post-chronic exposure to materials, drugs, and medical devices. We present data on acute CCM electrical stimulation effects on a functional and optimized coculture using commercially available hiPSC-derived cardiomyocytes and neurons. Moreover, this study provides an in vitro human heart model to evaluate neuronal innervation and cardiac ganglia regulation of contractility by applying CCM pulse parameters that closely resemble clinical setting. This ivNCC platform provides a potential tool for investigating aspects of cardiac and neurological device safety and performance.

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format. The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Human cardiomyocytes are more susceptible to irreversible electroporation by pulsed electric field than human esophageal cells.

Pulse electric field-based (PEF) ablation is a technique whereby short high-intensity electric fields inducing irreversible electroporation (IRE) are applied to various tissues. Here, we implemented a standardized in vitro model to compare the effects of biphasic symmetrical pulses (100 pulses, 1-10 µs phase duration (d), 10-1000 Hz pulse repetition rate (f)) using two different human cellular models: human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and human esophageal smooth muscle cells (hESMCs) cultured in monolayer format. We report the PEF-induced irreversibly electroporated cell monolayer areas and the corresponding electric field thresholds (EFTs) for both cardiac and esophageal cultures. Our results suggest marked cell type specificity with EFT estimated to be 2-2.5 times lower in hiPSC-CMs than in hESMCs when subjected to identical PEF treatments (e.g., 0.90 vs 1.85 kV/cm for the treatment of 100 pulses with d = 5 µs, f = 10 Hz, and 0.65 vs 1.67 kV/cm for the treatment of 100 pulses with d = 10 µs, f = 10 Hz). PEF treatment can result in increased temperature around the stimulating electrodes and lead to unanticipated thermal tissue damage that is proportional to the peak temperature rise and to the duration of the PEF-induced elevated temperatures. In our study, temperature increases ranged from less than 1°C to as high as 30°C, however, all temperature changes were transient and quickly returned to baseline and the highest observed ∆T returned to 50% of its maximum recorded temperature in tens of seconds.

Human in vitro assay for irreversible electroporation cardiac ablation.

Introduction: Pulsed electric field (PEF) cardiac ablation has been recently proposed as a technique to treat drug resistant atrial fibrillation by inducing cell death through irreversible electroporation (IRE). Improper PEF dosing can result in thermal damage or reversible electroporation. The lack of comprehensive and systematic studies to select PEF parameters for safe and effective IRE cardiac treatments hinders device development and regulatory decision-making. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been proposed as an alternative to animal models in the evaluation of cardiac electrophysiology safety. Methods: We developed a novel high-throughput in vitro assay to quantify the electric field threshold (EFT) for electroporation (acute effect) and cell death (long-term effect) in hiPSC-CMs. Monolayers of hiPSC-CMs were cultured in high-throughput format and exposed to clinically relevant biphasic PEF treatments. Electroporation and cell death areas were identified using fluorescent probes and confocal microscopy; electroporation and cell death EFTs were quantified by comparison of fluorescent images with electric field numerical simulations. Results: Study results confirmed that PEF induces electroporation and cell death in hiPSC-CMs, dependent on the number of pulses and the amplitude, duration, and repetition frequency. In addition, PEF-induced temperature increase, absorbed dose, and total treatment time for each PEF parameter combination are reported. Discussion: Upon verification of the translatability of the in vitro results presented here to in vivo models, this novel hiPSC-CM-based assay could be used as an alternative to animal or human studies and can assist in early nonclinical device development, as well as inform regulatory decision-making for cardiac ablation medical devices.

Acute effects of cardiac contractility modulation stimulation in conventional 2D and 3D human induced pluripotent stem cell-derived cardiomyocyte models.

Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical stimulations are delivered to the myocardium during the absolute refractory period to enhance cardiac function. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. In the present study, we sought to extend these results to human 3D microphysiological systems to develop a robust model to evaluate various clinical CCM pulse parameters in vitro. HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video (i.e., pixel) and force-based analysis. CCM pulses were assessed at varying electrical 'doses' using a commercial pulse generator. A robust CCM contractile response was observed for 3D ECTs. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed no contractile response. 3D ECTs displayed enhanced contractile properties including increased contraction amplitude (i.e., force), and accelerated contraction and relaxation slopes under standard acute CCM stimulation. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter-dependent manner by adjustment of CCM pulse delay, duration, amplitude, and number relative to baseline. The observed acute effects subsided when the CCM stimulation was stopped and gradually returned to baseline. These data represent the first study of CCM in 3D hiPSC-CM models and provide a nonclinical tool to assess various CCM device signals in 3D human cardiac tissues prior to in vivo animal studies. Moreover, this work provides a foundation to evaluate the effects of additional cardiac medical devices in 3D ECTs.

Acute effects of cardiac contractility modulation on human induced pluripotent stem cell-derived cardiomyocytes.

Cardiac contractility modulation (CCM) is an intracardiac therapy whereby nonexcitatory electrical simulations are delivered during the absolute refractory period of the cardiac cycle. We previously evaluated the effects of CCM in isolated adult rabbit ventricular cardiomyocytes and found a transient increase in calcium and contractility. In the present study, we sought to extend these results to human cardiomyocytes using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to develop a robust model to evaluate CCM in vitro. HiPSC-CMs (iCell Cardiomyocytes2 , Fujifilm Cellular Dynamic, Inc.) were studied in monolayer format plated on flexible substrate. Contractility, calcium handling, and electrophysiology were evaluated by fluorescence- and video-based analysis (CellOPTIQ, Clyde Biosciences). CCM pulses were applied using an A-M Systems 4100 pulse generator. Robust hiPSC-CMs response was observed at 14 V/cm (64 mA) for pacing and 28 V/cm (128 mA, phase amplitude) for CCM. Under these conditions, hiPSC-CMs displayed enhanced contractile properties including increased contraction amplitude and faster contraction kinetics. Likewise, calcium transient amplitude increased, and calcium kinetics were faster. Furthermore, electrophysiological properties were altered resulting in shortened action potential duration (APD). The observed effects subsided when the CCM stimulation was stopped. CCM-induced increase in hiPSC-CMs contractility was significantly more pronounced when extracellular calcium concentration was lowered from 2 mM to 0.5 mM. This study provides a comprehensive characterization of CCM effects on hiPSC-CMs. These data represent the first study of CCM in hiPSC-CMs and provide an in vitro model to assess physiologically relevant mechanisms and evaluate safety and effectiveness of future cardiac electrophysiology medical devices.

Growth in a biofilm sensitizes Cutibacterium acnes to nanosecond pulsed electric fields.

The Gram-positive anaerobic bacterium Cutibacterium acnes (C. acnes) is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. C. acnes can form biofilms; cells in biofilms are more resilient to antimicrobial stresses. Acne therapeutic options such as topical or systemic antimicrobial treatments often show incomplete responses. In this study we measured the efficacy of nanosecond pulsed electric fields (nsPEF), a new promising cell and tissue ablation technology, to inactivate C. acnes. Our results show that all tested nsPEF doses (250 to 2000 pulses, 280 ns pulses, 28 kV/cm, 5 Hz; 0.5 to 4 kJ/ml) failed to inactivate planktonic C. acnes and that pretreatment with lysozyme, a naturally occurring cell-wall-weakening enzyme, increased C. acnes vulnerability to nsPEF. Surprisingly, growth in a biofilm appears to sensitize C. acnes to nsPEF-induced stress, as C. acnes biofilm-derived cells showed increased cell death after nsPEF treatments that did not affect planktonic cells. Biofilm inactivation by nsPEF was confirmed by treating intact biofilms grown on glass coverslips with an indium oxide conductive layer. Altogether our results show that, contrary to other antimicrobial agents, nsPEF kill more efficiently bacteria in biofilms than planktonic cells.

Analysis of electrostimulation and electroporation by high repetition rate bursts of nanosecond stimuli.

Exposures to short-duration, strong electric field pulses have been utilized for stimulation, ablation, and the delivery of molecules into cells. Ultrashort, nanosecond duration pulses have shown unique benefits, but they require higher field strengths. One way to overcome this requirement is to use trains of nanosecond pulses with high repetition rates, up to the MHz range. Here we present a theoretical model to describe the effects of pulse trains on the plasma membrane and intracellular membranes modeled as resistively charged capacitors. We derive the induced membrane potential and the stimulation threshold as functions of pulse number, pulse duration, and repetition rate. This derivation provides a straightforward method to calculate the membrane charging time constant from experimental data. The derived excitation threshold agrees with nerve stimulation experiments, indicating that nanosecond pulses are not more effective than longer pulses in charging nerve fibers. The derived excitation threshold does not, however, correctly predict the nanosecond stimulation of cardiomyocytes. We show that a better agreement is possible if multiple charging time constants are considered. Finally, we expand the model to intracellular membranes and show that pulse trains do not lead to charge buildup, but can create significant oscillations of the intracellular membrane potential.

Electropermeabilization does not correlate with plasma membrane lipid oxidation.

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) layer, which enabled electroporation with micro- and submicrosecond pulses. No oxidation was observed with the electric field directed towards the ITO (cathode), even at field strengths much higher than that needed for permeabilization. Oxidation was readily detectable with the opposite polarity of pulses, but with the threshold higher than the permeabilization threshold. Moreover, a decrease in the medium conductance had opposite effects on permeabilization and lipid oxidation (it enhanced the former and suppressed the latter). We conclude that lipid oxidation can indeed occur at the plasma membrane after electric pulses, but it is not the cause of lasting membrane permeabilization.

Selective distant electrostimulation by synchronized bipolar nanosecond pulses.

A unique aspect of electrostimulation (ES) with nanosecond electric pulses (nsEP) is the inhibition of effects when the polarity is reversed. This bipolar cancellation feature makes bipolar nsEP less efficient at biostimulation than unipolar nsEP. We propose to minimize stimulation near pulse-delivering electrodes by applying bipolar nsEP, whereas the superposition of two phase-shifted bipolar nsEP from two independent sources yields a biologically-effective unipolar pulse remotely. This is accomplished by electrical compensation of all nsEP phases except the first one, resulting in the restoration of stimulation efficiency due to cancellation of bipolar cancellation (CANCAN-ES). We experimentally proved the CANCAN-ES paradigm by measuring YO-PRO-1 dye uptake in CHO-K1 cells which were permeabilized by multiphasic nsEP (600 ns per phase) from two generators; these nsEP were synchronized either to overlap into a unipolar pulse remotely from electrodes (CANCAN), or not to overlap (control). Enhancement of YO-PRO-1 entry due to CANCAN was observed in all sets of experiments and reached ~3-fold in the center of the gap between electrodes, exactly where the unipolar pulse was formed, and equaled the degree of bipolar cancellation. CANCAN-ES is promising for non-invasive deep tissue stimulation, either alone or combined with other remote stimulation techniques to improve targeting.

Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied by Immunogenic Cell Death in Murine Models of Lymphoma and Colorectal Cancer.

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of caspase-3/7 was increased fourteen-fold as compared with four-fold in EL-4 cells. Moreover, while nsPEF treatments induced the release of the ICD hallmark HMGB1 in both cell lines, extracellular ATP was detected only in CT-26. Finally, in vaccination assays, CT-26 cells treated with nsPEF or doxorubicin equally impaired the growth of tumors at challenge sites eliciting a protective anticancer immune response in 78% and 80% of the animals, respectively. As compared to CT-26, both nsPEF- and mitoxantrone-treated EL-4 cells had a less pronounced effect and protected 50% and 20% of the animals, respectively. These results support our conclusion that nsPEF induce ER stress, accompanied by bona fide ICD.