Tina Batista Napotnik’s research while affiliated with University of Ljubljana and other places

It is the interplay between various ion channels within the membranes of cardiomyocytes that is responsible for depolarization and repolarization of the heart cells. Cell-to-cell coupling between myocytes is mediated via gap junction proteins and is involved in overall propagation of action potentials throughout the heart. In this chapter, different phases of the action potential of heart cells are discussed, and the role and characteristics of the most important ion channels that are active during these phases are conferred. Differences in the expressions of the various ion and gap junction channels under normal and certain pathological conditions are described.

Electric pulses used in electroporation-based treatments have been shown to affect the excitability of muscle and neuronal cells. However, understanding the interplay between electroporation and electrophysiological response of excitable cells is complex, since both ion channel gating and electroporation depend on dynamic changes in the transmembrane voltage (TMV). In this study, a genetically engineered human embryonic kidney cells expressing NaV1.5 and Kir2.1, a minimal complementary channels required for excitability (named S-HEK), was characterized as a simple cell model used for studying the effects of electroporation in excitable cells. S-HEK cells and their non-excitable counterparts (NS-HEK) were exposed to 100 µs pulses of increasing electric field strength. Changes in TMV, plasma membrane permeability, and intracellular Ca²⁺ were monitored with fluorescence microscopy. We found that a very mild electroporation, undetectable with the classical propidium assay but associated with a transient increase in intracellular Ca²⁺, can already have a profound effect on excitability close to the electrostimulation threshold, as corroborated by multiscale computational modelling. These results are of great relevance for understanding the effects of pulse delivery on cell excitability observed in context of the rapidly developing cardiac pulsed field ablation as well as other electroporation-based treatments in excitable tissues.

Electroporation is a biophysical phenomenon involving an increase in cell membrane permeability to molecules after a high-pulsed electric field is applied to the tissue. Currently, electroporation is being developed for non-thermal ablation of cardiac tissue to treat arrhythmias. Cardiomyocytes have been shown to be more affected by electroporation when oriented with their long axis parallel to the applied electric field. However, recent studies demonstrate that the preferentially affected orientation depends on the pulse parameters. To gain better insight into the influence of cell orientation on electroporation with different pulse parameters, we developed a time-dependent nonlinear numerical model where we calculated the induced transmembrane voltage and pores creation in the membrane due to electroporation. The numerical results show that the onset of electroporation is observed at lower electric field strengths for cells oriented parallel to the electric field for pulse durations ≥10 µs, and cells oriented perpendicular for pulse durations ~100 ns. For pulses of ~1 µs duration, electroporation is not very sensitive to cell orientation. Interestingly, as the electric field strength increases beyond the onset of electroporation, perpendicular cells become more affected irrespective of pulse duration. The results obtained using the developed time-dependent nonlinear model are corroborated by in vitro experimental measurements. Our study will contribute to the process of further development and optimization of pulsed-field ablation and gene therapy in cardiac treatments.

Background: Irreversible electroporation is an energy form utilizing high-voltage pulsed electric field, leading to cellular homeostasis disruption and cell death. Recently, irreversible electroporation has shown promising results for the treatment of cardiac arrhythmias. However, reversible and irreversible effects of pulsed electric field on cardiac myocytes remain poorly understood. Here, we evaluated the influence of a monophasic single electric pulse (EP) on the contractility, Ca2+ homeostasis and recovery of cardiac myocytes. Methods: Isolated rat left ventricular myocytes were electroporated using single monophasic EP of different durations and voltages. Sarcomere length and intracellular Ca2+ were simultaneously monitored for up to 20 minutes after EP application in Fura-2 loaded left ventricular myocytes. Lethal voltage thresholds were determined using 100 µs and 10 ms pulses and by discriminating cell orientation with respect to the electric field. Results: Electroporation led to an immediate increase in intracellular Ca2+ which was dependent upon the voltage delivered to the cell. Intermediate-voltage EP (140 V, 100 µs) increased sarcomere shortening, Ca2+ transient amplitude, and diastolic Ca2+ level measured 1 minute post-EP. Although sarcomere shortening returned to pre-EP level within 5 minutes, Ca2+ transient amplitude decreased further below pre-EP level and diastolic Ca2+ level remained elevated within 20 minutes post-EP. Spontaneous contractions were observed after sublethal EP application but their frequency decreased progressively within 20 minutes. Lethal EP voltage threshold was lower in myocytes oriented perpendicular than parallel to the electric field using 100 µs pulses while an opposite effect was found using 10 ms pulses. Conclusions: Sublethal EP affected rat left ventricular myocytes contractility and disrupted Ca2+ homeostasis as a function of the EP voltage. Moreover, EP-induced lethality was preceded by a large increase in intracellular Ca2+ and was dependent upon the EP duration, amplitude and left ventricular myocytes orientation with respect to the electric field. These findings provide new insights into the effect of pulsed electric field on cardiac myocytes.

Electroporation (EP) is one of the successful physical methods for intracellular drug delivery, which temporarily permeabilizes plasma membrane by exposing cells to electric pulses. Orientation of cells in electric field is important for electroporation and, consequently, for transport of molecules through permeabilized plasma membrane. Uptake of molecules after electroporation are the greatest at poles of cells facing electrodes and is often asymmetrical. However, asymmetry reported was inconsistent and inconclusive—in different reports it was either preferentially anodal or cathodal. We investigated the asymmetry of polar uptake of calcium ions after electroporation with electric pulses of different durations, as the orientation of elongated cells affects electroporation to a different extent when using electric pulses of different durations in the range of 100 ns to 100 µs. The results show that with 1, 10, and 100 µs pulses, the uptake of calcium ions is greater at the pole closer to the cathode than at the pole closer to the anode. With shorter 100 ns pulses, the asymmetry is not observed. A different extent of electroporation at different parts of elongated cells, such as muscle or cardiac cells, may have an impact on electroporation-based treatments such as drug delivery, pulse-field ablation, and gene electrotransfection.

Exposure of cells to high voltage electric pulses increases transiently membrane permeability through membrane electroporation. Electroporation can be reversible and is used in gene transfer and enhanced drug delivery but can also lead to cell death. Electroporation resulting in cell death (termed as irreversible electroporation) has been successfully used as a new non-thermal ablation method of soft tissue such as tumours or arrhythmogenic heart tissue. Even though the mechanisms of cell death can influence the outcome of electroporation-based treatments due to use of different electric pulse parameters and conditions, these are not elucidated yet. We review the mechanisms of cell death after electroporation reported in literature, cell injuries that may lead to cell death after electroporation and membrane repair mechanisms involved. The knowledge of membrane repair and cell death mechanisms after cell exposure to electric pulses, targets of electric field in cells need to be identified to optimize existing and develop of new electroporation-based techniques used in medicine, biotechnology, and food technology.

In gene electrotransfer and cardiac ablation with irreversible electroporation, treated muscle cells are typically of elongated shape and their orientation may vary. Orientation of cells in electric field has been reported to affect electroporation, and hence electrodes placement and pulse parameters choice in treatments for achieving homogeneous effect in tissue is important. We investigated how cell orientation influences electroporation with respect to different pulse durations (ns to ms range), both experimentally and numerically. Experimentally detected electroporation (evaluated separately for cells parallel and perpendicular to electric field) via Ca2+ uptake in H9c2 and AC16 cardiomyocytes was numerically modeled using the asymptotic pore equation. Results showed that cell orientation affects electroporation extent: using short, nanosecond pulses, cells perpendicular to electric field are significantly more electroporated than parallel (up to 100-times more pores formed), and with long, millisecond pulses, cells parallel to electric field are more electroporated than perpendicular (up to 1000-times more pores formed). In the range of a few microseconds, cells of both orientations were electroporated to the same extent. Using pulses of a few microseconds lends itself as a new possible strategy in achieving homogeneous electroporation in tissue with elongated cells of different orientation (e.g. electroporation-based cardiac ablation).

Pulsed electric fields (PEFs) can be used to transiently increase cell membrane permeability in procedures ranging from gene therapy to tumor eradication. Although very efficient, PEF-based therapies generally require the use of invasive electrodes, which cause pain and tissue damage. An emerging noninvasive, contactless alternative to PEFs are High Intensity Pulsed Electromagnetic Fields (HI-PEMF), whereby the electric field inside the tissue is induced remotely by external pulsed magnetic field. However, one of the current major drawbacks of HI-PEMFs is their inferior efficiency compared to PEFs. In this study we present the proof-of-concept that by adding highly conductive 5 and 20 nm gold nanoparticles (Au NPs), we can significantly potentiate the permeabilizing effect of HI-PEMFs, making it possible to permeabilize up to 80% of the cells with minimal or no effect on cell survival, compared to negligible percentage of permeabilized cells using HI-PEMF alone. Experiments, conducted on Chinese Hamster Ovary cells and Escherichia coli, suggest that Au NPs act as distributed nanoelectrodes, locally enhancing the electric field induced at the plasma membrane. Our findings open up an avenue of possibilities for combining naked as well as functionalized Au NPs with HI-PEMFs for noninvasive, remotely controlled smart drug delivery applications.

Exposing cells to an electric field leads to electroporation of the cell membrane which has already been explored and used in a number of applications in medicine and food biotechnology (e.g. electrochemotherapy, gene electrotransfer, extraction of biomolecules). The extent of electroporation depends on several conditions, including pulse parameters, types of cells and tissues, surrounding media, temperature etc. Each application requires a specific level of electroporation, so it must be explored in advance by employing methods for detecting electroporation. Electroporation detection is most often done by measuring increased transport of molecules across the membrane, into or out of the cell. We review here various methods of electroporation detection, together with their advantages and disadvantages. Electroporation detection can be carried out by using dyes (fluorophores or colour stains) or functional molecules, by measuring the efflux of biomolecules, by impedance measurements and voltage clamp techniques as well as by monitoring cell swelling. This review describes methods of detecting cell membrane electroporation in order to help researchers choose the most suitable ones for their specific experiments, considering available equipment and experimental conditions.