483 REVIEW ARTICLE The electropermeome: cellular response to electroporation Copyright (c) 2022 Slovenian Medical Journal. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. The electropermeome: cellular response to electroporation Elektropermeom: celični odgovor na elektroporacijo Angelika Vižintin, Damijan Miklavčič Abstract The increased permeability of a cell membrane due to exposure of cells/tissues to an electric field is called electropora- tion. Electroporation induces a range of changes in the cell - from structural and chemical changes in the cell membrane, structural changes in proteins or protein complexes, transport of substances in and out of the cell, activation of signalling pathways, and repair mechanisms; it also triggers cell death under certain conditions. The term electropermeome is used to describe both the permeabilised cell during or immediately after the delivery of electrical pulses and all subsequent processes that remain active for some time after the increased transmembrane transport of substances for which the cell membrane is normally impermeable has ceased, i.e. even after the membrane has resealed. Electroporation is used in ma- ny areas, including tissue ablation, gene electrotransfer for plasmid delivery into cells and electrochemotherapy. Medical applications of electroporation are effective and safe, but the action of the electrical pulses can cause certain adverse side effects, notably muscle contractions and acute pain. Further elucidation of the underlying mechanisms of electroporation and the effects of individual electric field parameters on the electropermeome is crucial to optimise the parameters of electroporation and consequently the results of electroporation-based therapies. The aim of the present paper is to pro- vide a comprehensive overview of the mechanisms of electroporation and the electropermeome, i.e. the cellular response to electroporation. Izvleček Povečano prepustnost celične membrane zaradi izpostavitve celic oz. tkiv električnemu polju imenujemo elektroporacija. Povzroči vrsto sprememb v celici, od strukturnih in kemijskih sprememb v celični membrani, strukturnih sprememb protei- nov oz. proteinskih kompleksov, prenosa snovi v celice in iz njih do aktiviranja signalnih poti in popravljalnih mehanizmov; ob določenih pogojih sproži tudi celično smrt. S pojmom elektropermeom označujemo tako permeabilizirano celico med ali tik po dovajanju električnih pulzov kot tudi vse poznejše procese, ki ostanejo aktivni še nekaj časa potem, ko ni več mo- goče opaziti povečanega transmembranskega transporta snovi, za katere je celična ovojnica običajno neprepustna, torej tudi po času, ko že ugotavljamo, da je zaceljena. Elektroporacija se uporablja na številnih področjih, vključno z ablacijo tkiv, gensko elektrotransfekcijo za vnos plazmidov v celice ter elektrokemoterapijo. Aplikacije elektroporacije v medicini Laboratory of Biocybernetics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia Correspondence / Korespondenca: Damijan Miklavčič, e: Damijan.Miklavcic@fe.uni-lj.si Key words: electropermeabilization; electrical pulses; transmembrane transport; lipid peroxidation; cell death Ključne besede: elektropermeabilizacija; električni pulzi; transmembranski transport; peroksidacija lipidov; celična smrt Received / Prispelo: 10. 5. 2021 | Accepted / Sprejeto: 5. 4. 2022 Cite as / Citirajte kot: Vižintin A, Miklavčič D.. The electropermeome: cellular response to electroporation. Zdrav Vestn. 2022;91(11– 12):483–95. DOI: https://doi.org/10.6016/ZdravVestn.3267 eng slo element en article-lang 10.6016/ZdravVestn.3267 doi 10.5.2021 date-received 5.4.2022 date-accepted Physiology Fiziologija discipline Review article Pregledni znanstveni članek article-type The electropermeome: cellular response to electroporation Elektropermeom: celični odgovor na elektropo- racijo article-title The electropermeome Elektropermeom alt-title electropermeabilization, electrical pulses, transmembrane transport, lipid peroxidation, cell death elektropermeabilizacija, električni pulzi, trans- membranski transport, peroksidacija lipidov, celična smrt kwd-group The authors declare that there are no conflicts of interest present. Avtorji so izjavili, da ne obstajajo nobeni konkurenčni interesi. conflict year volume first month last month first page last page 2022 91 11 12 483 495 name surname aff email Damijan Miklavčič 1 Damijan.Miklavcic@fe.uni-lj.si name surname aff Angelika Vižintin 1 eng slo aff-id Laboratory of Biocybernetics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia Laboratorij za biokibernetiko, Fakulteta za elektrotehniko, Univerza v Ljubljani, Ljubljana, Slovenija 1 Slovenian Medical Journallovenian Medical Journal 484 PHYSIOLOGY Zdrav Vestn | November – December 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3267 1 Introduction Through a system of ion channels and pumps, cells maintain a difference in electrical potential between the inside and outside of the cell membrane, which is called transmembrane potential. In eukaryotic cells, this usu- ally ranges between -40 and -70 mV. The induced trans- membrane potential present during the cell’s exposure to the electric field is added to the transmembrane poten- tial. Exposing cells to a sufficiently strong electric field can impose a significantly higher transmembrane po- tential than the resting transmembrane potential. Such a high transmembrane potential and, as a result, a strong electric field in the membrane, leads to a series of chang- es, including structural membrane changes and modifi- cations of membrane molecules. Therefore, molecules to which the membrane is normally impermeable can pass through. Increased permeability of the cell membrane, i.e. permeabilization of the membrane, due to the expo- sure of cells/tissues to an electric field, is called electro- poration or also electropermeabilization (1). The electric field during electroporation is established by applying electrical pulses through electrodes that are in contact with the sample or tissue. Electroporation is used in many areas including tissue ablation, gene electrotransfer for plasmid deliv- ery into cells, and electrochemotherapy, which is a lo- cal method of cancer treatment using a combination of standard chemotherapy and short electrical pulses (2). Medical applications of electroporation are effective and safe. In electrochemotherapy and electroporation abla- tion, cell death is not mainly caused by thermal injury but by increased permeability of the cell membrane. Therefore, the healthy surrounding tissue is not injured by treatment (3,4). However, due to electrical pulses, some unwanted side effects can occur, particularly mus- cle contractions, which can cause unpleasant sensations and even pain; in some cases, it is necessary to coordi- nate the delivery of pulses with the electrocardiogram to prevent arrhythmias (e.g. ventricular fibrillation). To improve results of electroporation-based applications, it is crucial to evaluate the influence of individual pa- rameters – from electrical pulse parameters (e.g. voltage, so učinkovite in varne, vendar so zaradi delovanja električnih pulzov lahko prisotni tudi določeni neželeni stranski učinki, predvsem mišično krčenje in akutna bolečina. Za optimiziranje parametrov elektroporacije in s tem rezultatov na elektro- poraciji temelječih terapij je ključnega pomena nadaljnja razjasnitev osnovnih mehanizmov elektroporacije in vplivov po- sameznih parametrov električnega polja na elektropermeom. Namen prispevka je predstaviti celovit pregled mehanizmov elektroporacije ter elektropermeoma, tj. celičnega odgovora na elektroporacijo. duration and number of pulses) to electrode geometry and position – on success of treatment (2). Tradition- ally, electroporation procedures use monophasic pulses with a duration in the order of micro- and milliseconds, but in recent years the possibility of using nanosecond pulses and high-frequency biphasic pulses lasting only a few microseconds has been studied, as there it is pos- sible to overcome certain limitations that appear in con- ventional electroporation with monophasic milli- and microsecond pulses. Nanosecond pulses reduce muscle contraction (5) and cause less tissue heating due to less energy being delivered (6). This reduces the possibility of thermal injury while the use of biphasic pulses reduc- es muscle contractions (7,8) and arrhythmia risk (7,9). The comprehensive optimization of electrical pulse parameters for individual applications is limited by an incomplete understanding of the basic mechanisms of electroporation. The purpose of this paper is to provide a comprehensive overview of the cell membrane perme- ability increase mechanisms due to the action of elec- trical pulses and all subsequent changes and processes triggered by electroporation – from chemical changes in membrane lipids and modulation of protein function to changes in gene expression and protein synthesis, as well as the activation of cell death and the immune response. 2 Mechanisms of permeabilization of the cell membrane Experimental results show that an increase in mem- brane permeability can occur in less than 10 ns, suggest- ing a direct rearrangement of membrane components (10). The currently established explanation of electropo- ration is based on the formation of water pores in the lipid bilayer. Molecular dynamics simulations indicate that (with a sufficiently high potential on the bilay- er or a sufficiently high electric field) the pore forma- tion begins with the orientation of water molecules in the direction of the electric field and their penetration into the lipid bilayer from both (intra- and extracellu- lar) sides (Figure 1A). Water molecules, oriented in the 485 REVIEW ARTICLE The electropermeome: cellular response to electroporation direction of the electric field, are connected by hydrogen bonds into small clusters. These clusters, called water fingers, grow in size and increasingly protrude into the hydrophobic core of the lipid bilayer (Figure 1B) until they connect both sides (intra- and extracellular) and form a water channel (Figure 1C). Such a structure is called a hydrophobic pore. Phospholipids reorient in the presence of water channels by turning their polar head groups toward the resulting water channel to “shield” the nonpolar tails from water molecules. The reorientation of phospholipids stabilizes the pore, which at this stage is called a hydrophilic pore (Figure 1D). Stabilization of the pore allows even more water and other polar mole- cules to enter the water channel (1). In the absence of an electric field, the pores begin to close. Pore closing occurs in the reverse order of the analogous stages of pore formation. While the time re- quired for pore formation decreases exponentially with Figure 1: Pore formation in the lipid (phosphatidylcholine) bilayer. (A) orientation of water molecules in the direction of the electric field and penetration into the lipid bilayer, (B) the appearance of water fingers, hydrogen-bonded clusters of water molecules that protrude into the core of the lipid bilayer, (C) the joining of water fingers into a water channel called a hydrophobic pore that connects the intra- and extracellular side of the lipid bilayer, (D) reorientation of phospholipids by turning their polar heads towards the water channel, which at this stage is called a hydrophilic pore. The polar phosphatidylcholine heads are shown as green and white circles, but the lipid tails are not shown for clarity. 486 PHYSIOLOGY Zdrav Vestn | November – December 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3267 increasing electric field strength (11), the time required for the pores to close is practically independent of the strength of the electric field that triggered their forma- tion; the closing of pores in lipid bilayers in molecular dynamics simulations always takes from a few tens to a few hundred nanoseconds, which suggests that the pores are not stable. In simulations, the estimated time required for pore closure is several orders of magni- tude shorter than the experimentally determined time required for membrane closing (i.e. the time during which increased transmembrane transport is observed) (1). The increased permeability of the cell membrane is observed for a few minutes after the electric field is no longer present, even when using pulses with a duration of only a few nanoseconds. However, the duration of the increased permeability of the cell membrane depends on the temperature (12-15), which suggests that elec- troporation of cell membranes is a more complex pro- cess than the mere formation of short-lived pores in the lipid bilayer. Several studies indicate the importance of chemical changes in membrane lipids and modulation of protein function in increasing membrane permeabili- ty during electroporation (1). Electroporation causes a series of changes in the cell membrane (formation of lipid and protein pores, oxida- tion of membrane lipids), depolarization, formation of reactive oxygen species (ROS), release of ATP and K+ from the cell, influx of Ca2+ into the cytoplasm, entry of extracellular molecules into the cell, osmotic imbalance, protein reorganization or protein structural changes, including opening of ion channels, cytoskeleton disrup- tion, activation of various signalling pathways, changes in gene expression and protein synthesis, as well as acti- vation of several cellular repair mechanisms (Figure 2). Changes in the permeabilized cell membrane, as well as all subsequent processes that are active even when increased transmembrane transport of substances to which the cell membrane is normally impermeable is no longer observed, are denoted by the term electroper- meome (16). 2.1 Chemical changes of membrane lipids Electroporation pulses trigger the formation of ex- tra- and intracellular ROS (17-22). Lipid oxidation due to exposure to electrical pulses such as those used in electroporation alters the composition and properties of both lipid bilayers and cell membranes. Chemical changes in membrane lipids, particularly peroxidation, could explain the longer-lasting permeabilization of cell membranes after electroporation. Lipid peroxidation is the oxidative degradation of lipids. It involves the for- mation and breakdown of dioxygen adducts of unsatu- rated lipids called lipid hydroperoxides (Figure 3). The reaction is initiated by a strong oxidizing agent (e.g. hy- droxyl radical), which removes the weakly bound allylic hydrogen from the lipid. Further degradation of hydrop- eroxides, the primary products of lipid peroxidation, is a complex process in which many secondary products are formed, e.g. aldehydes, ketones, alcohols, hydrocarbons, esters, furans, lactones, and peroxides. Hydroperoxides and some of their decomposition products, e.g. muta- genic malondialdehyde (MDA), react with amino acids, DNA and membranes (23). It has been demonstrated that ROS concentration and the extent of lipid peroxidation increase with elec- tric field intensity, pulse duration and pulse number in bacterial, plant and animal cells as well as in liposomes, and that lipid peroxidation is associated with increased cell membrane permeability, time required for membra- neresealing, and cell damage (17-20,22,24). 2.1.1 Peroxidation of membrane phospholipids Cell membranes consist of a bilayer of phospholipids and sterols, in which proteins and other molecules are placed. The presence of oxidized lipids in lipid mem- branes disrupts the lipid order, leads to lateral expansion and thinning of the bilayer, lowers the temperature of the phase transition, changes the hydration of the bilayer, increases lipid mobility and the frequency of flip-flops, affects the lateral membrane organization and promotes the formation of membrane defects. Therefore, bilayers with oxidized lipids are significantly more permeable and conductive than unoxidized bilayers (25-29). Ex- periments showed the presence of conjugated dienes in the membranes of electroporated cells or vesicles, which indicates the presence of hydroperoxides, the prima- ry products of lipid peroxidation (17,22,24), as well as MDA, indicating the presence of secondary peroxida- tion products (24). Hydroperoxides are stable enough to be present in the lipid bilayer for some time after oxidation. Using molecular dynamics simulations, Rems et al (26) quan- tified the permeability and conductivity of a lipid bilayer with a different proportion of hydroperoxides, i.e. of pri- mary products of lipid peroxidation. They showed that even a small proportion (around 1%) of hydroperoxides affects the conductivity of the bilayer. However, they al- so found that the increase in the conductivity and ion permeability of the lipid bilayer due to the presence of hydroperoxides alone is too small to fully explain the 487 REVIEW ARTICLE The electropermeome: cellular response to electroporation Fi gu re 2 : E le ct ro pe rm eo m e sc he m e - c ha ng es a nd p ro ce ss es th at o cc ur in th e ce ll du rin g an d aft er e le ct ro po ra tio n. Am on g ot he r th in gs , e le ct ro po ra tio n ca us es th e fo rm at io n of li pi d an d pr ot ei n po re s, p er ox id at io n of m em br an e lip id s, o pe ni ng o f i on c ha nn el s an d N a+ /K + -A TP as e, in flu x of C a2 + i nt o th e cy to pl as m , A TP a nd K + r el ea se fr om th e ce ll an d re su lti ng o sm ot ic im ba la nc e, fo rm at io n of re ac tiv e ox yg en s pe ci es (R O S) , c yt os ke le to n di sr up tio n, ac tiv at io n of si gn al lin g pa th w ay s a nd re pa ir m ec ha ni sm s, a nd c ha ng es in g en e ex pr es si on a nd p ro te in sy nt he si s. 488 PHYSIOLOGY Zdrav Vestn | November – December 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3267 experimentally determined values. Spontaneous pore formation was not observed in lipid bilayers with hy- droperoxides (26,30,31). Phospholipids with aldehyde groups on acyl tails (sec- ondary products of phospholipid peroxidation) disrupt the lipid bilayer more than hydroperoxides. In experi- ments (27,32) and molecular dynamics simulations (28,30-33), a significant increase in membrane permea- bility with a certain proportion of phospholipids with al- dehyde groups was observed, as well as the spontaneous Figure 3: Scheme of lipid peroxidation reactions. The reaction is initiated by a strong oxidant, which removes a weakly bonded allylic hydrogen from the unsaturated fatty acid - this is how the alkyl fatty acid radical is formed. The addition of molecular oxygen to an alkyl radical can lead to the formation of a conjugated peroxyl radical. The latter abstracts the allylic hydrogen from another unsaturated fatty acid, leading to the formation of hydroperoxide, the primary product of lipid peroxidation, and a new alkyl radical. With a sufficient amount of molecular oxygen and non-oxidized unsaturated lipids, this step can be repeated many times, but it also competes with several termination reactions at the same time. Termination involves a reaction between two alkyl radicals, between two peroxyl radicals, between an alkyl and a peroxyl radical, or the reaction of an alkyl/peroxyl radical with a non-lipid substrate (e.g. phenolic antioxidants, ascorbate, glutathione, or amino acid residues). Further degradation of hydroperoxides, the primary products of lipid peroxidation, is a very complex process in which many secondary products are formed, some of which have important biological effects. 489 REVIEW ARTICLE The electropermeome: cellular response to electroporation organization of aldehydes into pores (27,30-34). The pores formed as a result of the presence of lipid perox- idation products are not the same as the pores formed in a non-oxidized lipid bilayer under the influence of an electric field. In molecular dynamics simulations, pores from lipid peroxidation products with aldehyde groups were opened for a few microseconds, and in the pres- ence of cholesterol for the entire duration of the simula- tion (5 µs) (33). 2.1.2 Oxidation of membrane sterols Sterols influence the conformational order of acyl lip- id chains, the thickness of the hydrophobic part of the membrane, and the lateral membrane organization and permeability. Cholesterol greatly reduces the membrane’s permeability for water, oxygen, ions, and other small mol- ecules. Most biological membranes are dominated by a single sterol. In mammalian cells, this is cholesterol (35). Oxidation of membrane cholesterol changes the structure of the membrane. Cholesterol can be oxidized enzymatically or non-enzymatically due to direct ROS action. Oxidized cholesterol derivatives with one or more additional oxygen functional groups are called ox- ysterols. Oxysterols can be divided into two groups: those in which the short nonpolar tail is oxidized, and those in which the tetracycline ring is oxidized. Oxysterols with an oxidized tail have a similar effect on membranes as cholesterol, but they cause the phospholipid tails to be less ordered and do not condense the lipid bilayer as much as cholesterol does. Rapid turnovers of oxysterols with oxidized tails increase membrane permeability (e.g. 25-hydroxycholesterol is known to increase membrane permeability to calcium ions and glucose). Oxysterols with an oxidized tetracycline ring, which can adopt a different conformation, disrupt the membrane structure more than oxysterols with oxidized tails, as they increase the mobility of phospholipid tails (35). There are few studies investigating sterol oxidation by electroporation. Kazmierska et al (36) observed a rel- atively low increase in the concentration of oxysterols (both those with an oxidized tail and those with an ox- idized tetracycline ring) in yolks exposed to electrical pulses. By increasing the number of electrical pulses, they measured a higher concentration of oxysterols. 2.2 Modulation of protein function Evidence of electroporation’s effect on proteins and their role in increasing membrane permeability can al- so be found in the literature. Both experiments on lipid bilayers and molecular dynamics simulations indicated that the presence of a protein channel in the lipid bilayer stabilizes the membrane, requiring a higher electric field strength for electroporation to occur. In simulations, the formation of larger pores near the channel was not ob- served (37,38). However, Azan et al (39,40) using con- focal Raman microspectroscopy demonstrated protein modifications in living cells exposed to electroporation pulses. Unfortunately, the method used does not allow for differentiation between the modification of mem- brane and cytoplasmic proteins. 2.2.1 Membrane proteins Submicrosecond pulses cause voltage-gated calcium channels to open via a mechanism that does not involve lipid bilayer pore formation, heating, or membrane de- polarization via voltage-gated sodium channels (41-43). Microsecond electroporation pulses, however, cause the Na+/K+-ATPase to open (44). Due to electroporation, cadherin in cell junctions is also lost (45,46). Cadherins are transmembrane proteins that play an important role in the formation of adherens junctions, a type of inter- cellular junction in epithelial and endothelial tissues. Using electric fields that induce electroporation, Rems et al. (47,48) observed pore formation in the volt- age-sensing domains of various voltage-gated channels in molecular dynamics simulations. In the simulations, the formation of the pore was followed by the unwinding of the voltage-sensing domain and the stabilization of the pore by membrane lipid heads. Such pores remained stable even until the end of the simulation (one micro- second after the electric field was no longer present), which is significantly longer than the pores in the lipid bilayer. Rems et al (47) concluded that in the case of ma- jor disruption of the protein channel structure, it cannot spontaneously fold back into its native conformation, but the cell repairs the damage through the mechanism of endocytic recycling. 2.2.2 Cytoskeleton Cytoskeleton proteins (actin filaments, intermediate filaments and microtubules) and related proteins affect membrane permeability – the formation and expansion of membrane pores and membrane closing after elec- troporation. Disruption of the actin filament network lowers the energy barrier for membrane pore forma- tion (15). Electroporation of cells or giant unilamel- lar vesicles with encapsulated actin did not cause large micrometre-diameter pores that were observed when 490 PHYSIOLOGY Zdrav Vestn | November – December 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3267 electroporation of empty vesicles (49). In cells and ves- icles with encapsulated actin, however, a longer lasting permeabilization of the lipid bilayer was observed than in cells incubated with a toxin that destabilizes actin fil- aments or in empty vesicles (15,50). Electroporation results in reversible disruption of the three-dimensional filamentous structures of actin, tubu- lin, and intermediate filaments, but not degradation of monomeric cytoskeletal proteins. The cytoskeleton reas- sembles within hours after electroporation (45,46,51,52). However, it is not yet entirely clear whether the destruc- tion of the cytoskeleton is a direct consequence of the ac- tion of electrical pulses or fields on cytoskeleton proteins or the result of ATP release from cells, increased con- centration of calcium ions in the cytoplasm, hydrolysis of phosphatidylinositol-4-5-bisphosphate (PIP2) and/or cell swelling due to electroporation (49). Pakhomov et al (53) showed that the disruption of actin filaments after electroporation with nanosecond pulses is the result of cell swelling. Contrarily, research in which pulses with different parameters were used indi- cated that actin filaments can collapse even without cell swelling or vesicle formation, which indicates the direct effects of electric pulses or fields on actin (45,50). Using atomic force microscopy, Louise et al (54) showed that cell swelling is mainly due to the destabilization of the interaction between cortical actin and the membrane due to electroporation pulses, but not to the depolymer- ization of actin filaments. This confirmed the findings on the membrane’s separation from the cytoskeleton under the influence of electrical pulses or fields (55). Using molecular dynamics simulations, Marracino et al (56) showed that high-voltage nanosecond electrical pulses cause changes in the conformation of the C-ter- minal end of β-tubulin and changes in the local electro- static properties of the GTPase domain and the bind- ing site for most molecules that bind to β-tubulin. Their findings suggest that nanosecond electrical pulses can physically affect microtubule dynamics. Chafai et al (57) also showed experimentally that nanosecond electrical pulses change the conformation of the C-terminal part of tubulin, which polymerizes into different structures; whether the modulation of tubulin self-organization is reversible or irreversible depends on the parameters of the electrical pulses. Modulation of tubulin-associated proteins (e.g. kinesin) could also alter the microtubule network dynamics. Using molecular dynamics simula- tions, Průša et al (58) showed that a 30 ns electrical pulse changes the contact surface between kinesin and tubulin as well as tubulin binding sites and nucleotide hydrolysis sites on kinesin. 3 Changes in gene expression and protein synthesis after electroporation Electroporation initiates a series of physiological cell responses, which is also reflected in gene expression and protein synthesis changes. Since electroporation puls- es induce ROS formation, it is expected that cells will respond to oxidative stress. In the yeast Saccharomyces cerevisiae, exposure to electrical pulses increased the ex- pression of genes for proteins involved in the response to oxidative stress (GLR1, SOD1, SOD2 and GSH1) (59). Michel et al (60), however, observed increased immuno- cytochemical staining with antibodies against superox- ide dismutase SOD-2 after electroporation, incubation with cisplatin, and a combination of electroporation and cisplatin (electrochemotherapy) in metastatic pancreatic cancer cells. An increase in SOD-2 gene expression after electroporation was also measured by Dovgan et al (61) in mesenchymal adipose-derived stromal cells and um- bilical-cord-derived stromal cells. By monitoring the expression of various genes after electroporation, Morotomi-Yano et al (62) showed that nanosecond electrical pulses present a different type of stress to cells than endoplasmic reticulum injury, ultra- violet light, or heat shock. Cells respond to physiological stress by activating various mechanisms. Since protein synthesis consumes a significant proportion of cellular building blocks and energy, it is highly regulated during stress responses. Electroporation causes phosphoryla- tion of the eukaryotic translation initiation factor eIF2a and dephosphorylation of the 4EBP1 protein, which indicates suppression of protein translation or protein synthesis in general (62), and to the reduced expression of genes involved in protein synthesis (63). Hojman et al (64) detected a reduced expression of genes involved in metabolism (e.g. genes for phosphoenolpyruvate car- boxykinase and dipeptidase) in murine muscles after electroporation, which suggests a reduction in catabo- lism. Electroporation also causes other changes: reduced expression of histones H2A and H4, which are crucial for chromatin organization (63,65), reduced expression of cytoskeletal protein genes (64), and changes in genes and proteins associated with cell death and immune re- sponse (66). 3.1 Cell death and immune response Cell death can be approximately divided into patho- logical (necrosis) and programmed. Until recently, apop- tosis was considered synonymous with programmed cell death, but in recent years other types of programmed 491 REVIEW ARTICLE The electropermeome: cellular response to electroporation cell death have been discovered, e.g. pyroptosis and necroptosis. Apoptosis can be triggered by several path- ways, which are mainly divided into extrinsic and intrin- sic pathways. The extrinsic pathway is mediated through receptors on the surface of the cell membrane (death re- ceptors). In the extrinsic pathway, caspase-8 is activated at the cell membrane and then directly activates effector caspases (caspase-3, -6 and -7). The intrinsic pathway, on the other hand, is mediated via mitochondria and is particularly fast, as all the factors are already present and only need activation. In the intrinsic pathway, disrup- tion of the balance between proapoptotic (e.g. Bid, Bax and Bak) and antiapoptotic (e.g. Bcl-2) proteins releas- es apoptosis-inducing proteins (e.g. cytochrome c and apoptosis-inducing factor, AIF) from the mitochondria, which activates caspases. Unlike apoptosis, activation of caspase-1, -4, 5- or -11 is key in pyroptosis, which then initiate the process of programmed cell death by cleav- ing the pore-forming protein gasdermin D. Key mark- ers of pyroptosis are caspase-1 activation and caspase-3 non-activation (the latter is associated with apoptosis). The activation of necroptosis is influenced by the activa- tion of the kinases RIPK3 and MLKL (67). Triggering of apoptosis after electroporation is main- ly mentioned in association with nanosecond pulses (68-70), but it has also been observed with electropo- ration with longer pulses. Ford et al (69) detected an electric field-dependent increase in the amount of acti- vated caspases-3, -6, -7, -8 and -9 in murine melanoma cells after electroporation with 3 ns pulses. However, no release of cytochrome c, AIF, or Smac/DIABLO from mitochondria was observed. Their findings suggest that nanosecond pulses trigger apoptosis through a pathway similar to the extrinsic activation pathway. In contrast, Beebe et al (68) observed in electroporation with 60 ns pulses that the initiation of apoptosis depends on the ac- tivation of caspases as well as on mitochondria, as they detected the release of cytochrome c into the cytoplasm in a T lymphocyte cell line. Zhang et al (71) monitored the expression of 17 genes related to apoptosis. After on- ly two hours after electroporation of breast cancer cells with microsecond pulses, changes in the expression of caspases and genes associated with death receptors were observed. The expression of caspase-3 was increased, while the expression of caspase-6, -7 and -9 and Bc1-2, Bid and FASLG were decreased. They concluded that the activation of apoptosis after electroporation was mainly through the intrinsic pathway. Since caspase-3 is involved in both the intrinsic and extrinsic pathways of apoptosis initiation, the increased expression of caspase-3 may indicate that the process of apoptosis has been initiated in the cells, but the pathway itself could not be determined. Zhang et al (72) detect- ed more caspase-3 in pancreatic cancer cells exposed to electroporation pulses, O’Brien et al (73) observed im- munohistochemical staining of cleaved caspase-3 only at the edge of the pancreatic ablation zone after electro- poration, Siddiqui et al (74) detected cleaved caspase-3 in the entire zone of liver ablation, and Mercadal et al (75) recorded increased expression of caspase-3 or -7 in pancreatic adenocarcinoma cells. All three studies indicated that apoptosis was initiated. Michel et al (60) observed increased immunocytochemical staining with anti-caspase-3 antibodies after incubation with cisplatin, microsecond pulse electroporation, and a combination of electroporation and cisplatin (electrochemotherapy) in metastatic pancreatic cancer cells. An electric current flowing through a conductor (e.g. cell suspension, tissue, and so on) causes it to be heated (i.e. Joule heating). With properly selected elec- trical pulse parameters, it is possible to achieve a small enough increase in temperature to prevent thermal injury to cells/tissue. Faroja et al (76) wanted to deter- mine whether high-energy pulses (i.e. a large number of pulses and/or a high electric field strength) can cause thermal injury to liver tissue. When the electropora- tion temperature did not exceed 39 °C, apoptotic cells with cleaved caspase-3 were observed and virtually no HSP70 heat shock proteins were detected (character- ized by their expression being greatly increased by heat stress or toxic chemicals). In contrast, in cells subject- ed to electroporation where the temperature exceeded 60°C, distinct expression of HSP70 and only minimal expression of caspase-3 was observed. Ben-David et al (77), however, observed differences in the immunohis- tochemical staining of cleaved caspase-3 and HSP70 in different tissues after electroporation: strong staining for cleaved caspase-3 and limited expression of HSP70 was detected in the liver, no staining was detected in muscle cells, while minimal staining for cleaved caspase-3 and a significant increase in HSP70 in the tissue surrounding the area where the electrical pulses were delivered were observed in the kidneys. Kanthou et al (45) did not de- tect an increased accumulation of HSP70 after electro- poration of umbilical cord endothelial cells, while Mla- kar et al (63) and Dovgan et al (61) after electroporation of melanoma cells and mesenchymal adipose-derived stromal cells and umbilical-cord-derived stromal cells observed increased expression of proteins from the heat shock protein family HSP70. Contrary to most of the literature, in the re- search carried out by Mercadal et al (75), pancreatic 492 PHYSIOLOGY Zdrav Vestn | November – December 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3267 adenocarcinoma cells died after electroporation via a pathway independent of caspase-3 or -7; however, Zhang et al (67) observed increased expression of cleaved caspase-1, gasdermin D, RIPK3, and MLKL and de- creased expression of cleaved caspase-3 six hours after liver electroporation. They concluded that the results in- dicate the activation of pyroptosis and necroptosis, but not apoptosis. Ringel-Scaia et al (78) observed changes in gene expression consistent with apoptosis immediate- ly after electroporation of breast cancer cells. Over time, however, they observed a change in gene expression to- wards inflammatory types of cell death and necrosis – after 24 hours, increased expression of genes associated with necrosis and pyroptosis was recorded. Pyroptosis is also associated with the regulation of pattern recog- nition receptors (PRR), so it is not surprising that they also observed increased expression of three networks associated with damage associated molecular patterns (DAMPs): ROS, ATP and HMGB1 signalling. However, they detected a decreased expression of genes related to immune system suppression, and an increased expres- sion of genes related to the inflammatory response. The authors also observed decreased expression of genes as- sociated with cell damage and increased expression of genes associated with regeneration. Peng et al (79) detected increased expression of genes related to apoptosis/necrosis (caspase-8, bcl-w, Mt2 and seven genes from the cytochrome P450 family) and in- creased expression of several chemokine genes, includ- ing MIP-1α, MIP-1β, MIP-1γ, IP-10 and MCP-2, four hours after electroporation in murine skeletal muscle. Heller et al (80) measured elevated mRNA levels for sev- eral inflammatory chemokines and cytokines (MIP-1α, MIP-1β, IP-10, IL-6, and inducible nitric oxide synthase) after electroporation of murine melanomas. They also detected increased levels of IL-1β and IL-6 proteins after electroporation. Goswami et al (81) studied the effect of microsecond electroporation pulses on triple negative 4T1 breast cancer cells. They measured increased mR- NA concentrations for IL-6 and tumour necrosis factor (TNF) and decreased mRNA concentrations for TSLP after electroporation. Lower expression of TSLP, which plays an important role in cancer progression, was also confirmed at the protein level. Zhang et al (71) observed a decreased expression of Ki-67 and TGF-β proteins af- ter electroporation of breast cancer cells. Ki-67 is used as a marker for dividing cells and is associated with tumour growth and invasion, and TGF-β expression correlates with tumour invasiveness. Mlakar et al (63), however, showed that electroporation of melanoma cells did not change the expression of the main tumour suppressor genes and oncogenes. All of these studies indicate that electroporation is a safe and non-carcinogenic method. 4 Conclusion Electroporation is a phenomenon that causes in- creased cell membrane permeability due to exposure of cells/tissues to an electric field. It causes a series of changes in the cell, including structural changes in the cell membrane, peroxidation of membrane lipids, influx of Ca2+ into the cytoplasm, ATP and K+ release from the cell, osmotic imbalance, cytoskeleton disruption, chang- es in gene expression and protein synthesis, formation of reactive oxygen species, activation of signalling pathways and repair mechanisms; it also triggers cell death under certain conditions. The term electropermeome refers to both a permeabilized cell and what happens during or immediately after the delivery of electrical pulses, as well as all subsequent processes that remain active for some time after increased transmembrane transport of substances to which the cell membrane is normally im- permeable is no longer observed, i.e. even after the time when we can already see that the membrane has resealed. Electroporation is used in many fields, including electrochemotherapy, tissue ablation, and gene electro- transfer. Electrochemotherapy has been used in clini- cal practice for more than 15 years and is included in guidelines and standard clinical practice in many Euro- pean countries for the treatment of various superficial tumours, including melanoma, squamous carcinoma and metastases of all histological types. Clinical studies have shown that electrochemotherapy is feasible, safe, and effective even for deep-seated tumours (2,3). While thermal techniques (radiofrequency and microwave ab- lation, cryoablation) are routinely used for soft tissue ablation, interest in irreversible electroporation ablation is growing. The latter is particularly interesting for use on anatomical sites where surgery and thermal ablation methods are not possible, e.g. due to the proximity of vital structures such as large blood vessels, intestine, and biliary or urinary tracts. Due to the predominantly non-thermal mechanism of action, ablation with irre- versible electroporation does not damage the surround- ing tissue. The efficacy and safety of irreversible electro- poration ablation have been demonstrated in numerous clinical studies for ablation of deep-seated liver, kidney, pancreas and prostate tumours, as well as for pulmonary vein isolation in the treatment of atrial fibrillation (4,82). 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