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. 2020 Mar 10;15(3):e0229948. doi: 10.1371/journal.pone.0229948

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation

Yoshimasa Hamada 1,2, Yuji Furumoto 3, Akira Izutani 3, Shusuke Taniuchi 1,2,4, Masato Miyake 1,2,4, Miho Oyadomari 1, Kenji Teranishi 3, Naoyuki Shimomura 3, Seiichi Oyadomari 1,2,4,*
Editor: Dong-Yan Jin5
PMCID: PMC7064201  PMID: 32155190

Abstract

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.

Introduction

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) at Ser51. The ISR is activated in response to several stress conditions, such as viral infection, heme deprivation, amino acid starvation, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress [1, 2]. The phosphorylation of eIF2α results in the inhibition of eIF2-GTP/Met-tRNAi ternary complex recycling, which is necessary for the initiation of mRNA translation, thereby reducing overall translation while selectively favoring the translation of proteins implicated in stress recovery, such as activating transcriptional factor 4 (ATF4) [3, 4]. ATF4 induces the expression of several genes involved in the regulation of redox balance, amino acid biosynthesis and transport to overcome the imposed stress and restore cellular homeostasis. There are four different eIF2α-specific kinases, namely, heme-regulated inhibitor (HRI), protein kinase double-stranded RNA-dependent (PKR), general control nonderepressible (GCN) 2, and PKR-like ER kinase (PERK), and each eIF2α kinase senses and responds to distinct cellular stresses, with some overlap in their activities [5].

As the ISR is an innate protective mechanism, dysregulation of ISR signaling has important pathologic consequences linked to inflammation [6], diabetes [7], cancer [8], and neurodegenerative diseases [9]. Furthermore, enhancement of ISR signaling has been suggested to have beneficial effects, further supported by genetic manipulation of the ISR pathway in mouse models of neurodegenerative diseases such as multiple sclerosis [10] and amyotrophic lateral sclerosis [9]. Hence, modulation of the ISR represents a promising therapeutic strategy, and recent encouraging advances have been made in this area through the development of small molecules to enhance ISR signaling. Indeed, several compounds have been described to activate the eIF2α kinases, 1H-benzimidazole-1-ethanol,2,3-dihydro-2-imino-a-(phenoxymethyl)-3-(phenylmethyl)-,monohydrochloride (BEPP) as a PKR activator [11], 1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(3,4-dichlorophenyl)urea (BTdCPU) as an HRI activator [12], halofuginone as a GCN2 activator [13] and CCT020312 as a PERK activator [14]. Halofuginone, for example, is a potent inhibitor of angiogenesis progression and is being evaluated in a clinical phase II trial [15].

Physiotherapy, such as electrotherapy, thermotherapy and phototherapy, has a place within clinical practice. Different modalities of therapy that activate the ISR in addition to drug therapy are worth developing. We previously reported that nanosecond pulsed electric fields (nsPEFs) induce eIF2α phosphorylation [16]. nsPEFs are characterized by ultrashort-duration and high-intensity electric fields [17]. Typical nsPEFs have a duration of 60–300 ns, with a rise time of 4–30 ns [18, 19]. Millisecond PEFs are commonly used in life sciences, especially for DNA transfection to generate pores on the cell membrane. On the other hand, nsPEFs can directly reach intracellular components without cell membrane destruction, and thus nsPEFs have emerged as a unique therapeutic tool for intracellular manipulation without any chemical intervention [20, 21]. Although nsPEFs are now recognized as a drug-free and purely electrical cancer therapy, the molecular mechanism of nsPEF action remains largely unclear.

In this study, we investigated which eIF2α kinase is responsible for nsPEF-induced eIF2α phosphorylation and how nsPEFs activate the responsible eIF2α kinase. Here, we present evidence that nsPEFs generate ROS that activate HRI, leading to eIF2α phosphorylation. Our results provide a molecular mechanism for the action of nsPEFs for research and therapeutic development.

Materials and methods

Cell culture and cell lines

SV40 large T-antigen immortalized mouse embryonic fibroblasts (MEFs) were cultured in DMEM-high glucose supplemented with 10% FBS (Giboco), 2 mM L-glutamine (Nakarai-Tesque, Japan), 55 μM 2-mercaptoethanol, and nonessential amino acids (Invitrogen) at 37°C under humidified conditions with 5% CO2. eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO MEF cells were previously established [22]. Wild type, HRI KO, and GCN2 KO in Hap1 cells were cultured in IMDM (HyClone) supplemented with 10% FBS, 55 μM 2-mercaptoethanol (Invitrogen) at 37°C under a humidified condition with 5% CO2. HRI KO and GCN2 KO in Hap1 cells were generated by CRISPR/Cas9 system.

Electrical devices for the generation of nsPEFs

A pulsed power generator, based on a Blumlein pulse-forming network (B-PFN) that generates nsPEFs, was designed and developed at Tokushima University. The pulsed power generator was composed of a B-PFN and a DC high-voltage power supply (ALE Model 102, Lambda-EMI, U.S.). The circuit constants C1 and L1 were 295 pF and 300 nH, respectively. The voltage and current of the output pulses were measured using a voltage probe (HVP-39pro, PINTEC, China) and current transformer (CURRENT MONITOR MODEL 110A, PEARSON ELECTRONICS, INC., U.S.), respectively, and the waveforms were monitored by an oscilloscope (DSO1024A, Agilent Technologies, U.S.). Under our experimental conditions, an electroporation cuvette with aluminum electrodes spaced 4 mm apart (Nepa Gene Co., Ltd., Japan) and filled with the cell suspension and silicon oil (Shin-Etsu Chemical Co., Ltd., Japan) resulted in an average pulse width at half maximum of approximately 70 ns (Fig 1A).

Fig 1. Phosphorylation of eIF2α is induced in WT MEF cells by 40 shots of nsPEFs with 70-ns duration and 30-kV/cm electric fields.

Fig 1

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(A) The circuit configuration of the B-PFN as an nsPEF generator. The right upper panel shows a photograph of the nsPEF delivery device with a 4-mm gap cuvette. The right lower panel shows typical waveforms of nsPEFs using a 4-mm gap cuvette. (B) Experimental protocol. Resuspended WT MEF cells (4 x 105) were loaded into a 4-mm gap cuvette and covered with 800 μL silicone oil. After the indicated nsPEF treatment, WT MEF cells were collected into a 1.5-mL tube and incubated at 37°C for 1 h followed by immunoblot analysis. (C) Representative immunoblots of phosphorylated eIF2α and total eIF2α in WT MEF cells 1 h after the indicated nsPEF treatment. An ER stressor Tg served as a positive control for eIF2α phosphorylation. (D) Densitometry quantification of phosphorylated eIF2α normalized to the total eIF2α level in WT MEF cells 1 h after the indicated nsPEF treatment. Error bars show the means ± SEM (n = 8, *P < 0.05).

Immunoblot analysis

Cells were lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% deoxycholic acid) with protease inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitor cocktail (Biotool). Immunoblot analysis was performed as previously described using Blocking One (Nacalai Tesque) or Blocking One-P (Nacalai Tesque) and WesternSure ECL Substrate (Li-Cor Biosciences). Protein was visualized by Ez-Capture II (ATTO Corp), and the band intensities were quantified using Image Studio software (LiCor Biosciences). The sources of antibodies were as follows: Phospho-Ser51-eIF2α (D9G8 #3398) (Cell Signaling Technology); eIF2α (D7D3 #5324) (Cell Signaling Technology); HRI (SC-30143) (Santa cruz); GAPDH (M171-3) (MBL); ATF4 (D4B8 #11815) (Cell Signaling Technology); ATF3 (SC-81189) (Santa cruz); CHOP (15204-1-AP) (Proteintech); XBP1s (D2C1F #12782) (Cell Signaling Technology); Ribophorin (Homemade).

ROS production detection

At the end of the treatment schedule, cells were incubated with 10 μM CM-H2DCFDA (Thermo Fisher) in culture media for 30 min. Then, cells were washed with PBS, and the cell pellets collected by trypsinization were resuspended in 10% FBS-supplemented DMEM and analyzed for intracellular ROS production by flow cytometry S3e (Bio-Rad). All experiments were performed in three independent replicates.

Cell viability assay

Cell viability was determined by WST-8 assay (Dojin Laboratory) according to the manufacturer's instructions. Briefly, WST-8 solution was added to cells in 96-well plates and the optical density of each well was read at 450 nm using a microplate reader EMax Plus (Molecular Devices) followed by incubation for 1, 2, and 4 h after nsPEF treatment.

Mitochondrial membrane potential measurements

The changes in mitochondrial membrane potential were assayed using using the lipophilic cationic probe JC-1 (Setareh Biotech). The cells were incubated with 5 μg/mL JC-1 dye in culture media for 1 h, subsequently washed with PBS and then resuspended in PBS. The samples were then analyzed using, cells were removed probe, resuspended in PBS The emitted green (JC-1 monomer) and red (JC-1 polymer) fluorescence were detected by a fluorescence microscope (Olympus) and were analyzed for mitochondrial membrane potential using ImageJ (NIH).

Statistical analysis

Statistical analysis was performed using Student’s t-test. Data are expressed as the mean ± SEM. A difference was considered to be statistically significant when the P value was less than 0.05, unless otherwise stated.

Results

Phosphorylation of eIF2α is induced in the WT MEF cells by 40 shots of nsPEFs with 70-ns duration and 30-kV/cm electric fields

Previous studies have demonstrated that 80-ns PEFs induce eIF2α phosphorylation, which is a hallmark of the ISR, in HeLa S3 cells [23]. In this study, we applied 70-ns PEFs, which did not cause significant heat generation or cell death to MEFs (Fig 1A and S1A and S1B Fig). PEFs result in discharge on the surface of the cell suspension, causing insulation breakdown. To avoid this, we placed 800 μl of silicone oil on 200 μl of the cell suspension, which helped to increase the electric field (Fig 1A and 1B). To determine the optimal conditions of nsPEF treatment for eIF2α phosphorylation, we employed two pulse numbers (20 or 40 shots) and two electric fields (30 kV/cm or 60 kV/cm) (Fig 1B). Under these conditions, there were no physiologically meaningful temperature shifts in the cell suspension or significant cell damage (S1A and S1C Fig). eIF2α phosphorylation induction was enhanced by 30-kV/cm nsPEFs to a greater extent than by 60-kV/cm nsPEFs (Fig 1C and 1D). Both 40 shots of nsPEFs and 200 nM thapsigargin (Tg) treatment, which is a well-validated ISR activator, constantly induced eIF2α phosphorylation. Therefore, 40 shots of 30-kV/cm nsPEFs were used for further studies.

HRI is responsible for eIF2α kinase ISR activation by 70-ns PEF treatment

To better understand the mechanism of eIF2α phosphorylation, we previously established 4KO cells, in which the four eIF2α kinase genes were deleted using CRISPR/Cas9-mediated genome editing, and single eIF2α kinase-rescued 4KO cell lines (single rescue 4KO cells), in which one of four eIF2α kinase genes was overexpressed in 4KO cells [22]. Because there is overlap in the type of stresses among each eIF2α kinase, the magnitude of the activation of the primary kinase overshadows that of the secondary kinase, making the latter kinase difficult to detect (Fig 2A). For overcoming this problem, single rescue 4KO cells are powerful tools for determining the eIF2α kinase responsible for ISR activation. We previously reported that the four known eIF2α kinases are sufficient for the ISR and that there are no additional eIF2α kinases in vertebrates. As expected, eIF2α phosphorylation was completely abolished when we applied nsPEFs to the 4KO cells (Fig 2B). We next used single rescue 4KO cells and found that the phosphorylation of eIF2α in response to nsPEFs was successfully recovered when HRI was expressed, indicating that of the eIF2α kinases, HRI is responsible for nsPEFs (Fig 2C). Activation of the ISR was further confirmed by induction of ISR downstream target genes such as ATF4 and ATF3 (S2D and S2E Fig). As we expected, proapoptotic factor CHOP and UPR marker XBP1s were not induced by nsPEF treatment, indicating that nsPEFs did not cause apoptosis or ER stress (S2D and S2E Fig). Overexpression of a single eIF2a kinase may not reflect normal physiological function. To exclude this possibility, we established single HRI KO and GCN2 KO cells using CRISPR/Cas9 system in human Hap1 cell, respectively. Induction of phosphorylated eIF2α by nsPEFs was observed in WT and GCN2 KO Hap1 cells, but not in HRI KO cells (Fig 2D). Furthermore, we confirmed that nsPEFs phosphorylated HRI in WT Hap1 cell by phos-tag SDS-PAGE as well as known a known HRI activator arsenite (Ars) did (Fig 2E and 2F, arrowheads). Thus, these data demonstrated that nsPEFs phosphorylates the eIF2α via HRI activation.

Fig 2. The eIF2α kinase HRI is responsible for nsPEF-induced eIF2α phosphorylation.

Fig 2

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(A) Various stressors phosphorylate eIF2α via activation of four eIF2α kinases, PERK, GCN2, HRI and PKR. (B) Representative immunoblots of phosphorylated eIF2α and total eIF2α in WT and 4KO MEF cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields). (C) Representative immunoblots of phosphorylated eIF2α and total eIF2α in WT, 4KO and single eIF2α kinase-rescued 4KO MEF cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields). Treatment with 200 nM Tg served as a positive control for eIF2α phosphorylation. (D) Representative immunoblots of HRI, phosphorylated eIF2α and total eIF2α in WT HRI KO and GCN2 KO Hap1 cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields). nsPEFs induced eIF2α phosphorylation in WT and GCN2 KO Hap1, but did not induce eIF2α phosphorylation in HRI KO Hap1. (E) Representative Phos-tag immunoblots of HRI and immunoblots of GAPDH in WT and HRI KO Hap1 cells 1 h after 2 mM arsenite treatment. The arrowhead indicated the phosphorylated form of HRI. (F) Representative Phos-tag immunoblots of HRI and immunoblots of GAPDH in WT and HRI KO Hap1 cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields). The arrowheads indicated the phosphorylated form of HRI.

ROS exposure activates the eIF2α kinase HRI to initiate ISR activation

Although HRI is well known to be activated by heme deficiency in immature erythroid cells [24], mRNA expression for HRI has been identified across a wide range of tissues [25]. ROS have been reported to act as an HRI activator [26] and HRI KO cells suffered from increased levels of ROS and apoptosis [27]. To verify previous reports, we analyzed eIF2α phosphorylation in single rescue 4KO cells using the ROS agent hydrogen peroxide (H2O2) and the ROS scavenger N-acetyl-L-cysteine (NAC). In the current study, eIF2α phosphorylation after H2O2 exposure was recovered in HRI-rescued 4KO cells (Fig 3A and 3B). Furthermore, blockade of ROS via administration of NAC led to reduced HRI-mediated eIF2α phosphorylation after H2O2 exposure (Fig 3A and 3B). Thus, these data demonstrated that ROS activate the ISR mainly by HRI activation.

Fig 3. The eIF2α kinase HRI is responsible for ROS-induced eIF2α phosphorylation.

Fig 3

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(A) Representative immunoblots of phosphorylated eIF2α and total eIF2α in WT, 4KO and single eIF2α kinase-rescued 4KO MEF cells 1 h after 1 mM H2O2 treatment with or without 10 mM NAC. (B) Densitometry quantification of phosphorylated eIF2α normalized to the total eIF2α level in WT, 4KO and single eIF2α kinase-rescued 4KO MEF cells 1 h after 1 mM H2O2 treatment with or without 10 mM NAC. Error bars show the means ± SEM (n = 4–9, **P < 0.01).

ROS-activated HRI phosphorylates eIF2α in response to 70-ns PEF treatment

nsPEF-induced HRI activation may be assumed to be mediated by ROS. Indeed, the generation of ROS by nsPEF treatment has been reported by several groups [28–30], but some contradicting results exist [31]. For example, 300-ns 45-kV/cm nsPEF to Jurkat cells [28], 100-ns 30-kV/cm nsPEF to BxPC-3 cells [29] and 100-ns 40-kV/cm nsPEF to B16f10 or Panc-1 cells [30] increased intracellular ROS. On the other hand, 300-ns and 40-, 50- or 60-kV/cm nsPEF to E4 squamous cells did not increase intracellular ROS [31]. To examine the contribution of ROS on nsPEF-induced HRI activation, we analyzed eIF2α phosphorylation in each single rescue 4KO cell line after 70-ns PEF treatment with or without NAC. As expected, eIF2α was significantly phosphorylated by nsPEF treatment in WT and HRI-rescued 4KO cells but not in PERK-, PKR- or GCN2-rescued 4KO cells (Fig 4A and 4B). NAC treatment decreased nsPEF-induced eIF2α phosphorylation in WT and HRI-rescued 4KO cells to almost basal levels, indicating that nsPEF-induced ROS are a main cause of nsPEF-induced HRI activation (Fig 4A and 4B). To further confirm the nsPEF-induced ROS generation, we monitored the intracellular ROS content using the CM-H2DCFDA fluoroprobe, a membrane-permeable form of a ROS indicator (Fig 4C). nsPEF treatment elevated the ROS level in both WT and 4KO cells, and NAC attenuated ROS generation produced by nsPEFs (Fig 4C and S2C Fig). Mitochondria are considered as the main source of ROS in the cell. Therefore, we monitored mitochondrial membrane potential using JC-1 dye but the mitochondrial membrane potential was not found to be affected by nsPEFs treatment (S2A and S2B Fig). Altogether, our data strongly suggest that nsPEFs generate ROS, which increase eIF2α phosphorylation via HRI activation (Fig 4D).

Fig 4. ROS-activated HRI phosphorylates eIF2α during nsPEF treatment.

Fig 4

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(A) Representative immunoblots of phosphorylated eIF2α and total eIF2α in WT, 4KO and single eIF2α kinase-rescued 4KO MEF cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields) with or without 10 mM NAC. (B) Densitometry quantification of phosphorylated eIF2α normalized to the total eIF2α level in WT, 4KO and single eIF2α kinase-rescued 4KO MEF cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields) with or without 10 mM NAC. Error bars show the means ± SEM (n = 5–7, *P < 0.05, **P < 0.01). (C) Representative flow cytometric profiles of intracellular ROS levels in nsPEF-treated WT MEFs with or without 10 mM NAC using the CM-H2DCFDA fluoroprobe. (D) Proposed schematic model of the phosphorylation of eIF2α by nsPEF treatment. nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields) generates intracellular ROS, which increases eIF2α phosphorylation via HRI activation.

Discussion

Dysregulation of ISR signaling contributes to a wide range of pathological conditions linked to inflammation [6], diabetes [7], cancer [8], and neurodegenerative diseases [9]. Thus, modulation of the ISR may hold promise for a new therapeutic tool to treat various forms of human disease. Here, we present studies showing that 70-ns 30-kV/cm nsPEF treatment induced ISR activation in WT MEF cells. Using both 4KO cells and single rescue 4KO cells, we found that HRI is activated by nsPEF treatment and that activated HRI phosphorylates eIF2α. Furthermore, using the ROS indicator CM-H2DCFDA and the ROS scavenger NAC, we demonstrated that nsPEF-generated ROS are required for HRI activation.

Mitochondria are considered the main source of ROS in the cell, and mitochondrial dysfunction is often associated with increased ROS production [32]. The distinct effect of nsPEFs from classical plasma membrane electroporation is the opening of nanopores into intercellular organelles, such as the mitochondria, nucleus and ER [19]. The nsPEF-induced release of cytochrome c from the mitochondria into the cytoplasm suggests that the mitochondria are intracellular targets for nsPEFs [33]. Indeed, nsPEF-induced permeabilization of mitochondrial membranes has been reported [34]. ROS production from mitochondria may depend on several factors, such as proton motive force or the redox state of the NADH pool, but the molecular mechanism of ROS production by nsPEFs remains to be elucidated.

nsPEF-triggered ROS production has been observed by several [28–30] groups, but one report indicated that nsPEFs have no or minimal effects on ROS production [31]. This discrepancy can be explained by cell-type differences. Pakhomova et al. reported that ROS production increased with time after treatment in nsPEF-sensitive Jurkat cells but remained stable in nsPEF-resistant U937 cells under the same conditions of 300-ns 45-kV/cm nsPEFs [28]. In the case of E4 squamous cells, ROS production was not observed under the same 300-ns duration with electric field strength from 0 to 60 kV/cm [31]. These results suggest that nsPEF-induced ROS production is cell-type dependent, probably due to different intercellular antioxidant activity or different mitochondrial vulnerability to nsPEFs.

However, different nsPEF conditions may lead to different cellular consequences. We previously reported that PERK and GCN2 are involved in eIF2α phosphorylation caused by 80-ns 20-kV/cm nsPEFs [23], demonstrating that nsPEF-induced eIF2α phosphorylation was reduced but not abolished in PERK/GCN2 double-KO MEFs, indicating that HRI is also involved in nsPEF-induced eIF2α phosphorylation. However, in the present study, we did not observe PERK or GCN2 activation in PERK- or GCN2-rescued 4KO MEF cells under 70-ns 40-kV/cm nsPEF treatment. Because PERK and GCN2 are known to be activated by ER stress [35], 80-ns 20-kV/cm nsPEF treatment may form nanopores in the ER membrane, as well as in the mitochondria membrane, thereby causing ER stress. Thus, we assume that intracellular membranes may be affected differentially by nsPEF conditions. Optimized nsPEF conditions such as pulse duration, rise time, pulse amplitude and number for organelle-specific effects are worthy of further exploration.

nsPEFs have attracted much attention during the last decade because they may represent a drug‐free therapy. The therapeutic use of nsPEFs allows the manipulation of cell fate in two distinct ways: first, nsPEF treatment may lead to cell death; second, nsPEFs may enhance cellular function. For instance, nsPEF therapy has been proven effective in treating cancer in a mouse xenograft model such as melanoma [36], basal cell carcinoma [37] and pancreatic carcinoma [38]. nsPEFs initiate apoptosis in a nonthermal manner, presumably through mitochondrial and caspase-dependent mechanisms. However, nsPEFs regulate diverse biological effects, such as enhancement of chondrogenic differentiation [39], endothelial cell proliferation [40], and damage-free excitation of peripheral nerves [41] and cardiomyocytes [42]. The double-edged sword of nsPEF effects may depend on cell type or cell type-specific signaling pathways. Activation of the ISR could also decide cell fate (either survival or apoptosis) depending on the magnitude of eIF2α phosphorylation. Our results indicated that the two-faced nature of nsPEFs is mediated in part through ISR activation. More detailed mechanism is needed to be investigated in near future.

Supporting information

S1 Fig. nsPEFs did not affect the medium temperature and cell proliferation.

(A) Measurements of medium temperature before and after nsPEFs treatment. Error bars show the means ± SEM (n = 6, *P < 0.05). (B) Representative images and quantification of viability in WT or 4KO cells at 4 h with mock or nsPEF treatment. Cell viability was detected using WST-8 reagent, and the values are shown as the mean as the mean ± SEM.

(PDF)

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S2 Fig. nsPEFs induced the ISR activation without mitochondrial membrane potential.

(A) Representative images of JC-1 dye stained cells treated with nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields) or a mitochondrial uncoupler FCCP used as positive control. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of mitochondrial membrane potential. (B) Quantification of Green (JC-1 monomer)/Red (JC-1 polymer) fluorescence ratio in cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields) (n = 30, **P < 0.01, n.s. = not significant). (C) Quantification of intracellular ROS levels in nsPEF-treated WT or 4KO MEFs using the CM-H2DCFDA fluoroprobe. Error bars show the means ± SEM (n = 3–5, *P < 0.05, n.s. = not significant). (D) Representative immunoblots of ATF4, ATF3, CHOP, XBP1s, and Ribophorin 1 h after treatment with the nsPEFs and 2 μg/mL Tm in WT Hap1 cells. (E) Densitometry quantification of ATF4, ATF3, CHOP, and XBP1s expression were normalized to the Ribophorin expression as the mean + SEM (n = 3, *P < 0.05, **P < 0.01, n.s. = not significant).

(PDF)

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S1 Data

(PPTX)

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Acknowledgments

We thank C. Kimura (Tokushima University) for help with manuscript preparation and Oyadomari laboratory members for helpful discussion.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by JSPS KAKENHI Grant Number JP19H0285310 (S.O.).

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Decision Letter 0

Dong-Yan Jin

3 Sep 2019

PONE-D-19-21632

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation

PLOS ONE

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**********

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Reviewer #1: The manuscript by Hamada et al described the study of nanosecond pulsed electric fields (nsPEFs) on integrated stress response (ISR). They concluded that HRI is the eIF2alpha kinase responsible for the ISR activation by nsPEF-mediated oxidative stress. However, the scope of manuscript is very limited and preliminary. The second arem of ATF4 induction was not investigated in this manuscript. Their study did not increase our knowledge on the mechanisms as how and why nsPEFs may be useful in clinical practice. There is also a concern on the sole approach of using overexpression of single eIF2a kinase. Studies using single KO of each kinase shall be included, if indeed HRI is the sole eIF2a kinase responsible. It is important to note that overexpression of single eIF2a kinase may not be physiological due to the high level of kinase achieved.

The specific comments are:

1. Citations on the literature of HRI are inadequate; missing several important papers on the activation of HRI by oxidative stress and protection of cells against oxidative insults.

2. Fig. 1C, Fig 4A and B

Why is eIF2aP in 4KO+HRI so much lower than WT?

It seems that GCN2 also contributes to increased eIF2aP upon nsPEFs.

3. Fig. 2A, Similarly to Fig. 1C, why is Why is eIF2aP in 4KO+HRI so much lower than WT upon H2O2 treatment.

4. Fig. 4C, The FACS plots for ROS measurement shall be presented

Reviewer #2: The integrated stress response (ISR), a mechanism by which cells modulate protein synthesis in response to cellular stress, is controlled by the phosphorylation of translation initiation factor eIF2alpha. Four eIF2 kinases (HRI, GCN2, PERK, and PKR) each sense and are activated in response to different cellular stresses, and integrate on the shared eIF2alpha substrate. HRI, for instance, has been reported to be activated by elevated ROS in multiple cells types and heme deprivation in erythroid cells. In this paper, the authors utilize previously engineered MEF cells, which express each eIF2 kinase individually, to identify the kinase responsible for eIF2alpha phosphorylation in response to nsPEFs, a potential therapeutic tool for ISR activation. The authors report that 30 kV/cm nsPEFs promote the generation of ROS, and that cells expressing HRI as the sole eIF2 kinase display with heightened levels of eIF2alpha phosphorylation. Moreover, reduction of ROS levels by addition of a ROS scavenger, decreases eIF2alpha phosphorylation in the HRI expressing cells. Taken together, these results suggest that HRI is the eIF2 kinase activated in response to 30 kV/cm nsPEFs and reveal that the method of HRI activation in these conditions relies upon elevated ROS levels. It remains unclear, however, if HRI performs this function in a wild-type state in which all four eIF2 kinases are expressed at their endogenous levels, or only in these single kinase overexpression cell lines.

In general, the results are convincing and well described. I have a few comments that the authors should address to strengthen the conclusions of the manuscript.

1. Page 3, line 71: reference 11 does not support the statement that “enhancement of ISR signaling has been suggested to have beneficial effects,” and should be removed as a reference from this sentence as it is written.

2. Figures 1D, 3B, 4B, and 4C: why were SEM recorded rather than SD? The Materials and Methods indicate that data are represented as the mean +/- SD.

3. Page 8, line 153 and page 8, lines 158-160. What data support the two statements that PEFs did not result in meaningful temperature shifts or changes in cell viability?

4. Figure 3: The immunoblot for eIF2alpha phosphorylation in the 4KO+GCN2 cell line does not reflect the quantification provided. Is there another image that better represents the quantification?

5. Are ROS levels elevated to similar levels in response to nsPEFs in the WT, 4KO, 4KO+HRI, 4KO+PKR, 4KO+PERK, and 4KO+GCN2 cells lines?

6. The main conclusion of the manuscript is that nsPEFs generate ROS that activate HRI, leading to eIF2alpha phosphorylation. However, HRI activation under these conditions has not been demonstrated. A direct demonstration of activation/auto-phosphorylation of HRI would strengthen the conclusion.

7. While the data clearly indicate that eIF2alpha phosphorylation is induced to the greatest extent in the 4KO+HRI overexpression cell line (as compared to the other eIF2 kinase rescue lines), it is unclear to me if HRI serves to phosphorylate eIF2 in response to nsPEFs in the natural cellular state (i.e. when HRI and the other eIF2 kinases are expressed together at their endogenous levels). One strategy to asses this would be the individual knockdown or knockout of HRI from the WT cell line, followed by the assessment of eIF2alpha phosphorylation in response to PEFs.

Reviewer #3: In this manuscript the authors show that nanosecond pulsed electric fields (nsPEFs) activate the integrated stress response in mouse embryo fibroblasts (MEFs) via ROS-dependent HRI activation. The results are convincing and interesting, but the story is not quite complete. The ISR is usually activated by unfolded proteins in the cytosol, ER, mitochondria, or nucleus. These unfolded proteins can be induced by ROS, so it seems likely that ROS trigger HRI and eIF2alpha phosphorylation via unfolded proteins. The authors should present data to support or refute this idea. Detailed comments follow.

1. What happens to the wt MEFs after they are exposed to this dose of nsPEF? To the 4KO MEFs?

2. Is it possible that the molecular mechanism(s) that couple nsPEF to the ISR will be different in different cell types? (Do the authors have any evidence for this?)

3. The authors previously implicated PERK and GCN2 in nsPEF-induced ISR. Here they suggest that the discrepancy between that conclusion and the one defended here has to do with dose. Do they have direct evidence for this?

4. Does this dose of nsPEF cause cytochrome c release? A decrease in mitochondrial membrane potential?

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: David J. McConkey

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PLoS One. 2020 Mar 10;15(3):e0229948. doi: 10.1371/journal.pone.0229948.r002

Author response to Decision Letter 0

21 Jan 2020

We are grateful to the Editor and the reviewers for their critical comments and useful suggestions that have helped us to improve our paper. As indicated in the point-by-point responses below, we have taken all these comments and suggestions into account in the revised version of our manuscript.

We hope that the revised version of our paper is now suitable for publication in PLoS One and we look forward to hearing from you at your earliest convenience.

Reviewer #1

Major comments are:

1. The manuscript by Hamada et al described the study of nanosecond pulsed electric fields (nsPEFs) on integrated stress response (ISR). They concluded that HRI is the eIF2alpha kinase responsible for the ISR activation by nsPEF-mediated oxidative stress. However, the scope of manuscript is very limited and preliminary. The second arm of ATF4 induction was not investigated in this manuscript.

We confirmed the ATF4 induction in Hap1 cells with statistical significance (Sup. Fig. 2D and E).

2. Their study did not increase our knowledge on the mechanisms as how and why nsPEFs may be useful in clinical practice.

As this reviewer pointed out, we omitted the usefulness of nsPEFs in clinical practice in order to focus on mode of mechanism of nsPEFs in this paper.

3. There is also a concern on the sole approach of using overexpression of single eIF2a kinase. Studies using single KO of each kinase shall be included, if indeed HRI is the sole eIF2a kinase responsible. It is important to note that overexpression of single eIF2a kinase may not be physiological due to the high level of kinase achieved.

We established HRI KO and GCN2 KO in Hap1 cells using CRISPR/Cas9 system, and we can confirm the HRI is responsible kinase for eIF2α phosphorylation by nsPEFs exposure (Fig. 2D-F).

The specific comments are:

4. Citations on the literature of HRI are inadequate; missing several important papers on the activation of HRI by oxidative stress and protection of cells against oxidative insults.

According to the reviewer’s suggestions, we cited two important paper demonstrating that an essential role of HRI for red blood cells survival in heme deficiency (PMID:15931390) and HRI activation by oxidative stress (PMID: 22498744).

5. In Fig 4A and B, why is eIF2aP in 4KO+HRI so much lower than WT? It seems that GCN2 also contributes to increased eIF2aP upon nsPEFs.

We assumed that this lower phosphorylation level could be caused by lower expression level of HRI compared with that of WT. Unfortunately, we can’t validate the level of HRI overexpression compared with endogenous HRI due to lack of proper commercially available antibody to detect mouse HRI.

6. In Fig. 2A, Similarly to Fig. 1C, why is Why is eIF2aP in 4KO+HRI so much lower than WT upon H2O2 treatment.

It is the same reason above mentioned.

7. In Fig. 4C, The FACS plots for ROS measurement shall be presented.

We included the representative FACS plot for ROS measurement (Fig. 4C).

 

Reviewer #2:

Major comments are:

The integrated stress response (ISR), a mechanism by which cells modulate protein synthesis in response to cellular stress, is controlled by the phosphorylation of translation initiation factor eIF2alpha. Four eIF2 kinases (HRI, GCN2, PERK, and PKR) each sense and are activated in response to different cellular stresses, and integrate on the shared eIF2alpha substrate. HRI, for instance, has been reported to be activated by elevated ROS in multiple cells types and heme deprivation in erythroid cells. In this paper, the authors utilize previously engineered MEF cells, which express each eIF2 kinase individually, to identify the kinase responsible for eIF2alpha phosphorylation in response to nsPEFs, a potential therapeutic tool for ISR activation. The authors report that 30 kV/cm nsPEFs promote the generation of ROS, and that cells expressing HRI as the sole eIF2 kinase display with heightened levels of eIF2alpha phosphorylation. Moreover, reduction of ROS levels by addition of a ROS scavenger, decreases eIF2alpha phosphorylation in the HRI expressing cells. Taken together, these results suggest that HRI is the eIF2 kinase activated in response to 30 kV/cm nsPEFs and reveal that the method of HRI activation in these conditions relies upon elevated ROS levels. It remains unclear, however, if HRI performs this function in a wild-type state in which all four eIF2 kinases are expressed at their endogenous levels, or only in these single kinase overexpression cell lines. In general, the results are convincing and well described. I have a few comments that the authors should address to strengthen the conclusions of the manuscript.

We thank this reviewer for appreciating our paper properly.

The specific comments are:

1. Page 3, line 71: reference 11 does not support the statement that “enhancement of ISR signaling has been suggested to have beneficial effects,” and should be removed as a reference from this sentence as it is written.

As this reviewer pointed out, we omitted the reference.

2. Figures 1D, 3B, 4B, and 4C: why were SEM recorded rather than SD? The Materials and Methods indicate that data are represented as the mean +/- SD.

We unified the statistical analysis into SEM.

3. Page 8, line 153 and page 8, lines 158-160. What data support the two statements that PEFs did not result in meaningful temperature shifts or changes in cell viability?

According to the reviewer’s suggestions, we monitored the cell viability (Sup. Fig. 1B）and the temperature shifts（Sup. Fig. 1A）after nsPEFs treatment. There is no adverse effect of nsPEFs on both cell viability and cell culture temperature.

4. Figure 3: The immunoblot for eIF2alpha phosphorylation in the 4KO+GCN2 cell line does not reflect the quantification provided. Is there another image that better represents the quantification?

We performed the immunoblot analysis again and the representative image is now shown (Fig. 3A and B).

5. Are ROS levels elevated to similar levels in response to nsPEFs in the WT, 4KO, 4KO+HRI, 4KO+PKR, 4KO+PERK, and 4KO+GCN2 cells lines?

According to the reviewer’s suggestions, we measured the ROS levels again, and the level of ROS was elevated even in 4KO cells（Sup. Fig. 2C）

6. The main conclusion of the manuscript is that nsPEFs generate ROS that activate HRI, leading to eIF2alpha phosphorylation. However, HRI activation under these conditions has not been demonstrated. A direct demonstration of activation/auto-phosphorylation of HRI would strengthen the conclusion.

According to the reviewer’s suggestions, we performed phos-tag analysis of HRI. The HRI phosphorylation was induced by nsPEFs treatment as well as arsenite treatment (Fig. 2E and F).

7. While the data clearly indicate that eIF2alpha phosphorylation is induced to the greatest extent in the 4KO+HRI overexpression cell line (as compared to the other eIF2 kinase rescue lines), it is unclear to me if HRI serves to phosphorylate eIF2 in response to nsPEFs in the natural cellular state (i.e. when HRI and the other eIF2 kinases are expressed together at their endogenous levels). One strategy to asses this would be the individual knockdown or knockout of HRI from the WT cell line, followed by the assessment of eIF2alpha phosphorylation in response to PEFs.

According to the reviewer’s suggestions, we established HRI KO and GCN2 KO in Hap1 cells using CRISPR/Cas9 system, and we can confirm the HRI is responsible kinase for eIF2α phosphorylation by nsPEFs exposure (Fig. 2 D).

 

Reviewer #3:

Major comments are:

In this manuscript the authors show that nanosecond pulsed electric fields (nsPEFs) activate the integrated stress response in mouse embryo fibroblasts (MEFs) via ROS-dependent HRI activation. The results are convincing and interesting, but the story is not quite complete. The ISR is usually activated by unfolded proteins in the cytosol, ER, mitochondria, or nucleus. These unfolded proteins can be induced by ROS, so it seems likely that ROS trigger HRI and eIF2alpha phosphorylation via unfolded proteins. The authors should present data to support or refute this idea. Detailed comments follow.

We thank this reviewer for appreciating our paper properly.

The specific comments are:

1. What happens to the wt MEFs after they are exposed to this dose of nsPEF? To the 4KO MEFs?

We monitored the cell viability (Sup. Fig. 1B）and the cell culture medium temperature（Sup. Fig. 1A）under our condition of nsPEFs treatment. There is no significant effect of nsPEFs on both the cell viability and the cell culture medium temperature after the nsPEFs treatment.

2. Is it possible that the molecular mechanism(s) that couple nsPEF to the ISR will be different in different cell types? (Do the authors have any evidence for this?)

To exclude the possibility that our findings were specific to mouse fibroblasts, we examine the effect of nsPEFs in human Hap1 cells. The HRI-mediated ISR activation was induced also in Hap1 cells by the nsPEFs treatment, showing that ROS-mediated HRI activation is at least common in mouse fibroblasts and human Hap1 cells (Fig. 2 D-F, Sup. Fig. 2 D and E).

3. The authors previously implicated PERK and GCN2 in nsPEF-induced ISR. Here they suggest that the discrepancy between that conclusion and the one defended here has to do with dose. Do they have direct evidence for this?

Together with the above-mentioned point, we appreciate the thoughtful comments from this reviewer. Consistent with the previous our reports, nsPEFs induced the ISR. However, several factors such as s duration, number of pulses and intensity could lead to different biological processes. This could be attributed to varied phospholipid composition of organelle membrane among the different cell type, and this is an interesting question but beyond the limits of the present paper.

4. 4. Does this dose of nsPEF cause cytochrome c release? A decrease in mitochondrial membrane potential?

Change in mitochondrial membrane potential is known as a critical upstream signal of cytochrome C release. Therefore, we measured the mitochondrial membrane potential using JC-1 dye. We found that there was no significant change of mitochondrial membrane potential under our condition of nsPEFs treatment (Sup. Fig. 2A).

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Decision Letter 1

Dong-Yan Jin

10 Feb 2020

PONE-D-19-21632R1

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation

PLOS ONE

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Reviewers' comments:

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Reviewer #2: All comments have been addressed

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Reviewer #1: The revised manuscript by Hamada et al has addressed all the concerns from the previous review. In particular, the single KO cell lines of HRI and GCN2 were generated and studied. In Furthermore, Activation of HRI in vivo in the Wt Hap1 cells was also demonstrated by phos-tag immunoblot analysis. One minor comment is that T-HRI band in Fig. 2E and F shall be unphophorylated HRI not total HRI. So, it shall be changed to HRI.

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PLoS One. 2020 Mar 10;15(3):e0229948. doi: 10.1371/journal.pone.0229948.r004

Author response to Decision Letter 1

18 Feb 2020

We agreed with the reviewer 1. The phosphorylation states of HRI on Phos-tag SDS-PAGE are now indicated on the right side of the blot with arrowheads. We thank the reviewer for bringing it to our attention.

Attachment

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Decision Letter 2

Dong-Yan Jin

19 Feb 2020

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation

PONE-D-19-21632R2

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Reviewers' comments:

Acceptance letter

Dong-Yan Jin

25 Feb 2020

PONE-D-19-21632R2

Nanosecond pulsed electric fields induce the integrated stress response via reactive oxygen species-mediated heme-regulated inhibitor (HRI) activation

Dear Dr. Oyadomari:

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. nsPEFs did not affect the medium temperature and cell proliferation.

    (A) Measurements of medium temperature before and after nsPEFs treatment. Error bars show the means ± SEM (n = 6, *P < 0.05). (B) Representative images and quantification of viability in WT or 4KO cells at 4 h with mock or nsPEF treatment. Cell viability was detected using WST-8 reagent, and the values are shown as the mean as the mean ± SEM.

    (PDF)

    Click here for additional data file. (312.1KB, pdf)
    S2 Fig. nsPEFs induced the ISR activation without mitochondrial membrane potential.

    (A) Representative images of JC-1 dye stained cells treated with nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields) or a mitochondrial uncoupler FCCP used as positive control. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of mitochondrial membrane potential. (B) Quantification of Green (JC-1 monomer)/Red (JC-1 polymer) fluorescence ratio in cells 1 h after nsPEF treatment (40 shots of 70-ns duration and 30-kV/cm electric fields) (n = 30, **P < 0.01, n.s. = not significant). (C) Quantification of intracellular ROS levels in nsPEF-treated WT or 4KO MEFs using the CM-H2DCFDA fluoroprobe. Error bars show the means ± SEM (n = 3–5, *P < 0.05, n.s. = not significant). (D) Representative immunoblots of ATF4, ATF3, CHOP, XBP1s, and Ribophorin 1 h after treatment with the nsPEFs and 2 μg/mL Tm in WT Hap1 cells. (E) Densitometry quantification of ATF4, ATF3, CHOP, and XBP1s expression were normalized to the Ribophorin expression as the mean + SEM (n = 3, *P < 0.05, **P < 0.01, n.s. = not significant).

    (PDF)

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    S1 Data

    (PPTX)

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    Attachment

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    Attachment

    Submitted filename: Response to Reviewers (Hamada et al).docx

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    Attachment

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    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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