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

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the [alpha] subunit of eukaryotic translation initiation factor 2 (eIF2[alpha]). Four eIF2[alpha] 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 eIF2[alpha] kinase is activated by nsPEF treatment. To assess the responsible eIF2[alpha] kinase, we used previously established eIF2[alpha] kinase quadruple knockout (4KO) and single eIF2[alpha] kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2[alpha] phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2[alpha] phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H.sub.2 DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2[alpha] phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2[alpha] kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.