Adult human dermal fibroblasts exposed to nanosecond electrical pulses exhibit genetic biomarkers of mechanical stress

Abstract

BackgroundExposure of cells to very short (<1 µs) electric pulses in the megavolt/meter range have been shown to cause a multitude of effects, both physical and molecular in nature. Physically, nanosecond electrical pulses (nsEP) can cause disruption of the plasma membrane, cellular swelling, shrinking and blebbing. Molecularly, nsEP have been shown to activate signaling pathways, produce oxidative stress, stimulate hormone secretion and induce both apoptotic and necrotic death. We hypothesize that studying the genetic response of primary human dermal fibroblasts exposed to nsEP, will gain insight into the molecular mechanism(s) either activated directly by nsEP, or indirectly through electrophysiology interactions.MethodsMicroarray analysis in conjunction with quantitative real time polymerase chain reaction (qRT-PCR) was used to screen and validate genes selectively upregulated in response to nsEP exposure.ResultsExpression profiles of 486 genes were found to be significantly changed by nsEP exposure. 50% of the top 20 responding genes coded for proteins located in two distinct cellular locations, the plasma membrane and the nucleus. Further analysis of five of the top 20 upregulated genes indicated that the HDFa cells’ response to nsEP exposure included many elements of a mechanical stress response.ConclusionsWe found that several genes, some of which are mechanosensitive, were selectively upregulated due to nsEP exposure. This genetic response appears to be a primary response to the stimuli and not a secondary response to cellular swelling.General significanceThis work provides strong evidence that cells exposed to nsEP interpret the insult as a mechanical stress.

Nanosecond pulsed electric field exposure does not induce the unfolded protein response in adult human dermal fibroblasts

“…The results of our experiments did not show any significant change in the expression of Caspase‐4 post‐exposure to nsPEFs at the tested parameters. These results mirror the microarray analysis provided by Roth et al []. Interestingly, no significant increase in Caspase‐4 expression was observed post‐exposure to TM, a drug known to induce ER stress.…”

“…In short, a custom Blumlein line pulser composed of two parallel plates connected via spark gap chamber pressurized with Sulfur Hexafluoride (SF 6 ) gas was used to deliver the 10-ns PEF to a metal-walled cuvette containing cells. To better correlate our results with previously observed microarray results [Kolb et al, 2006;Roth et al, 2017], cell exposure parameters were mirrored: 100, 150 kV/cm 10-ns pulses at a repetition rate of 1 Hz. Each nsPEF waveform was acquired using a high-speed oscilloscope (TDS3052B, Tektronix, Wilsonville, OR) ( Fig.…”

“…Microarray data previously collected and published [Roth et al, ] were mined for genetic markers of ER stress and UPR. The change in expression level of transcripts between the baseline and experimental array (Log Ratio) and P ‐value results indicated that there was a significant increase in the expression of ATF6 and PERK as compared to the sham (Table ).…”

“…Consequently, this change in molecular environment would not allow for proper protein folding, generating ER stress and activation of the UPR. Therefore, as a starting point for determining if ER stress occurs following nsPEF exposure, we mined prior microarray data [Roth et al, 2017] for genetic indicators of upregulation of the UPR. The microarray data indicated that both ATF6 and PERK were significantly upregulated in response to nsPEF exposure.…”

“…To better understand the stress(es) imposed on cells during nsPEF exposure, we previously provided a transcript profile, utilizing microarray analysis, for adult Human Dermal Fibroblasts (HDFa) exposed to 100 nsPEFs at a duration of 10-ns and an electric field of 150 kV/cm 4 h post-exposure [Roth et al, 2017]. The current study expands this work by focusing only on a stress pathway specifically related to ER function to determine if nsPEF-induced stress affects the functionality of the ER in a meaningful way.…”