Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration

Abstract

Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to μsPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.

Characterization of Ablation Thresholds for 3D-Cultured Patient-Derived Glioma Stem Cells in Response to High-Frequency Irreversible Electroporation

“…In the case of comparing thresholds in collagen and HA, cell-ECM interactions may furthermore play an important role, making comparison across scaffolding more challenging. Additionally, other signaling changes upon ephrin-activation may also be important in the context of IRE or H-FIRE induced cell death (e.g., altered cytoskeleton integrity [68]), such that the ephrin effect on thresholds observed in previous work may not have been purely due to morphological changes. It should also be noted that, while cell size and morphology are crucial factors in determining the onset of electroporation, they are not the only important aspects for resulting cell death thresholds.…”

Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9+ T Cells

“…Electroporation of cells can be irreversible, when it disrupts the plasma membrane, causing loss of cell homeostasis and leading to cell death. On top, applied electric fields, even if they are transient, can reach the mitochondrion, which harbors the electron transport chain and can release cytochrome C, which would affect the metabolic capacities and again induce cell death ( 34 ). Thus, conditions have to be acquired, which allow the cells to fully recover from the transient perturbation.…”

Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog

“…Oxygen Consumption Rate (OCR) of saponin-permeabilized cells was assayed with an Agilent Seahorse XFe96 analyzer, and basal respiration, reserve respiratory capacity and maximal respiratory capacity were calculated as described elsewhere [ 28 ]. Oxygen consumption of saponin-permeabilized cells [ 29 ] was also measured by Oroboros O2k respirometry [ 30 , 31 ]. Cellular ATP pool was measured using Mitochondrial ToxGlo Assay (Promega Corporation, Madison, WI).…”