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Title:
Distinct Effects of Nanosecond Pulsed Electric Fields (nsPEFs) on Mitochondrial Structure and Function
Authors:
Beebe. Stephen J. - Frank Reidy Research Center for Bioelectrics;
Asadipour, Kamal - Frank Reidy Research Center for Bioelectrics and Department of Electrical and Computer Engineering;
Potter, Lucas - Frank Reidy Research Center for Bioelectrics and Department of Electrical and Computer Engineering;
Ruedlinger, Brittany - Frank Reidy Research Center for Bioelectrics;
Lai, Nicola - Department of Electrical and Computer Engineering.
Abstract:
Background and Objectives: Nanosecond pulsed electric fields (nsPEFs) stimulate structural and functional changes in plasma membranes (PM) and especially in intracellular organelles. As a regulator of cell fate, we focused on responses of mitochondria as a nsPEF Receptor.
Methods: include determination of the ΔΨm with TMRE, ROS with MitoSox, and integrity of the OMM and the IMM with impermeable molecules cytochrome c and NADH, respectively.
Results: Cyclosporin A attenuated the loss of mitochondrial membrane potential (ΔΨm) but did not affect cell death, suggesting some limited role for cyclophilin D on the mitochondrial permeability transition pore (mPTP) in response to nsPEFs. Although nsPEFs coincidently dissipated the ΔΨm and elevated mitochondrial ROS (mROS), both of which were enhanced by Ca2+, there were significant losses in ΔΨm without significant ROS production Furthermore, decreases in ΔΨm occurred before there were increases in mROS (5±2 min vs. 10±2). Trolox was used as a cell-permeable analogue of vitamin E that is used as a standard for measuring the antioxidant capacity of complex mixtures. While Trolox significantly inhibited the increase in mROS and the loss of ΔΨm; However, a significant loss of ΔΨm occurred in the presence of trolox under conditions where there were insignificant increases in ROS in the absence of Trolox. To evaluate nsPEF effects on mitochondrial membrane integrity, we used cytochrome c and NADH as impermeable molecules to the outer mitochondrial membrane (OMM) and inner (IMM), respectively. NsPEFs disrupted the OMM causing a large increase in O2 consumption but not the IMM, indicating that NsPEF-induced loss in the ΔΨm was independent of IMM electro-permeabilization but could be due to effects on mPTP through effects on the OMM. Nevertheless, nsPEFs does have effects on IMM function since they attenuate O2 consumption as a measure of electron transport (ET) in the mitochondrial electron transport chain (ETC) at least at Complex I in intact and permeabilized cells. Similar results occurred in isolated mitochondria, suggesting nsPEF can have direct effects on mitochondria. Interestingly, the effects of nsPEFs on increases in mROS were synergistic with the complex I inhibitor rotenone, suggesting that nsPEFs and rotenone act at different sites in the ETC, presumably in complex I.
Conclusions: These studies show diverse effects of nsPEFs on mitochondria, including attenuating ET (O2 consumption) and causing a loss of ΔΨm without affecting IMM electro-permeability. NsPEFs can have direct effects on isolated mitochondria with disruptive effects on the OMM in cells, either of which may be related to effects of nsPEFs on the mPTP causing a loss of ΔΨm. Furthermore, these data suggest that the increase in ROS may be the result of nsPEFs on the mPTP and loss of ΔΨm and not the cause of it. Overall, these studies show diverse effects of nsPEFs on mitochondria, including attenuating ET and dissipating loss of ΔΨm without causing a loss of IMM permeability.
Keywords:
Refs:
Topic 1:
1. Biological responses (molecular, subcellular, cellular and intercellular)
Topic 2:
12. Biomedical applications
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