Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration
Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration
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DOI：
10.1016/j.bpj.2018.04.047
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发表时间：
2018-06-19
影响因子：
3.4
通讯作者：
Verbridge, Scott S.
中科院分区：
文献类型：
--
作者：
Goswami, Ishan;Perry, Justin B.;Verbridge, Scott S.
临床上使用的是微秒脉冲宽度（μ sPEFs）的脉冲电场；即不可逆电穿孔/纳米刀用于软组织肿瘤消融。不可逆电穿孔中使用的 μ sPEF 脉冲参数（0.5-1 kV/cm，80-100 个脉冲，类似于每个 100 μ s，1 Hz 频率）可能会由于细胞外膜的破坏和随后电场的穿透而导致细胞内形成内部电场。内部场可能会破坏电压敏感的线粒体，尽管研究文献对于 mu sPEF 是否会发生这种破坏还相对不清楚。这项研究报告了临床使用的 mu sPEF 参数对活细胞线粒体呼吸的影响。使用高通量安捷伦 Seahorse 机器，观察到包含 80 个振幅为 600 或 700 V/cm 的脉冲的 mu sPEF 暴露不会改变在暴露过夜后恢复后测量的 4T1 细胞中的线粒体呼吸。为了记录 mu sPEF 暴露后线粒体功能的变化，使用高分辨率呼吸测量法通过对谷氨酸-苹果酸和 ADP 的响应来测量电子传递链状态，并通过对羰基氰化物-对三氟甲氧基苯腙的响应来测量线粒体膜电位。除了测量线粒体对 mu sPEF 暴露的直接反应外，还对使用洋地黄皂苷透化的细胞和通过药物 latrunculin B 处理而因肌动蛋白解聚而导致细胞骨架受损的细胞进行了测量。前一种处理用作对照，以梳理质膜透化的影响，而后者用于研究如果 mu sPEF 影响线粒体所锚定的细胞骨架，则可能对线粒体产生的间接影响。根据结果得出的结论是，在测试的脉冲参数内，μ sPEF 本身不会阻碍线粒体生理学，但可以通过损害肌动蛋白来影响线粒体。据我们所知，肌动蛋白解聚后线粒体对 mu sPEF 的敏感性为癌症治疗提供了一条新途径。
Pulsed electric fields with microsecond pulse width (mu sPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The mu sPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, similar to 100 mu s 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 mu sPEFs. This investigation reports the influence of clinically used mu sPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that mu sPEF 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 mu sPEF 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-trifluoromethoxphenylhydrazone. In addition to measuring immediate mitochondrial responses to mu sPEF 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 mu sPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, mu sPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to mu sPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.