Aon Miguel A, Cortassa Sonia, Maack Christoph, O'Rourke Brian
Institute of Molecular Cardiobiology, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21205, USA.
J Biol Chem. 2007 Jul 27;282(30):21889-900. doi: 10.1074/jbc.M702841200. Epub 2007 May 31.
Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with DeltaPsi(m) and the NADH/NAD(+) redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible DeltaPsi(m) depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in DeltaPsi(m) to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4'-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, DeltaPsi(m) depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized DeltaPsi(m) and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.
线粒体膜电位（ΔΨm）去极化会导致缺血后心脏的细胞死亡以及电和收缩功能障碍。线粒体活性氧生成与清除之间的失衡先前被认为与内膜阴离子通道（IMAC）的激活有关，IMAC不同于通透性转换孔（PTP），是心肌细胞对代谢应激的第一反应。谷胱甘肽氧化还原对GSH/GSSG与ΔΨm以及NADH/NAD⁺氧化还原状态平行振荡。在此我们表明，还原型谷胱甘肽的耗竭是心肌细胞中线粒体同步振荡的另一种触发因素，并且中间的GSH/GSSG比值会导致可逆的ΔΨm去极化，尽管广泛的硫醇氧化会诱导不可逆的PTP激活。响应二硫苏糖醇的线粒体功能障碍分阶段发生，从ΔΨm振荡发展为持续去极化，同时伴有GSH的耗竭。4'-氯地西泮（一种IMAC抑制剂）可消除线粒体振荡，而环孢素A则无效。在皂素通透的心肌细胞中，硫醇氧化还原状态被系统地钳制在GSH/GSSG比值范围从300:1到20:1。在150:1 - 100:1的比值下，ΔΨm可逆地去极化，并且一种基质定位的荧光标记物得以保留；然而，将GSH/GSSG降低至50:1会使ΔΨm不可逆地去极化，并诱导活性氧生成、NAD(P)H氧化以及基质成分丧失的最大速率。通过抑制GSH摄取、NADPH依赖性谷胱甘肽还原酶或NADH/NADPH转氢酶，线粒体对GSH的敏感性会发生改变，这表明基质GSH的再生或补充至关重要。结果表明，GSH/GSSG氧化还原状态控制着心肌细胞中由硫醇氧化触发的线粒体离子通道（PTP之前的IMAC）的顺序开放。
