Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119992, Russia.
Oxid Med Cell Longev. 2020 Feb 10;2020:3631272. doi: 10.1155/2020/3631272. eCollection 2020.
A new mitochondria-targeted probe MitoCLox was designed as a starting compound for a series of probes sensitive to cardiolipin (CL) peroxidation. Fluorescence microscopy reported selective accumulation of MitoCLox in mitochondria of diverse living cell cultures and its oxidation under stress conditions, particularly those known to cause a selective cardiolipin oxidation. Ratiometric fluorescence measurements using flow cytometry showed a remarkable dependence of the MitoCLox dynamic range on the oxidation of the sample. Specifically, MitoCLox oxidation was induced by low doses of hydrogen peroxide or organic hydroperoxide. The mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1), which was shown earlier to selectively protect cardiolipin from oxidation, prevented hydrogen peroxide-induced MitoCLox oxidation in the cells. Concurrent tracing of MitoCLox oxidation and membrane potential changes in response to hydrogen peroxide addition showed that the oxidation of MitoCLox started without a delay and was complete during the first hour, whereas the membrane potential started to decay after 40 minutes of incubation. Hence, MitoCLox could be used for splitting the cell response to oxidative stress into separate steps. Application of MitoCLox revealed heterogeneity of the mitochondrial population; in living endothelial cells, a fraction of small, rounded mitochondria with an increased level of lipid peroxidation were detected near the nucleus. In addition, the MitoCLox staining revealed a specific fraction of cells with an increased level of oxidized lipids also in the culture of human myoblasts. The fraction of such cells increased in high-density cultures. These specific conditions correspond to the initiation of spontaneous myogenesis , which indicates that oxidation may precede the onset of myogenic differentiation. These data point to a possible participation of oxidized CL in cell signalling and differentiation.
一种新的线粒体靶向探针 MitoCLox 被设计为一系列对心磷脂 (CL) 过氧化敏感的探针的起始化合物。荧光显微镜报告说，MitoCLox 选择性地积累在各种活细胞培养物的线粒体中，并在应激条件下氧化，特别是那些已知会导致选择性心磷脂氧化的条件。使用流式细胞术的比率荧光测量显示，MitoCLox 动态范围对样品氧化的依赖性非常显著。具体来说，MitoCLox 氧化是由低剂量的过氧化氢或有机过氧化物诱导的。先前已显示，线粒体靶向抗氧化剂 10-(6'-质体醌基)癸基三苯基膦 (SkQ1) 可选择性保护心磷脂免受氧化，它可防止过氧化氢诱导的细胞中 MitoCLox 氧化。同时追踪 MitoCLox 氧化和膜电位变化对过氧化氢添加的反应表明，MitoCLox 的氧化没有延迟地开始，并在第一个小时内完全进行，而膜电位在孵育 40 分钟后开始下降。因此，MitoCLox 可用于将细胞对氧化应激的反应分为单独的步骤。MitoCLox 的应用揭示了线粒体群体的异质性；在活的内皮细胞中，在靠近细胞核的地方检测到一小部分圆形、脂质过氧化水平升高的线粒体。此外，MitoCLox 染色还揭示了在人成肌细胞培养物中，具有增加的氧化脂质水平的特定细胞分数。这种细胞的分数在高密度培养物中增加。这些特定条件对应于自发肌发生的启动，这表明氧化可能先于成肌分化的开始。这些数据表明氧化的 CL 可能参与细胞信号转导和分化。
