Inhibition of complex I of the electron transport chain causes O₂⁻·-mediated mitochondrial outgrowth

Abstract

-Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O 2 Ϫ ⅐ production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O 2 Ϫ ⅐ production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY 581/591 . Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O 2 Ϫ ⅐ to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O 2 Ϫ ⅐ production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O 2 Ϫ ⅐ production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O 2 Ϫ ⅐ to prevent alterations in mitochondrial morphology. rhodamine 123; video-rate confocal microscopy; superoxide; MitoQ HIGHLY AEROBIC CELLS from brain, heart, muscle, liver, kidney, and endocrine tissue depend on the ATP-generating capacity of their mitochondria to meet energetic demands. Acute changes in cellular energy consumption are met through feedback and/or feedforward regulation of enzymes involved in aerobic ATP production, whereas chronic changes lead to alterations in mitochondrial capacity and/or architecture (3,22,35,36).Marked changes in the structure of the cellular mitochondrial network are observed during differentiation, cellular senescence, and apoptosis, whereas subtle rearrangements occur during cellular growth and division (56).Mitochondria generate ATP through oxidative phosphorylation (OXPHOS), and defects in this system lead to decreased energy production, increased formation of O 2 Ϫ ⅐ and derived reactive oxygen species such as hydrogen peroxide and ⅐OH, and the release of death-promoting factors (44,47,56). Defects occur in a wide variety of degenerative diseases, aging, and cancer and primarily affect tissues that have high energy requirements and are unable to adapt to conditions of reduced mitochondrial energy supply. Cells that can survive under such conditions, ...

Life cell quantification of mitochondrial membrane potential at the single organelle level

“…To develop and validate our protocol we used human skin fibroblasts, which are relatively large cells that display an extremely flat morphology (\3 lm in the axial direction; (6)). Given these morphological properties, human skin fibroblasts are almost intrinsically ''confocal'' and in extension to our previous confocal microscopy studies (6,16,17), we here demonstrate that mitochondria in these cells can be appropriately visualized by nonconfocal epifluorescence videomicroscopy. Although TMRM is generally considered the fluorescent cation most sensitive to changes in Dw, correct quantitative measurements require the absence of TMRM autoquenching, leakage/extrusion, and photobleaching.…”

“…Ideally, the COR images should be noise-free and lack nonmitochondrial background staining. We previously used R123 pulse-loading, confocal microscopy, and image averaging to meet these requirements (6,16,17). Here, we demonstrate that also epifluorescence images of TMRM-stained fibroblasts display mitochondria-specific signals and a signal-to-noise ratio that is sufficiently high to allow correct image binarization (Fig.…”

“…To determine the TMRM intensity of individual mitochondrial objects, the BC-corrected (COR) image was first converted into a binary (BIN) image. A stepby-step summary of the calculations involved in this process is depicted in Figure 1B (panel I ''image binarization'') and has been described in detail previously (6,16,17). Briefly, the COR image is contrast optimized using a linear contrast stretch (LCS), which reassigns the gray values in the image to cover the entire available range (i.e., from 0 to 255).…”

YY1 is indispensable for Lgr5 + intestinal stem cell renewal

“…6 A and B). Mitochondrial dysfunction has been shown to increase generation of reactive oxygen species (ROS) and DNA damage (47)(48)(49)(50)(51). Indeed, immunohistochemistry revealed elevated levels of 8-hydroxyguanosine, an antigen formed on DNA oxidation (Fig.…”

Regulation and Quantification of Cellular Mitochondrial Morphology and Content

“…[161,162]). Similarly, when mitochondrial function was chronically inhibited by rotenone, a specific blocker of the first ETC complex (CI), mitochondrial length and degree of branching dose-dependently increased [37,163]. In the light of the above, and given the fact that mitochondrial shape and mass also can differ within and between the same cell type (e.g.…”