1Microscopical Imaging Center and 2Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, and 3Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Medical Center, Nijmegen, The Netherlands; and 4Medical Research Council Dunn Human Nutrition Unit, Cambridge, United Kingdom
Submitted 10 December 2004 ; accepted in final form 10 January 2005
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ABSTRACT |
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rhodamine 123; video-rate confocal microscopy; superoxide; MitoQ
Mitochondria generate ATP through oxidative phosphorylation (OXPHOS), and defects in this system lead to decreased energy production, increased formation of O2· and derived reactive oxygen species such as hydrogen peroxide and ·OH, 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, such as cancer cells, show a variety of adaptations, including upregulation of glucose transport, glycolysis, and lactate formation (38). At the same time, these cells show a marked reduction in mitochondrial content (10) and OXPHOS capacity (46), whereas the mitochondrial reticulum is largely perinuclear (49). Cancer cells that are forced to grow on galactose and glutamine readily switch from anaerobic to aerobic energy production (43). This adaptation is accompanied by an increase in OXPHOS protein, a decrease in mitochondrial matrix pH, a more oxidized matrix redox state, an increase in the amount of cristae but no increase in mitochondrial mass, and a more extended mitochondrial network. Similar observations were initially reached in budding yeast, in which a change of substrate induced a threefold increase in mitochondrial volume (16). Qualitative and/or quantitative changes in the mitochondrial reticulum are also observed under pathological conditions that are caused by inherited mutations in mitochondrial DNA or in nuclear OXPHOS genes (8, 20) and suggest a tight relationship between mitochondrial structure and function (11).
Recent insights suggest that O2· anions, formed as a byproduct of the OXPHOS process, may activate specific redox-sensitive signaling pathways (13). Evidence has been provided that these pathways control uncoupling of protein-mediated proton conductance (7). In addition, these pathways are implicated in mitochondrial biogenesis (29, 32, 33) and regulation of cellular antioxidant capacity (40). Failure to make the appropriate changes is thought to lead to increased O2· production, which, if not properly balanced by the cell's antioxidant mechanisms, may cause structural and functional damage to polyunsaturated fatty acids in membrane lipids, proteins, and DNA. There is good evidence that increased oxygen radical formation is the cause of atherosclerosis and possibly also of the major neurodegenerative and chronic inflammatory diseases (21). Moreover, increased radical formation has been implicated in aging (17) and apoptosis (31). However, in the majority of diseases in which tissue damage occurs, increased radical formation is regarded as a consequence rather than a cause (21). Human mitochondrial complex I (NADH:ubiquinone oxidoreductase; EC: 1.6.5.3 [EC] ) is the largest multisubunit assembly of the OXPHOS system, comprising 39 nuclear encoded and 7 mitochondrially encoded subunits (23). Malfunction of this complex is associated with a wide variety of clinical syndromes (47). To enhance the understanding of the pathophysiology of these diseases, with the final aim of developing new treatment strategies to stabilize or even cure them, we have studied genetically characterized human complex I-deficient fibroblast cell lines as a model for OXPHOS system disease with the knowledge that these cells are glycolytic (41). In doing so, we recently showed that agonist-induced mitochondrial Ca2+ accumulation and ensuing ATP production are significantly decreased in skin fibroblasts derived from patients with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex (53).
Pham et al. (39) reported that mitochondrial morphology and dynamics are altered in skin fibroblasts from patients with mitochondrial complex I deficiency. Similar observations were made with regard to control fibroblasts treated for 5 min with the complex I inhibitor rotenone (40 µM). Studies with mitochondrial membranes isolated from patient fibroblasts showed that NADH-stimulated mitochondrial O2· formation is increased in human complex I deficiency and that 10 µM rotenone readily increases formation of this radical in control membranes (40). Together with observations that exogenous application of hydrogen peroxide increases mitochondrial mass in human lung fibroblasts (29), these findings suggest a causal relationship between increased mitochondrial O2· formation and alterations in mitochondrial reticulum and dynamics in complex I deficiency. However, no definitive proof has yet been offered.
In the present report, we show that a sustained increase in mitochondrial O2· production brought about by chronic inhibition of complex I of the electron transport chain (100 nM rotenone, 72 h) causes a marked increase in mitochondrial length and branching.
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MATERIALS AND METHODS |
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Quantitative analysis of mitochondrial morphology by video-rate laser-scanning confocal microscopy. Stock solutions of the lipophilic cation rhodamine 123 (R123), Mitotracker Green FM (MG), and Mitotracker Red CMXRos (MR; all from Molecular Probes, Leiden, The Netherlands) were freshly prepared in dimethyl sulfoxide (DMSO) before each measurement. Fibroblasts were incubated in culture medium containing 200 µM R123 or 5 µM MR or MG for 40 s (R123), 3 min (MR), or 20 min (MG) at 20°C. After being loaded, cells were thoroughly washed with HEPES-Tris medium containing (in mM) 132 NaCl, 4.2 KCl, 1 CaCl2, 1 MgCl2, 5.5 D-glucose, and 10 HEPES, pH 7.4. For confocal imaging, coverslips were mounted in an incubation chamber and placed on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan) attached to an Oz confocal microscope (Noran Instruments, Middleton, WI). Measurements were performed at 20°C. The light from an argon ion laser (488 nm; Omnichrome, Chino, CA) was delivered to the cells using a x40 oil-immersion planapochromat lens objective [numerical aperture (NA), 1.4; Nikon]. For all dyes, fluorescence emission light was directed through a 500-nm LP barrier filter (Chroma Technology, Brattleboro, VT) and quantified using a photomultiplier tube (Hamamatsu Photonics, Bridgewater, NJ). Given the flat morphology of the fibroblasts (<3 µm in the axial direction), slit settings were chosen such that axially each cell was entirely present within the confocal volume (27). This prevented exclusion of mitochondrial structures from the image and guaranteed an optimal fluorescence signal at minimal laser intensity. Hardware and image acquisition were controlled using Intervision software (version 1.5; Noran Instruments) run under IRIX 6.2 on an Indy workstation (Silicon Graphics, Mountain View, CA) equipped with 512 Mb of memory. Before image acquisition, brightness and contrast settings were optimized using a custom-made lookup table that colored the upper and lower 10 gray levels red and blue, respectively. Images (512 x 480 pixels) were collected at 30 Hz with a pixel dwell time of 100 ns. To reduce random noise, images were averaged in real time using the running average algorithm of the Intervision Acquisition software with a window size of 32. This acquisition protocol, in combination with the low mitochondrial mobility at 20°C, effectively prevented distortion of the image by mitochondrial movement. Images were recorded from a cross-shaped area transecting the center of the coverslip and converted to tagged image file format using a Silicon Graphics O2 workstation running IRIX 6.5. Quantitative analysis of mitochondrial morphology was performed using Image Pro Plus 4.5 (Media Cybernetics, Silver Spring, MD) as described in RESULTS.
Quantitative analysis of mitochondrial O2· production using digital imaging microscopy. Fibroblasts were incubated in HEPES-Tris medium containing 10 µM hydroethidine (HEt; Molecular Probes) for 10 min at 37°C. HEt is an uncharged compound that readily enters the cell. Within the cell, it reacts with O2· to form the fluorescent and positively charged product ethidium (Et) (15). The reaction was stopped by thoroughly washing the cells with PBS to remove excess HEt. For quantitative analysis of Et emission signals, coverslips were mounted in an incubation chamber placed on the stage of an inverted microscope (Axiovert 200 M; Carl Zeiss, Jena, Germany) equipped with a Zeiss x40/1.3 NA fluor lens objective. Et was excited at 490 nm using a monochromator (Polychrome IV; TILL Photonics, Gräfelfing, Germany). Fluorescence emission was directed using a 525DRLP dichroic mirror (Omega Optical, Brattleboro, VT) through a 565ALP emission filter (Omega Optical) onto a CoolSNAP HQ monochrome charge-coupled device camera (Roper Scientific, Vianen, The Netherlands). The image-capturing time was 100 ms. Routinely, 10 fields of view per coverslip were analyzed. Hardware was controlled using Metafluor 6.0 software (Universal Imaging, Downingtown, PA). Quantitative image analysis was performed using Metamorph 6.0 software (Universal Imaging) as described in RESULTS.
Quantitative analysis of the extent of lipid peroxidation by video-rate laser-scanning confocal microscopy. The extent of lipid peroxidation was quantified using the fluorescent ratio probe C11-BODIPY581/591 (Molecular Probes). Upon oxidation, the red emitting form of the dye (595 nm) is converted into a green emitting form (520 nm), which results in an increase in green-to-red emission ratio (14). Cells were incubated in HEPES-Tris medium containing 4 µM C11-BODIPY581/591 for 30 min at 37°C. After being washed thoroughly, images were collected using the Oz confocal system as described above. Green and red fluorescence emission signals were separated using a 560DM dichroic mirror and appropriate band-pass filters (535D20 and 580LP; Chroma). Quantitative image analysis was performed as described in RESULTS.
Statistics and data analysis. Numerical values were visualized using Origin Pro 6.1 software (OriginLab, Northampton, MA), and values from multiple experiments were expressed as means ± SE. Statistical significance was assessed using Student's t-test.
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RESULTS |
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All three dyes revealed extensive mitochondrial reticula as well as individual mitochondrial filaments (Fig. 1, AC, left). Next, images were segmented using three gray-level intervals (050, 51150, and 151255; see columns below intensity scale in Fig. 1). For only R123, a discrete range of gray levels was associated exclusively with mitochondrial structures (Fig. 1A, third image from left). When R123-stained cells were treated with 1 µM concentration of the protonophore carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP), the mitochondrial staining pattern was acutely lost, thus confirming the mitochondrial localization of the dye (data not shown).
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Chronic treatment with rotenone increases O2· production in human skin fibroblasts. Measurement of the effect of rotenone on O2· formation in living cells has yielded conflicting results. Increases were observed in the human osteosarcoma-derived cell line 143B (4), mesencephalic neurons (37), and HL-60 cells (31), whereas decreases occurred in hepatocytes (55), cultured mice hippocampal neurons (45), and monocytes and macrophages (30). In this study, we used hydroethidine (HEt), a redox-sensitive probe that is widely used to measure mitochondrial O2· production in living cells (5, 6, 15, 57). HEt is a cell-permeant compound that is oxidized by O2· to its positively charged product Et. Digital imaging microscopy of fibroblasts loaded with 10 µM HEt for 10 min and thoroughly washed to remove nonoxidized HEt, revealed the presence of Et in both nucleoli and a widespread network of tubular structures present in the cytosolic compartment (Fig. 5A). The intensity of fluorescence emission did not change during 10 min of illumination at 0.2 Hz as demonstrated by a linear fit with a slope of 4.4·105 ± 2.3·106 (P < 0.001). This indicates that excess HEt was effectively removed, Et did not leak out of the cell, and photobleaching and/or photoactivation did not occur during at least 10 min of image acquisition. The mitochondrial nature of the tubular structures was demonstrated by the immediate loss of Et fluorescence upon addition of the mitochondrial uncoupler FCCP (1 µM; Fig. 5B). This confirms that mitochondrial Et accumulation depends on mitochondrial membrane potential (5, 6, 15).
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MitoQ prevents rotenone-induced mitochondrial outgrowth without affecting rotenone-induced O2· formation. To investigate the mechanism underlying rotenone-induced mitochondrial outgrowth, we applied the antioxidant mitoquinone (MitoQ) (25). MitoQ is a ubiquinone derivative that is mitochondria-targeted by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Chronic treatment of human fibroblasts with MitoQ for 72 h did not alter mitochondrial morphology (Fig. 6A) or number (not shown). At a concentration of 10 nM, however, MitoQ abolished the effect of chronic rotenone treatment on mitochondrial outgrowth. Figure 6B shows that MitoQ alone had no effect on mitochondrial O2· production. In addition, Fig. 6 shows that MitoQ did not inhibit rotenone-induced O2· formation. These findings demonstrate that MitoQ acts downstream of O2· to inhibit rotenone-induced mitochondrial outgrowth.
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DISCUSSION |
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The data presented show that chronic treatment of fibroblasts with 100 nM rotenone for 72 h decreased complex I activity by 80% and caused significant mitochondrial outgrowth. Importantly, the alterations in mitochondrial shape were not the result of changes in cell cycle phase. This finding is in agreement with the findings of previous studies showing that 100 nM rotenone did not affect cell growth and viability of human B lymphoma cells (1). The morphological parameters analyzed were aspect ratio (AR), which is a measure of mitochondrial length, and form factor (F), which is a combined measure of mitochondrial length and degree of branching. Both parameters are independent of objective or zoom factor and therefore are most suited for comparison between different microscopes and cell types. To minimize the effects of phototoxicity and mitochondrial movement, images were acquired at video speed (30 Hz) and averaged in real time. For statistical evaluation, we calculated the average values of AR, F, and Nc (the number of mitochondria per cell) for each field of view. These averages were not influenced by the small number of partially imaged mitochondria, because typically 80500 mitochondria per field of view were present. Day-to-day variations in AR, F, and Nc were effectively corrected by expressing each value as a percentage of the corresponding control value recorded on the same day. Because our R123 staining procedure was very short (40 s) and because image analysis can be automated allow for future application of this protocol in the rapid screening of many mitochondrial structures.
Chronic treatment with 100 nM rotenone for 72 h caused a marked increase in form factor but did not significantly alter the aspect ratio or the number of mitochondrial structures. These findings show that human skin fibroblasts respond to chronic complex I inhibition with the formation of a more complex mitochondrial reticulum. Chronic treatment with 15 nM rotenone tended to increase both AR and F, suggesting that the effect of rotenone is dose dependent. When the concentration of rotenone was increased to 100 nM, however, the effect of chronic rotenone treatment on AR decreased rather than increased, whereas the effect on F increased further to reach statistical significance. Pham et al. (39) recently reported that human skin fibroblasts displayed increased amounts of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms upon acute (5 min) treatment with 40 µM rotenone. We did not observe such aberrations in mitochondrial morphology in our study, indicating that mitochondria respond completely differently depending on the concentration of the inhibitor and the duration of treatment. Chronic treatment of fibroblasts with 5 µM rotenone caused massive cell death (data not shown), demonstrating the inability of these cells to cope with relatively high inhibitor concentrations. It should be noted, however, that the relatively low concentration of 100 nM rotenone used in the present study decreased the activity of complex I by 80%. Moreover, acute addition of 100 nM rotenone caused an immediate increase in the rate of O2· production (data not shown), indicating that the inhibitor acts instantaneously. These observations suggest that the cytotoxicity of the higher rotenone concentrations is not directly related to its inhibitory effect on complex I activity and that therefore the results obtained with these concentrations should be treated with caution.
Several studies have shown that high concentrations of rotenone or exogenously added H2O2 can induce apoptosis (1, 12, 26, 31, 51). In general, this induction is accompanied by permeabilization of the mitochondrial inner membrane, opening of the permeability transition pore, and dissipation of the mitochondrial membrane potential (18). It has been demonstrated that chronic (36 h) rotenone treatment induces apoptosis by enhancing mitochondrial reactive oxygen production in HL-60 cells (31). However, the lowest concentration of rotenone that produced a significant increase in the percentage of apoptotic cells was 200 nM. In agreement with this finding, the present study did not show any adverse effects of chronic treatment with 100 nM rotenone on cell growth and viability. To the contrary, rotenone-treated fibroblasts displayed an increase in complexity of the mitochondrial network, suggesting the induction of an adaptive response. A similar increase was observed in cancer cells that were forced to grow on galactose and glutamine (43). Importantly, the latter study showed that the increase in complexity of the mitochondrial reticulum was accompanied by an increase in OXPHOS protein, indicating the adaptive nature of this response. These findings support the existence of a tight relationship between mitochondrial structure and function (11).
The rotenone-induced increase in mitochondrial outgrowth was completely prevented by cotreatment with MitoQ, a mitochondria-targeted derivative of coenzyme Q10 (25, 44). Given its very large hydrophobicity, MitoQ is preferentially adsorbed to the matrix-facing leaflet of the inner mitochondrial membrane, with the triphenylphosphonium moiety at the membrane surface at the level of the fatty acid carbonyls and the alkyl chain and ubiquinol moiety inserted into the hydrophobic core of the lipid bilayer (2). The inhibitory effect of MitoQ on rotenone-induced mitochondrial outgrowth suggests that an increase in mitochondrial oxidative stress is the primary cause of this cellular response.
MitoQ completely blocked rotenone-induced outgrowth and lipid peroxidation but had no effect on the rotenone-induced increase in mitochondrial O2· formation. The latter finding shows that MitoQ exerts its effect downstream of this O2·. It has been demonstrated that O2· produced upon complex I inhibition are released into the mitochondrial matrix (9, 50). The rotenone-induced increase in O2· production found in the present study might very well be the basis of the rotenone-induced increase in H2O2 production observed in previous studies (4, 31, 51). The observation that exogenous application of H2O2 increases mitochondrial mass in human lung fibroblasts (29) indeed suggests the involvement of matrix manganese superoxide dismutase in the mechanism of action of rotenone. Recent work concerning the mechanism by which O2· activates mitochondrial uncoupling proteins has suggested that O2· releases ferrous iron from iron-sulfur center-containing enzymes, which reacts with hydrogen peroxide, produced by the action of manganese superoxide dismutase, to form the ·OH. This radical then extracts a hydrogen atom from an unsaturated fatty acyl chain of a phospholipid to generate carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate the uncoupling proteins (34). Because MitoQ reacts mainly with lipid peroxidation products (Murphy M, unpublished observations), the present findings may suggest that rotenone acts through these products to increase mitochondrial outgrowth. However, further research is required to define the exact site of action of MitoQ. The present findings support recent insights that intracellular oxidants may act as specific signaling molecules under both physiological and pathological conditions (17).
The present study shows that 100 nM rotenone causes a twofold increase in the rate of mitochondrial O2· production in intact human skin fibroblasts. In agreement with this finding, NADH-stimulated mitochondrial O2· formation was found to be increased in mitochondrial membranes isolated from complex I-deficient human skin fibroblasts (40, 42). These findings support the existence of a site of electron leakage upstream from the rotenone binding site (19). A considerably higher concentration of 10 µM rotenone was used to evoke a significant increase in NADH-stimulated mitochondrial O2· formation in mitochondrial membranes isolated from control human skin fibroblasts (40). Therefore, the present method of determining the accumulation of Et in intact cells incubated for a short period in the presence of HEt is highly sensitive and most suitable for quantification of the rate of mitochondrial O2· production in patient fibroblasts.
In conclusion, the data presented herein are compatible with the existence of a O2·-induced mechanism of mitochondrial outgrowth that is activated at subapoptotic levels of complex I inhibition and leads to a possibly adaptive increase in the complexity of the mitochondrial reticulum. Importantly, we have shown that this mechanism is activated at pathological levels of complex I inhibition. Detailed analysis of mitochondrial morphology in patient fibroblasts will reveal whether this mechanism is activated or whether activation of this mechanism is impaired in human complex I deficiency.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of F. H. van der Westhuizen: School for Chemistry and Biochemistry, Potchefstroom University for Christian Higher Education, Potchefstroom, South Africa.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* W. J. H. Koopman and S. Verkaart contributed equally to this study.
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