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Mitochondrial superoxide (IJBCB)

Mitochondrial superoxide (IJBCB)

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The International Journal of Biochemistry & Cell Biology 40 (2008) 1792–1805

Oxidative stress caused by blocking of mitochondrial Complex I H+ pumping as a link in aging/disease vicious cycle
Andrea Dlaskov´ 1 , Lydie Hlavat´ 1 , Petr Jeˇ ek ∗ a a z
Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, V´deˇ sk´ 1083, Prague 14220, Czech Republic ı n a Received 12 December 2007; received in revised form 14 January 2008; accepted 14 January 2008 Available online 19 January 2008

Abstract Vulnerability of mitochondrial Complex I to oxidative stress determines an organism’s lifespan, pace of aging, susceptibility to numerous diseases originating from oxidative stress and certain mitopathies. The mechanisms involved, however, are largely unknown. We used confocal microscopy and fluorescent probe MitoSOX to monitor superoxide production due to retarded forward electron transport in HEPG2 cell mitochondrial Complex I in situ. Matrix-released superoxide production, the un-dismuted surplus (Jm ) was low in glucose-cultivated cells, where an uncoupler (FCCP) reduced it to half. Rotenone caused a 5-fold Jm increase (AC50 2 M), which was attenuated by uncoupling, membrane potential ( Ψ m ), and pH-collapse, since addition of FCCP (IC50 55 nM), valinomycin, and nigericin prevented this increase. Jm doubled after cultivation with galactose/glutamine (i.e. at obligatory oxidative phosphorylation). A hydrophobic amiloride that acts on the ND5 subunit and inhibits Complex I H+ pumping enhanced Jm and even countered the FCCP effect (AC50 0.3 M). Consequently, we have revealed a new principle predicting that Complex I produces maximum superoxide only when both electron transport and H+ pumping are retarded. H+ pumping may be attenuated by high protonmotive force or inhibited by oxidative stress-related mutations of ND5 (ND2, ND4) subunit. We predict that in a vicious cycle, when oxidative stress leads to higher fraction of, e.g. mutated ND5 subunits, it will be accelerated more and more. Thus, inhibition of Complex I H+ pumping, which leads to oxidative stress, appears to be a missing link in the theory of mitochondrial aging and in the etiology of diseases related to oxidative stress. © 2008 Elsevier Ltd. All rights reserved.
Keywords: In situ mitochondrial superoxide production; MitoSOX; Proton-pumping NADH:quinone oxidoreductase; HEPG2 cells; Oxidative stress; Aging

1. Introduction Complex I (H+ -pumping NADH:quinone oxidoreductase) is an essential component of the mitochondrial respiratory chain (Brandt, 2006; Lenaz, Baracca, Fato,

Corresponding author. Fax: +420 296442488. E-mail addresses: dlaskova@biomed.cas.cz (A. Dlaskov´ ), a hlavata@biomed.cas.cz (L. Hlavat´ ), jezek@biomed.cas.cz (P. Jeˇ ek). a z 1 These authors contributed equally to the work. 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.01.012

Genova, & Solaini, 2006; Lenaz, Fato, Formiggini, & Genova, 2007; Yagi & Matsuno-Yagi, 2003), participating not only in cell respiration, but also in cellular/organismal reactive oxygen species homeostasis (Brand et al., 2004; Jeˇ ek & Hlavat´ , 2005), apoptoz a sis initiation or modulation (Ott, Gogvadze Orrenius, & Zhivotovsky, 2007), and O2 sensing (Piruat & Lopez-Barneo, 2005). This huge 46 subunit mammalian complex is vulnerable to oxidative stress, hence it is one of the factors that determines lifespan, pace of aging, susceptibility to oxidative stress-related diseases,

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and certain mitopathies (Bai et al., 2004; Bourges et al., 2004; Chomyn & Attardi, 2003; Smigrodzki & Khan, 2005). This is so because Complex I comprises 7 of 13 proteins encoded by the mitochondrial genome (membrane arm subunits ND1-6, and ND4L, among which ND2, ND4, and ND5 act in H+ pumping; Gemperli, Schaffitzel, Jakob, & Steuber, 2007; Nakamaru-Ogiso, Boo Seo, Yagi, & Matsuno-Yagi, 2003; Nakamaru-Ogiso, Sakamoto, Matsuno- Yagi, Myioshi, & Yagi, 2003). Thus, mtDNA mutations in regions encoding Complex I subunits, induced by continuous inevitable mitochondrial superoxide (O2 •− ) production, might initiate a vicious cycle due to further enhanced O2 •− production brought on by the now impaired function of Complex I or other respiratory machinery elements (Muller, Lustgarten, Jang, Richardson, & Van Remmen, 2007). The progressive increase in mitochondria-derived oxidative stress may also contribute to numerous diseases including atherosclerosis, hypertension, ischemia-reperfusion injury, inflammation, cystic fibrosis, cancer, type-2 diabetes, and neurodegenerative diseases (e.g. multiple sclerosis, Parkinson’s and Alzheimer’s disease) (Brand et al., 2004; Jeˇ ek & Hlavat´ , 2005). Despite the suggesz a tion that mitochondria-derived oxidative stress impacts many disease states and aging, it is not understood why Complex I-related mtDNA mutations lead to oxidative stress. Notably, we lack a high-resolution structure of Complex I, a detailed mechanism of O2 •− production within the Complex I, and a detailed pathway of electron transport and H+ pumping (Brandt, 2006; Brand et al., 2004; Grivennikova & Vinogradov, 2006; Ohnishi & Salerno, 2005). Scant direct evidence exists for the dependence of mitochondrial O2 •− formation in intact cells on the mitochondrial inner membrane potential ( Ψ m ) or on the entire protonmotive force ( p = Ψ m + pH, in mV) (Jeˇ ek & Hlavat´ , 2005). There are no indications of z a attenuation of in situ mitochondrial O2 •− production by uncoupling or by other means of p modulation despite clear demonstrations of such phenomenon in isolated mitochondria (Brand et al., 2004; Jeˇ ek & Hlavat´ , z a 2005). Rather, when uncouplers like carbonyl cyanide p(trifluoro-methoxy)phenylhydrazone (FCCP) are added to culture cells, remodeling (De Vos, Allan, Grierson, & Sheetz, 2005) or fragmentation/fission of mitochondrial network (Benard et al., 2007; Duvezin-Caubet et al., 2006; Ishikara, Fujita, Oka, & Mihara, 2006; Lyamzaev et al., 2004; Pletjushkina et al., 2006), apoptosis (Aronis et al., 2003; Dispersyn, Nuydens, Connors, Borgers, & Geerts, 1999), or changes in gene expression (Desquiret et al., 2006; Kuruvilla et al., 2003) are observed.

It is still un-clear whether Complex I derived O2 •− generation is sensitive to Ψ m . Complex I derived O2 •− production due to both forward (Lambert & Brand, 2004a) and reverse electron transport diminishes with decreasing pH in isolated skeletal muscle mitochondria (Lambert & Brand, 2004a, 2004b). The reverse electron transport is inhibited by rotenone. Complex III (ubiquinol-cytochrome c oxidoreductase) can also produce O2 •− as shown in vitro in isolated glutathionedepleted nonphosphorylating rat heart mitochondria respiring with succinate at state 4. These mitochondria exhibited a 55% decrease in H2 O2 formation when Ψ m decreased by only 10% (Korshunov, Skulachev, & Starkov, 1997). About 30–43% (Starkov & Fiskum, 2003; Starkov et al., 2004) of the levels of state 4 H2 O2 generation was maintained in the phosphorylating state, i.e. state 3. Thus, H+ backflux to the matrix, ensured either by ATP synthase or by uncoupling, attenuates O2 •− production. Still, the relative contributions of Complexes I and III to overall O2 •− formation in mitochondria is not known (Jeˇ ek & Hlavat´ , 2005; Muller, z a Liu, & Van Remmen, 2004). Nevertheless, almost 100% of Complex I O2 •− production is released to the matrix (Brand et al., 2004; Muller et al., 2004). In this work, we used confocal microscopy with the mitochondrial superoxide indicator MitoSOX Red to assess excessive matrix O2 •− release in situ. We found that Complex I O2 •− production is enhanced only and exclusively when both electron transport and H+ pumping are retarded. Since inhibition of Complex I H+ pumping usually results from oxidative stress-induced mutations in mtDNA encoding subunits ND2, ND4, and ND5, the fact that this inhibition produces further oxidative stress represents a missing link in a vicious cycle of aging or oxidative stress-related diseases. 2. Materials and methods 2.1. Cell cultivation The human hepatocellular carcinoma cell line HEPG2 (ECACC 85011430) was cultivated at 37 ◦ C in humidified air with 5% CO2 in DMEM (Gibco, cat. no. 11995-065; contains no glucose) supplemented with 3 mM glutamine, 5% FCS (Biochrome, cat. no. S0113), 10 mM HEPES, 100 IU/ml penicillin, and 100 g/ml streptomycin. The added carbon source was either 25 mM glucose or 10 mM galactose (with glucose-free dialyzed FCS, PAA, cat. no. A15-107). The latter is referred to as oxphos conditions (cells), because cells rely largely on oxidative phosphorylation (Rossignol et al., 2004). The doubling time for glc and oxphos cells was

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on average 23.5 and 39.4 h, respectively. A stable cell line HEPG2-mRoGFP was selected by Geneticin after transfection with a pcDNA vector encoding mRoGFP (obtained from Dr. Rossignol, University of Bordeaux 2, France). For confocal microscopy, HEPG2 cells were cultured for 2 days on glass coverslips coated with polyl-lysine. 2.2. Confocal microscopy The confocal inverted fluorescent microscope Leica SP2 AOBS DM IRE2 HC Fluo TCS 1-B (an objective PL APO 100 × /1.40–0.70 oil) was used with an Argon laser (488 nm/20 mW, 514 nm/20 mW) for excitation of cells in a thermostable sample chamber set to 37 ◦ C, supplied from a CO2 incubator, to mimic cultivation conditions. A pinhole 1 Airy unit was used to set confocal conditions. 2.3. Semiquantitative analysis of Jm using confocal microscopy Loading/incubation of cells with 4.1 M MitoSOX Red for 15 min at 37 ◦ C was done exactly the same way for each experiment and all reagents were added thereafter. Excitation was at 514 nm and emission was detected between 580 (or 610) and 679 nm. A series of images was taken after the addition of a reagent to the cells, typically each 30 s for the next 20 min. Regions of the interest corresponding to mitochondria of approximately 15–20 cells per slide were selected using Ellipse software (ViDiTo, Kosice, Slovakia) so that nearly an equal pixel number was analyzed in each image. Changes in integrated fluorescence intensity at these loci were quantified from plots of fluorescence in selected area vs. time. Slopes were determined by linear regressions. 2.4. Response of MitoSOX Red spectra to superoxide and/or DNA in vitro and in situ The fluorescent probe MitoSOX Red, a triphenylphosphonium-(TPP)-conjugated dihydroethidine, should indicate O2 •− , in addition to other reactive oxygen species. In a cell-free system, where O2 •− was generated by xanthine/xanthine oxidase for 30 s, a substantial increase in both ∼610 and ∼663 nm peaks of MitoSOX emission spectra was observed at 500 nm excitation (Fig. 1a vs. b), as recorded on a Fluorolog 322 (Spex–Jobin–Yvon–Horiba) fluorometer with double grating monochromators for both excitation and emission. Addition of double-stranded herring DNA increased fluorescence up to eightfold and the emission maximum shifted slightly to 593 nm (a shoulder shifted

to ∼655 nm, Fig. 1a). Both the MitoSOX Red free and DNA-intercalated forms were sensitive to O2 •− as indicated by the lack of high emission increase when SOD was present (Fig. 1a). Higher signal intensities with SOD (Fig. 1a) than in the absence of enzyme system (Fig. 1b) reflect its optical interference. Excitation spectra of MitoSOX Red without DNA and independently of the presence of O2 •− exhibited a maximum at 468 nm with a small peak at 378 nm, whereas in the presence of DNA two equally obvious peaks at 510 and 390 nm were observed (data not shown). In situ, MitoSOX Red should detect surplus mitochondrial O2 •− generation released to the matrix (Jm ), that is to say, that portion that is not consumed by the Mn-superoxide dismutase (MnSOD). O2 •− reacts with the dihydroethidine moiety, forming a highly fluorescent complex of 2-OH-dihydroethidine, whereas ethidium, as a positively charged entity, can arise from spontaneous oxidation (Georgiou, Papapostolou, Patsoukis, Tsegenidis, & Sideris, 2005; Mukhopadhyay, Rajesh, Yoshihiro, Haslo, & Pachem, 2007; Robinson et al., 2006; Zhao et al., 2003, 2005; Zielonka, Zhao, Xu, & Kalyanaraman, 2005). The positively charged TPP moiety of MitoSOX Red enables its high accumulation in the mitochondrial matrix. Its ability to intercalate into mtDNA (Fig. 2) allows for a sufficient portion of MitoSOX Red to be retained in the matrix even in the absence of Ψ m . Due to the existing order of magnitude of Jm (pmol O2 •− l−1 min−1 ), this “dark” portion is not consumed during the typical 20-min experiment and can still react with O2 •− . Hence, the probe indicates Jm for many more minutes even after the Ψ m collapse (see Predicted MitoSOX Red response). Note, that once it has interacted with O2 •− and becomes fluorescent, the probe is not quenched. Therefore, only an increase in fluorescence with time is expected when unreacted probe is available. The probe reacting with O2 •− during the loading procedure establishes a certain fluorescence level enabling confocal imaging (plus a small background of non-specifically oxidized probe, e.g. due to incident laser light); moreover as a significant portion of it intercalates into mtDNA, it is not expulsed from the matrix upon collapse of p ( Ψ m ). Indeed, we did not observe a drop in fluorescence upon FCCP addition. 2.5. Predicted MitoSOX Red response Consider the external medium compartment, having a volume Vext , layered on a coverslip with cells of the overall total volume Vcell , equal to the volume of cytoplasm Vcyt plus the volume of mitochondrial matrix Vm (the volume of the intermembrane space is

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Fig. 1. Emission spectra of MitoSOX Red. (a) The emission spectra of MitoSOX Red were measured in the presence or absence of superoxide. Emission spectra (2 nm slit width) at excitation at 500 nm (10 nm slit width) of MitoSOX Red (7 M) in PBS either with (upper spectra) or without (lower spectra) dsDNA (43 g/ml isolated from herring) were recorded after 30 s incubation with xanthine oxidase (0.025 U, giving 25 nmol superoxide per min; total 12.5 nmol) and 286 M xanthine (solid lines) or with 0.028 U of CuZnSOD (dotted lines, absence of superoxide). (b) The emission spectra of MitoSOX Red were measured without xanthine oxidase and xanthine. Only MitoSOX Red (lower spectra) or MitoSOX Red and DNA (upper spectra) were present. Background buffer light scattering was subtracted from all spectra. Emission maxima were at 610 nm (shoulders ∼663 nm) without DNA and at 593 nm (shoulders ∼655 nm) with DNA.

neglected). In our case Vext was 1000 or 2000 l, Vcell was 0.24 l (for 200,000 HEPG2 cells and assuming an average cell volume of 1.2 pl, see Wehner, Lawonn, & Tinel, 2002), and Vm is 0.024 or 0.12 l, if one assumes mitochondria represent 10 or 50% of cell volume, respectively. Cells have plasma membrane potential Ψ p with a typical magnitude of 60 mV and a Ψ m typically 120–180 mV. We neglect ethidium formation and so we assume that MitoSOX Red is bearing a single positive charge on the TPP moiety. Under these conditions, redistribution of MitoSOX Red between the external medium compartment and the matrix will be given by Eq. (1), derived by Rafael and Nicholls (1984), while neglecting concentration of MitoSOX in the cytosol: ψp + ψm = (2.303RT/F ) log{AVcyt cm /Vm cext } (1) where R and F are the gas and Faraday constants, respectively, T the absolute temperature, A accounts for the difference in activities in different compartments, and c stands for the MitoSOX concentration in the matrix, cm = nm /Vm , or the external compartment, cext = next /Vext , where n is the probe amounts in picomoles. Using a Ψ p of 60 mV, Ψ m of 180 mV, 2.303RT/F as 60, and Vcyt /Vm equal to 10 or 2, and A

equal to 1, then log{10Vext nm /Vm next } = 4 Vext nm /Vm next = 1000 nm + next = ntotal (2) (3) (4)

Considering mitochondria as only 10% of the cell volume (Vm of 0.024 l) and at a ctotal of 4.1 M and a Vext of 2000 l, one can calculate nm to be 97 pmol and cm to be 4.04 mM; considering the mitochondria as 50% of the cell volume (Vm of 0.120 l), then nm would be 464 pmol and cm 3.87 mM. The calculated amount of cm and nm is important with respect to possible order of magnitude of mitochondrial O2 •− production. Our quantification of H2 O2 release in isolated rat liver mitochondria gave 60 pmol min−1 mg mitochondrial protein−1 . If SOD dismuted 90% of this production, 6 pmol min−1 mg−1 would remain. Assuming 1 mg mitochondrial protein per 1 l of Vm matrix volume, we estimated O2 •− production of 0.144 and 0.72 pmol O2 •− min−1 for Vm 0.024 and 0.12 l, respectively. Consequently, it would take 675 and 644 min, respectively, for that amount of MitoSOX Red residing in the matrix to be converted by O2 •− into the fluorescent form. When one considers that in the presence of FCCP or other agents collapsing Ψ m , there would be a three orders of magnitude lower

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amount of MitoSOX Red in the matrix, one would expect a problem in the inability of such a low probe amount to indicate O2 •− for a sufficiently long time. This obstacle is compensated for by the fact that a significant portion of MitoSOX Red is intercalated into mtDNA. With 10% of intercalated probe in mtDNA, 9.7 and 46.4 pmol still remains after the FCCP addition, allowing for the possibility of observation for the first 67 and 64 min, respectively. With a higher fraction of probe intercalated or a lower excessive O2 •− release rates, higher observation times would be possible. For high intercalation, the intensity scale for MitoSOX emission is similar in both the case of a high Ψ m and zero Ψ m . 2.6. Cell in situ Ψ m monitoring

In situ Ψ m was monitored with tetramethylrhodamine ethyl ester (TMRE, Molecular Probes) using confocal microscopy. Cells were stained with 10 nM TMRE and excited at 514 nm in time-lapse sequences. Emission was detected between 580 and 650 nm. 2.7. Total cellular SOD, CuZnSOD, and MnSOD activities Cellular activities of SOD, CuZnSOD, and MnSOD were measured using an SOD assay activity kit (Cayman) according to the manufacturer’s instructions. The assay utilizes a tetrazolium salt to detect O2 •− generated by xanthine oxidase and hypoxanthine. Addition of KCN to the assay inhibits CuZnSOD, resulting in detection of only MnSOD activity. 2.8. Quantification of electron transport complexes The amount of electron transport complexes was quantified either by running blue native PAGE gels (4–13% gradient separation gel), enabling separation of digitonin-solubilized mitochondrial complexes (Wittig, Braun, & Schagger, 2006) or by immunoblotting using the Human Total OXPHOS Complexes Detection kit (MitoScience) after separating mitochondrial proteins by SDS-PAGE. Band quantification was performed by densitometry using the Sciom Image software beta 4.02 Win 1. 3. Results 3.1. Subcellular localization of MitoSOX Red To verify that the O2 •− -sensitive probe MitoSOX Red localizes to the mitochondria, we have incubated

Fig. 2. Localization of MitoSOX in HEPG2 cells. (a–c) Confocal images of MitoSOX Red in glc HEPG2 cells (a, red) expressing mRoGFP (b, green) and magnified merged images (c); staining for 15 min with 4.1 M MitoSOX. Excitation for MitoSOX was set at 514 nm and for mRoGFP at 488 nm. The contrast of the original Tiff images was enhanced by 30% and of the merged image by 70%. (d–f) Confocal images of MitoSOX Red (d, red) and DNA stain SYTO (e, green) in glc HEPG2 cells and the magnified merged image (f). HEPG2 cells were stained with 0.25 M SYTO for 10 s and subsequently with 4.1 M MitoSOX for 15 min; excitation for SYTO was set at 488 nm. The contrast of the merge Tiff image was enhanced by 40%.

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human hepatocellular carcinoma (HEPG2) cells expressing matrix-targeted redox-sensitive GFP (mRoGFP) with MitoSOX. Fig. 2a–c shows high, about 99.8%, co-localization of MitoSOX and mRoGFP signals (mRoGFP taken as 100%) in the confocal plane. We also co-incubated HEPG2 cells with MitoSOX Red and SYTO, a stain for both nuclear DNA and mtDNA (Fig. 2d–f). SYTO only partially (82–87%) co-localized with the more spatially distributed MitoSOX Red. If SYTO exclusively stained mtDNA, ∼80% emission intensity of MitoSOX within the mitochondrial SYTOpositive region would imply that free MitoSOX was >20%, because intercalated MitoSOX has a higher quantum yield (Fig. 1). Thus MitoSOX was either intercalated into mtDNA or free in the matrix aqueous compartment. 3.2. HEPG2 cells cultivated with glucose do not undergo significant oxidative stress We measured in situ rates of surplus O2 •− release (i.e. unconsumed by Mn-superoxide dismutase) into O2 the mitochondrial matrix (Jm ). In HEPG2 cells cultivated with 25 mM glucose (glc cells) the MitoSOX emission signal increased only slightly (Figs. 3, 4, and 5a, no addition), indicating no significant surplus O2 •− production. This suggests that MnSOD can dismute the majority of O2 •− produced by the respiratory chain and released to the matrix. The already slow Jm rates of the glc cells were further decreased by ∼50% upon addition of an uncoupler (FCCP) after probe loading (Figs. 3, 4, and 5a). Oligomycin activated Jm more than fourfold, while establishing the nonphosphorylating state 4.
•−

increase was substantially prevented. It was reduced on average 4.5-fold, frequently down to the basal Jm of intact glc cells (Figs. 3, 4, and 5a). An apparent IC50 was 55 nM, consistent with AC50 s (Table 1) for activation of glc cell respiration without rotenone. Due to sufficient MitoSOX intercalation into mtDNA, the p collapse by FCCP did not completely deplete the unreacted probe from the matrix, hence MitoSOX responses to matrix O2 •− were apparent. This was further supported by an experiment where H2 O2 was added after FCCP, restoring a substantial increase in fluorescence (Fig. 4). Although caused by hydroperoxyl radical, the restoration clearly indicates that sufficient amount of unreacted probe existed in the matrix to be oxidized even after p collapse. 3.4. Conversion of pH into rotenone-induced Jm in situ Ψ m decreases

Nigericin is a K+ /H+ antiporter with a strict 1:1 stoichiometry that converts the whole pH component of p into Ψ m while increasing Ψ m . Addition of 10 nM nigericin to glc cells did not affect magnitudes of Jm (Fig. 5a). Nigericin also prevented the high Jm values induced by rotenone (Fig. 5a), but glc cell respiration was not significantly affected by up to 1 M nigericin (Table 1). 3.5. Ψ m collapse decreases rotenone-induced Jm in situ Valinomycin-mediated K+ uniport short-circuits Ψ m and converts whole p into pH. Valinomycin (10 nM) abolished high rotenone-induced Jm in glc cells but also slightly increased basal Jm (Fig. 5a). Glc cell respiration was activated up to 3.8-fold (1.4-fold with rotenone) with AC50 s 3–6 nM, but was inhibited by >1 M valinomycin, which caused MitoSOX release into the cytoplasm and apoptosis and/or necrosis. 3.6. The Jm pattern in HEPG2 cells relying on oxidative phosphorylation When HEPG2 cells were cultivated in galactose/glutamine and had to rely on oxidative phosphorylation, oxphos cells (Rossignol et al., 2004), the Jm pattern was identical to that of glc cells under all conditions tested, with the exception of FCCP treatment (Fig. 5a and Table 1). Except for the rotenone, the basal Jm values of oxphos cells were on average 1.8-fold higher compared with glc cells, corresponding to their higher Ψ m (Fig. 5b). Addition of 20 M rotenone led to an

3.3. Uncoupling attenuates rotenone-induced Jm in situ Addition of 20 M rotenone to glc cells after the probe equilibration markedly enhanced Jm (Figs. 3, 4, and 5a) without inducing cell death. Twenty micromoles of rotenone increased Jm on average 5.5-fold in glc cells with an AC50 of 2 M (Fig. 5a), providing excessive oxidative stress. IC50 s for inhibition of normal (state 3) cell respiration by rotenone ranged from 34 to 65 nM (but was ∼3 M with FCCP, Table 1). About 13% (20–30% with FCCP) of state 3 respiration remained at rotenone >1 M. This reflects the fact that rotenone does not prevent succinate-supported respiration in situ. TMRE indicated that Ψ m was kept rather high with rotenone (data not shown). When 1 M FCCP was added prior to rotenone but after MitoSOX Red loading, the rotenone-induced Jm

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Fig. 3. First and last confocal images in a series accessing the rates of fluorescence increases of MitoSOX Red. HEPG2 glc cells were stained with 4.1 M MitoSOX for 15 min and images were taken every 30 s (the first images are displayed in the left panels) up to 20 min (the last images are displayed in the right panels). Additions were as follows: no addition (a and b); 20 M rotenone (c and d); 1 M FCCP (e and f); 20 M rotenone plus 1 M FCCP (g and h).

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with higher state 3 respiration. Oxphos cells had higher MnSOD activity (Table 1). TMRE staining and in some cases MitoSOX imaging also indicated a distinct mitochondrial morphology (Fig. 6) of the oxphos-cultivated HEPG2 cells characterized by an interconnected network of mitochondrial reticulum of thin tubules with a few bulkier spherical mitochondria in contrast to less frequent thicker tubules and frequent predominant bulky mitochondria within the reticulum or solitary mitochondria observed under glucose cultivation (Fig. 6b–d). 3.7. Inhibition of Complex I H+ pumping enhances superoxide production and abolishes its uncoupler-mediated suppression Because rotenone-induced O2 •− production originates entirely from Complex I, and as uncoupling may accelerate residual H+ pumping in rotenoneinhibited Complex I, we further studied the effects of an H+ -pumping inhibitor, hydrophobic amiloride, 5-(Nethyl-N-isopropyl) amiloride (EIPA), known to bind to the membrane subunit ND5 (Gemperli et al., 2007; Nakamaru-Ogiso, Boo Seo, et al., 2003; NakamaruOgiso, Sakamoto, et al., 2003). Addition of 1 M EIPA approximately doubled the basal Jm in both glc and oxphos cells (Fig. 7) independently of FCCP without affecting respiration, thus directly activating O2 •− production (not trivially via respiration increase). EIPA did not prevent, but instead activated by ∼10%, the already high rotenone-induced Jm (Fig. 7). Surprisingly, EIPA at 0.5–1 M (Fig. 7, AC50 < 0.3 M EIPA) prevented suppression by FCCP of rotenone-induced Jm in both

Fig. 4. Derivation of Jm in mitochondria in situ. HEPG2 glc cells were stained with 4.1 M MitoSOX Red for 15 min. Images were taken every 30 s (see Fig. 8) and were processed to produce derived traces for the integral intensity of MitoSOX Red fluorescence within mitochondrial matrix vs. time. Jm values were determined from the slopes of the traces. Arrow, 500 M H2 O2 was added during the time course of glc cells with FCCP to show the ability of MitoSOX to be oxidized within mitochondria after their de-energization.

equally high Jm in oxphos cells and glc cells (Fig. 5a). The amount of Complex I in oxphos cells was maximally 110% of glu cells (Table 1). Details of differences between glc and oxphos cells are summarized in Table 1. Higher oxidative phosphorylation of oxphos cells was reflected by: (i) ∼2-fold higher average respiration of 106 cells, (ii) ∼30–40 mV higher Ψ m, as indicated by on average 4.2-fold higher initial TMRE emission (n = 6), and (iii) higher autofluorescence excited at 488 nm reflecting either a higher flavoprotein content or higher oxidation of flavoproteins (Huang, Heikal, & Webb, 2002); both are consistent

Fig. 5. (a) Relative Jm values of oxphos- (dashed columns) and glucose-cultivated HEPG2 cells (black columns). Twelve to 20 image series (5–6 series for effects of 10 nM nigericin, “Nig”, and valinomycin, “Val”) for each reagent or combination, such as shown in Fig. 2, were performed, and Jm values were calculated from the slopes of the integral intensity vs. time and finally normalized to the Jm values obtained for 20 M rotenone (100%). Where indicated, 1 M FCCP was added. The t-test yielded significant differences (p < 0.1) between the calculated relative Jm values for FCCP or no additions (only oxphos) vs. rotenone + FCCP; for no additions vs. FCCP (only glc), vs. rotenone + FCCP, or vs. rotenone + valinomycin (only oxphos). (b) Ratios of Jm for oxphos- vs. glucose-cultivated HEPG2 cells. The t-test yielded significant differences between ensembles of Jm (absolute rates) for oxphos- vs. glucose-cultivation as indicated: *p < 0.1; **p < 0.01; ***p < 0.001.

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Table 1 Comparison of glucose- and galactose/glutamine-cultivated HEPG2 cellsa Parameter Surplus superoxide production released to the matrix Jm (state 3)/Jm (state 4) (n = 3) Rotenone activation (AC50 ) Prevention of rotenone activation by FCCP (IC50 ) Respiration State 3 respiration per 106 cells (pmol O2 s−1 ) (n = 50) State 3/state 4 (state 4: n = 6) Rotenone inhibition in state 3 (IC50 ) Rotenone inhibition after FCCP (IC50 ) FCCP activation in state 3 (AC50 ) FCCP activation in state 4 (AC50 ) FCCP activation after rotenone (AC50 ) EIPA inhibition Valinomycin activation (AC50 Glc: up to 30 nM, oxphos: up to 80 nM) Valinomycin inhibition (IC50 ) Nigericin activation up to100 nM Nigericin inhibition (IC50 ) Other parameters Total SOD activity (U/ml, n = 5) CuZnSOD activity (U/ml, n = 5) MnSOD activity (U/ml, n = 5) Complex I content from BN-PAGE, n = 4, Glc cells set to 100%
a When

Glc cells 0.2 ± 0.1 2 M 55 nM 51 ± 17 4 ± 0.5 68, 64, and 34 nM (13% remaining) 3 M (20–30% remaining) 25, 57, 144, and 220 nM 132 and 450 nM 186 nM No inhibition even at >1 mM 3, 5, and 6 nM >500 and >1000 nM <10% activation >1000 and >500 nM 0.07 ± 0.02 0.02 ± 0.01 0.04 ± 0.02 100 ± 9

Oxphos cells 0.3 ± 0.1 3 M 70 nM 97 ± 24 5.6 ± 1.6 74, 40, and 20 nM (5% remaining) 100 nM (2% remaining) 26, 38, 65, 265, and 300 nM 127 and 428 nM 200 nM No inhibition even at >1 mM 2 and 2.5 nM >1000 nM and >10 M <3% activation >1000 nM and >10 M 0.16 ± 0.05 0.05 ± 0.03 0.08 ± 0.05 113 ± 11

for a reagent, an apparent IC50 or AC50 is mentioned, it has been calculated from a corresponding Hill plot. When several numbers are shown, those originate from repeated estimations. State 4 has been established by 1 g ml−1 oligomycin.

glc and oxphos cells, in many cases returning Jm to rotenone-induced values. 4. Discussion Our principal findings on the mechanism of O2 •− formation within the mitochondrial Complex I led us to predict significant consequences for mitochondriaderived oxidative stress. We hypothesize that the missing link in the mitochondrial theory of aging and etiology of diseases originating from oxidative stress lies in our finding that inhibition of Complex I H+ pumping leads to elevated O2 •− production, and hence to oxidative stress, given also by cascade of downstream products of O2 •− (Brand et al., 2004; Jeˇ ek & Hlavat´ , z a 2005). This link has been clearly demonstrated for the ND5 subunit via studies with its inhibitor, the hydrophobic amiloride derivative EIPA (Gemperli et al., 2007; Nakamaru-Ogiso, Boo Seo, et al., 2003; NakamaruOgiso, Sakamoto, et al., 2003). ND5 participation in H+ pumping is derived from its sequence homology with ancient Na+ /H+ antiporters which provides the ability of Escherichia coli ND5 homolog NuoL to pump Na+ . Such a Na+ pumping was found to be inhibited by EIPA

(Gemperli et al., 2007). Moreover, Complex I-related H+ pumping of isolated rat liver mitochondria was recently found to be inhibited by EIPA without inhibiting respiration (Dlaskov´ et al., unpublished). a Our hypothesis explains why severe consequences may result from slight initial oxidative modifications. They lead to oxidative damage of mtDNA thereby to mutated subunits ND2, ND4, and ND5, and hence to the impaired H+ pumping. This provides further oxidative stress and further oxidative damage of mtDNA, thus representing ever accelerating amplification by a vicious spiral (cycle) leading to aging, diseases, and cell death. Oxidatively damaged subunit proteins may be replaced by new protein expression at a sufficient turnover, whereas oxidative damage of mtDNA leads to mutated subunits and, at high enough levels, is lethal (Sato, Nakada, & Hyashi, 2006; Smigrodzki & Khan, 2005). Indeed, severe diseases such as Leber’s hereditary optic neuropathy, mitochondrial encephalopathy lactic acidosis stroke-like episodes, Leigh syndrome, and Parkinson’s disease originate from oxidative modifications or inherited mutations of the mtDNA coding region for the H+ pumping subunit ND5 (Bourges et al., 2004; Chomyn & Attardi, 2003; Parker & Parks,

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Fig. 6. Morphological differences in the mitochondrial network between glucose- and oxphos-cultivated HEPG2 cells. (a and b) Visualization using TMRE of oxphos- (a) and glucose-cultivated (b) HEPG2 cells. Cells were incubated with 50 nM TMRE for 10 min, then images were taken. The fluorescence intensity was actually ∼4-fold higher in (a), since a higher photomultiplier gain was used for (b). (c–f) Visualization using MitoSOX Red prior to (c and e) and after 20 min incubation with 20 M rotenone (d and f) for glc (c and d) and oxphos cells (e and f). Image pair (c–f) have the same scale.

2005; Smigrodzki, Parks, & Parker, 2004). Even if mitochondria perhaps defend against such oxidative stress by complementation, i.e. fusing into a network or reticulum (Chan, 2006; Sato et al., 2006), a threshold of point of no return likely exists. The vicious cycle may happen also with oxidatively mutated coding regions in mtDNA for 22 tRNAs and 12S and 16S ribosomal RNAs, which obviously affect expression of the whole

mtDNA, and with oxidatively mutated mitochondriacoded subunits of Complex III (cyt b) and ATP synthase (ATP6/8). Concerning a mechanism of O2 •− production within Complex I (Brandt, 2006; Brandt, Kerscher, Drose, Zwicker, & Zickermann, 2003; Lambert & Brand, 2004a; Yagi & Matsuno-Yagi, 2003), our data suggest that not only retardation of electron transport (mostly

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Fig. 7. EIPA elevates Jm and attenuating effect of FCCP on rotenonemediated induction is prevented by EIPA. Figure illustrates Jm in oxphos- (dashed columns) vs. glucose-cultivated HEPG2 cells (black columns). Three to six image series for each reagent or combination were measured and Jm values were normalized to those obtained with 20 M rotenone (100%). Where indicated, 1 M EIPA or 1 M FCCP was present. AC50 for abolishing FCCP attenuation of rotenoneinduced Jm was <0.3 M EIPA. The t-test yielded significant (p < 0.01) differences between Jm values with EIPA or EIPA + FCCP vs. no addtions.

pathophysiological) within Complex I must occur, but that the simultaneous additional retardation of H+ pumping is essential for high O2 •− formation. Decreased H+ pumping may result from the pathophysiological impairment due to mutated ND2, ND4, and ND5 subunits or is

manifested physiologically as feedback suppression by p or Ψ m (Fig. 8). Indeed, either rotenone-mediated electron transport retardation at Δp ∼0 (uncoupling, Fig. 5a) or the inhibition of H+ pumping itself by the ND5-bound inhibitor EIPA (Gemperli et al., 2007; Nakamaru-Ogiso, Boo Seo, et al., 2003; NakamaruOgiso, Sakamoto, et al., 2003) led only to intermediate O2 •− formation (Fig. 7), perhaps resulting from fully reduced flavin as seen for isolated Complex I (Kussmaul & Hirst, 2006). Rotenone-binding in proximity to the Q-site (ubiquinone binding site) highly retards electron transport throughout the peripheral arm of Complex I (Brandt, 2006; Brandt et al., 2003; Lambert & Brand, 2004a; Yagi & Matsuno-Yagi, 2003), which results in the formation of longer-lived semiquinone species having a higher probability of reacting with oxygen and thus forming O2 •− . Even with retarded electron transport, a high rate of O2 •− formation is reached only when H+ pumping is also attenuated by a high p (Fig. 8a) or by high Ψ m . When the p feedback retardation of H+ pumping is released by uncoupling and H+ pumping is re-accelerated, O2 •− is not formed in excess, even if inhibition by rotenone persists (Fig. 8b). This view is independently supported by the fact that EIPA, which doubled basal Jm and slightly increased the rotenoneinduced Jm , strongly overcame the suppressive effect of FCCP on rotenone-induced O2 •− formation (Fig. 7). Blockage of H+ pumping through EIPA-mediated inhibition (Gemperli et al., 2007; Nakamaru-Ogiso, Boo Seo, et al., 2003; Nakamaru-Ogiso, Sakamoto, et al., 2003)

Fig. 8. Principles for superoxide production within Complex I. The schema illustrates O2 •− production by Complex I, which takes place in high rates only when electron transfer is highly retarded (e.g. by rotenone) and when H+ pumping also is slowed down. The latter can happen physiologically at high p (a) or pathophysiologically when H+ pumping is inhibited (c). With H+ pumping intact but at p zero, O2 •− production ceases (b). When substrate oxidation is faster than the Q cycle or electron flow within the remaining respiratory chain, the resulting NADH “overflow” acts similarly as a relative retardation of electron flow, which together with high p generates an intermediate O2 •− production (d). Conformational changes are depicted by zigzag lines.

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was sufficient to act in concert with electron flow blocked at the Q-site by rotenone (Fig. 8c). We predict that a mild uncoupling causing reacceleration of electron flow through the respiratory chain may have an “antioxidant” role in vivo but only in cases where the H+ pumping subunits are not extensively damaged. We also interpret valinomycin prevention of the rotenone-induced Jm as a direct consequence of a collapsed Ψ m on Complex I H+ pumping, i.e. as released “feedback pressure” of H+ pumping required for high O2 •− formation (Fig. 8). In conclusion, during aging and pathogenesis, oxidative modifications of nuclear-coded subunits or mutations resulting from previous oxidative stress in mitochondria-coded subunits of Complex I are serious when occur in residues (or influence them) participating either in electron transport pathway or in pathway including transfer of conformation changes and H+ pumping. The resulting elevated O2 •− formation provides further accelerating oxidative stress in a vicious cycle especially when the damage occurs in both pathways. The above schemes are grounded in the structure of Complex I and reflect a yet unknown H+ pumping mechanism. The consensus view is that the H+ pumping within Complex I (stoichiometry 4H+ per 2 e− ) is provided by long-range conformational changes occurring when an electron leaves the redox center N2 located on the PSST subunit on a peripheral arm in proximity to the matrix side of the membrane (Brandt, 2006). Conformational changes relay redox changes between the pH-dependent mid redox potential of redox center N2 (−150 mV) and ubiquinone (QH2 /Q, +90 mV), residing at the Q-site, to H+ transport across the entire membrane-associated part of the complex (Brandt, 2006). One can speculate that p or Ψ m slow down these changes, which also delay redox changes leading to longer-lived semiquinones that react with oxygen to form O2 •− . These conformation changes are also blocked by EIPA (Nakamaru-Ogiso, Boo Seo, et al., 2003; Nakamaru-Ogiso, Sakamoto, et al., 2003) and thus its effect fits into our interpretation. Complex I is likely an important player in homeostasis of reactive oxygen species (Kussmaul & Hirst, 2006), significantly contributing to O2 •− release into the mitochondrial matrix in vivo. Even if Complex III probably contributed to our measured Jm values (Muller et al., 2004), since only rotenone-sensitive portions are unequivocally attributed to the Complex I, we may deduce a Complex I role from our comparison of cells relying on oxidative phosphorylation, which exhibited >2-fold Jm compared to glucose-cultivated cells. Thus the basal Jm in oxphos cells was higher by the same factor as their respiration increased (up to twofold), but was still

rather low due to a sufficient MnSOD capacity (MnSOD activity actually rose, Table 1). An increased NADH or NADH/NAD+ ratio (Kussmaul & Hirst, 2006) leading to a high electron flow exceeding output electron flow on Complex I and higher Ψ m are likely causes (Fig. 8d). We speculate that electron transport within Complex I (and maybe also Complex III) in oxphos cells is relatively more retarded (Fig. 8d) due to higher substrate oxidation resulting in accumulating NAD(P)H levels and faster respiration, thereby faster H+ pumping and creating higher p (confirmed by higher TMRE emission). As a result, the Jm is higher than in glc cells (Fig. 5b), although Complex I content is similar. The substrate load (NADH/NAD+ ratio) in oxphos cells should be high enough that compensation by the increased H+ backflow via FO ATP synthase (Desquiret et al., 2006; Rossignol et al., 2004) and doubled MnSOD content cannot attenuate such elevated O2 •− formation. In turn, the situation in glucose-cultivated cells (moderate levels of oxidative phosphorylation) resembles the effect of calorie restriction (Muller et al., 2007). Thus fast electron flux via the whole respiratory chain at a high substrate pressure produces more O2 •− than when slower flux occurs at the same relative retardation (same oxidation/reduction states). Moreover, we have shown that uncoupling attenuates O2 •− production in situ, at least for basal Jm of glc cells, and that Jm is >4-fold and >3-fold higher at state 4 in glc and oxphos cells, respectively. We have also demonstrated that MitoSOX Red is a useful indicator of mitochondrial oxidative stress. This is due to the fact that it principally increases its fluorescence only when excessive O2 •− production takes place, i.e. production that cannot be efficiently dismuted by matrix MnSOD. Thus we can predict that in situ mitochondria of any cells with insufficient MnSOD capacity or any cells where excessive O2 •− is produced by the respiratory chain due to disease or aging processes will be indicated by increased MitoSOX emission with time. This increase will thus indicate excess O2 •− production especially when a high Ψ m is established. Comparing only fluorescence levels (Georgiou et al., 2005; Mukhopadhyay et al., 2007; Robinson et al., 2006; Zhao et al., 2003, 2005; Zielonka et al., 2005) could be misleading due to unknown contribution of non-specific oxidation and extent of intercalation into mtDNA. Acknowledgements Supported by grants NR/7917-6 from the Czech Ministry of Health; IAA500110701 and AV0Z50110509 from the Academy of Sciences; 301/05/0221 (to P.J.) and 303/05/P100 (to L.H.) from the Grant Agency of

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