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Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria

Department of Biochemistry and Molecular Biology and CNR Institute of Biomembranes and Bioenergetics, University of Bari, Bari, Italy

¹ Correspondence: E-mail: g.paradies{at}biologia.uniba.it

	ABSTRACT

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Several lines of evidence indicate that mitochondria-mediated reactive oxygen species (ROS) generation is a major source of oxidative stress in the cell. Release of cytochrome c from mitochondria is a central event in apoptosis induction and appears to be mediated by ROS. Dissociation of cytochrome c from the IMM, where it is bound to cardiolipin, represents a necessary first step for cytochrome c release. In the present study, the role of ROS and cardiolipin in the release of cytochrome c from rat liver mitochondria was investigated. ROS were produced by mitochondria oxidizing succinate in the nonphosphorylating state. Cytochrome c was quantitated by a new, very sensitive and rapid reverse-phase HPLC method. We found that succinate-supported ROS production resulted in a release of cytochrome c from mitochondria and a parallel loss of cardiolipin content. These effects were directly and significantly correlated and also abolished by ADP, which prevents succinate-mediated ROS production. The ROS-induced cytochrome c release was independent from MPT and appears to involve VDAC. It is suggested that mitochondrial-induced ROS production promotes cytochrome c release from mitochondria by a two-steps process, consisting of the dissociation of this protein from cardiolipin, followed by permeabilization of the outer membrane, probably by interaction with VDAC. The data may help clarify the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol and the role of ROS and cardiolipin in this release.—Petrosillo, G., Ruggiero, F. M., Paradies, G. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria.

Key Words: ROS • cytochrome c release • mitochondrial dysfunction

	INTRODUCTION

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Mitochondria play a crucial role in the apoptosis induction. Release of cytochrome c from mitochondria appears to be a central event in the induction of the apoptosis cascade that ultimately leads to programmed cell death (1 2 3) . Nevertheless, the mechanism underlying cytochrome c release from mitochondria is still not fully understood. Cytochrome c release from mitochondria that triggers caspase activation appears to be largely mediated by reactive oxygen species (4) . However, the exact mechanism by which reactive oxygen species (ROS) promote cytochrome c release and subsequent apoptotic process remains unclear.

It has been shown that cytochrome c release from mitochondria is preceded by its dissociation from the inner mitochondrial membrane (IMM) (5) . Cytochrome c is bound to the outer surface of the inner membrane phospholipids, primarily to cardiolipin molecules. The binding of cytochrome c to cardiolipin has been studied extensively and some molecular aspects of this interaction have been elucidated (6 7 8 9) . Cardiolipin is rich in unsaturated fatty acids (90% represented by linoleic acid), which appear to be essential for its interaction with cytochrome c in order to anchor the protein to the membrane. It would be expected that oxidative damage to cardiolipin by ROS may disturb the interaction of cytochrome c with this phospholipid at the level of the inner mitochondrial membrane and that this, in turn, would induce the dissociation of cytochrome c from the membrane, enabling its release into the extramitochondrial space. Accordingly, a loss of molecular interaction between cytochrome c and cardiolipin due to the lipid peroxidation has been reported (10) . In addition, it has been found that changes in the cardiolipin content, due to oxidative damage (11 , 12) or to alteration in its biosynthetic pathway (13) , trigger the release of cytochrome c from mitochondria in the apoptotic process. A role for cardiolipin in the apoptotic process is also supported by results showing that this phospholipid provides specificity for targeting of tBid to mitochondria (14) . tBid has been shown to promote cytochrome c release from mitochondria and liposomes (15) . These findings clearly point to a central role for cardiolipin homeostasis in programmed cell death.

It has recently been reported that an increase in ROS generation by the mitochondrial respiratory substrates induces cytochrome c release from mitochondria (16) . This release occurs without apparent disruption of the mitochondrial outer membrane. It has also been found that superoxide anion induces rapid and massive cytochrome c release from mitochondria, which is independent of mitochondrial pore transition (MPT). This O₂^·– induced release of cytochrome c is mainly due to voltage-dependent anion channel (VDAC)-dependent selective permeabilization of mitochondrial outer membrane (17) .

An early event in the release of cytochrome c from mitochondria is its dissociation from the IMM, where it is bound to cardiolipin. We recently reported that mitochondria-mediated ROS production induces the dissociation of cytochrome c from submitochondrial particles via cardiolipin peroxidation (18) . The present study was performed with the aim of exploring the mechanism whereby ROS and cardiolipin promote the release of cytochrome c from intact mitochondria. While most of the data concerning cytochrome c release from mitochondria has been obtained from Western blot analysis or absorbance spectroscopy, here, a new, very rapid and sensitive method to quantify cytochrome c based on reverse-phase high-pressure liquid chromatography (RP-HPLC) was used. We found that ROS produced at the level of mitochondrial respiratory chain induce the release of cytochrome c from mitochondria. This release may occur by a two-step process wherein cytochrome c is first dissociated from cardiolipin, then released in the extramitochondrial environment.

	MATERIALS AND METHODS

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Rat liver mitochondria were isolated in a medium of 250 mM sucrose, 10 mM Tris-HCl, 1 mM EGTA, pH 7.4, by differential centrifugation of liver homogenates essentially as described previously (19) . Mitochondria were resuspended in 250 mM sucrose, 10 mM Tris-HCl (pH 7.4) and stored in ice.

Mitochondrial protein concentration was measured by the Biuret method using serum albumin as standard.

Fluorometric determination of mitochondrial H₂O₂ production
The rate of mitochondrial hydrogen peroxide production was estimated by measuring the linear fluorescence increase induced by H₂O₂ oxidation of dichlorofluorescin to the fluorescent dichlorofluorescein in the presence of horseradish peroxidase (20) . Rat liver mitochondria (0.5 mg protein) were suspended in 2.5 mL of a medium of 100 mM sucrose, 75 mM KCl, 5 mM Tris, pH 7.4, supplemented with 1 mM phosphate, 10 µM EGTA, 7.5 µg horseradish peroxidase, 1 µM dichlorofluorescin. The production of hydrogen peroxide was induced by addition of 10 mM succinate as substrate. The amount of H₂O₂ produced was calculated by measuring the fluorescence changes upon addition of known amounts of H₂O₂.

Measurement of mitochondrial content of cytochrome c
Mitochondrial cytochrome c content was determined spectrophotometrically at _550-540 nm of reduced minus oxidized difference spectra (21) .

Detection of cytochrome c release
Cytochrome c content in the supernatant was determined by HPLC using a 5 mm C4 reverse-phase column (150x4.6 mm) on an HP series 1100 HPLC chromatograph, essentially as described in ref 22 . A gradient of 20% acetonitrile in water with trifluoroacetic acid (0.1% vol: vol) to 60% acetonitrile in water with trifluoroacetic acid (0.1% vol: vol) over 12 min with a flow rate of 1 mL/min was used. Absorption at 393 nm was used for detection.

Measurement of mitochondrial cardiolipin content
Cardiolipin content was determined by the HPLC technique as described previously (23) . Lipids from liver mitochondria were first extracted with chloroform/methanol, then phospholipids were separated by the HPLC with an Altex ultrasil-Si column (4.6x250). The chromatographic system was programmed for gradient elution using two mobile phases: solvent A, hexane/2-propanol (6: 8, v/v) and solvent B, hesane/2-propanol/water (6: 8:1.4, v/v/v). The percentage of solvent B in solvent A was increased in 15 min from 0% to 100%. Flow rate was 2 mL/min and detection at 206 nm.

Lipid peroxidation
Lipid peroxidation was estimated by appearance of conjugated dienes as follows. Lipids were extracted from mitochondria by the Bligh and Dyer procedure (24) . Lipid extracts from 1 mg of protein were dissolved in 2.5 mL of chloroform:methanol (1:1) and absorption spectra were followed between 210 and 310 nm (25) with a Perkin-Elmer Lambda 3B spectrophotometer.

Mitochondrial swelling assays
To measure the mitochondrial swelling that accompanies opening of the permeability transition pore, mitochondria (0.5 mg/3 mL) were suspended in a reaction medium consisting of 100 mM sucrose, 75 mM KCl, 5 mM Tris, pH 7.4, 10 µM EGTA, and 1 mM phosphate; the mitochondrial swelling was monitored continuously at 540 nm.

	RESULTS

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It has been shown that addition of succinate to aerobic mitochondria in the nonphosphorylating state causes a large -dependent H₂O₂ production that arises from O₂^·– due to the reverse electron transport from complex II through coenzyme Q to complex I (26 , 27) . Figure 1 shows the production of H₂O₂ in mitochondria supplemented with 10 mM succinate. Addition of ADP or protonophor p-trifluoro-methoxycarbonylcyanide phenylhydrazone (FCCP) completely abolished the succinate-supported H₂O₂ production (traces a, b) while oligomycin, an inhibitor of ATP synthase, prevented the ADP effect (trace d). Rotenone also abolished H₂O₂ production (trace c), confirming that this production is mainly ascribed to the membrane potential-supported reverse electron transfer from succinate to complex I, as previously reported (27 , 28) .

View larger version (17K): [in this window] [in a new window]   Figure 1. H2O2 production in rat liver mitochondria. The mitochondrial production of H2O2 was measured as described in Materials and Methods. 10 mM succinate was used as substrate. Where indicated, 2 mM ADP (trace a), 1 µM FCCP (trace b), 1.5 µM rotenone (trace c), and 2mM ADP plus 1.5 µM oligomycin were added.

The most commonly used method for examining cytochrome c release from mitochondria is immunoblotting with anti-cytochrome c antibodies. Recently, a new method was presented in the literature for cytochrome c detection and quantification based on RP-HPLC (22) . Figure 2 shows the linearity of the response of peak area vs. the amount of cytochrome c injected. The limit of quantitation of cytochrome c by this method was 0.5 pmol.

View larger version (11K): [in this window] [in a new window]   Figure 2. Cytochrome c detection by RP-HPLC. RP-HPLC cytochrome c determination was carried out as described in Materials and Methods. Horse heart cytochrome c (0.5–10 pmol) was injected in 20 µL aliquots and the peak area was derived from integrating the chromatographic peak.

The release of cytochrome c was measured in mitochondria supplemented with succinate (under conditions of ROS production) in the presence of 10 µM EGTA to chelate free Ca²⁺. The mitochondrial cytochrome c content as quantified by spectroscopic analysis amounted to 0.21 ± 0.02 nmol/mg protein. As shown in Fig. 3 , 10 min exposure of mitochondria to 10 mM succinate resulted in a release of 34 ± 3 pmol of cytochrome c per milligram of protein. In the control (absence of succinate), a small but detectable amount of cytochrome c (5 pmol) was liberated due probably to unspecific partial damage of outer mitochondrial membrane. The release of cytochrome c induced by the succinate-supported H₂O₂ production could be abolished by the addition of ADP, which prevents -dependent ROS formation (27) . A similar effect was obtained with rotenone. Addition of cyclosporin A, which inhibits mitochondrial pore transition, did not prevent the ROS-induced cytochrome c release. By contrast, the addition of 4,4’-diisothiocyanatostilbene- 2,2’-disulfonic acid (DIDS), reported to be a VDAC blocker (29) , inhibited ROS-induced cytochrome c release. This effect of DIDS could be due to an impairment of succinate utilization by mitochondria rather than to an interaction with VDAC. We therefore tested the effect of DIDS on succinate-supported respiration and succinate-supported H₂O₂ production in mitochondria. Both these processes were not significantly affected by DIDS (data not shown).

View larger version (32K): [in this window] [in a new window]   Figure 3. Cytochrome c release in mitochondria supplemented with succinate and effect of ADP, rotenone, cyclosporin A, and DIDS. Mitochondria were incubated at 37°C for 10 min in a reaction medium consisting of 100 mM sucrose, 75 mM KCl, 5 mM Tris, pH 7.4, 10 µM EGTA, and 1 mM phosphate in the absence (control) or presence of 10 mM succinate. Where indicated, 2 mM ADP, 1.5 µM rotenone, 1 µM cyclosporin A, or 50 µM DIDS were added 1 min before addition of succinate. After incubation, mitochondria were centrifuged and the supernatant was withdrawn, filtered, then injected into the HPLC for determination of cytochrome c content. All values are expressed as mean ± SE of 4 separate experiments.

Cytochrome c is bound to cardiolipin at the level of the IMM. Cardiolipin is localized almost exclusively in the inner mitochondrial membrane (30) . Using the dye nonylacridine, a compound that binds specifically with cardiolipin, 57% of this phospholipid was shown to be present in the outer leaflet of the IMM (31) . Increased production of ROS has been associated with early changes in intramitochondrial cardiolipin distribution during apoptosis (32) . ROS may alter the interaction between cytochrome c and cardiolipin at the level of the IMM by attacking double bonds of fatty acid constituents of cardiolipin. The content of cardiolipin was measured in mitochondria supplemented with succinate by a very sensitive HPLC technique set up in our laboratory with a quantitation limit of 0.5 nmol (23) . As shown in Fig. 4 , exposure of mitochondria for 10 min to 10 mM succinate resulted in a loss of cardiolipin content compared with untreated mitochondria. Addition of ADP to succinate supplemented mitochondria prevented this cardiolipin loss.

View larger version (42K): [in this window] [in a new window]   Figure 4. Loss in the cardiolipin content in mitochondria supplemented with succinate and prevention by ADP. Mitochondria were incubated at 37°C for 10 min in the same reaction medium described in the legend of Fig. 3 in the absence (control) or presence of 10 mM succinate. Where indicated, 2 mM ADP was added before addition of succinate. Cardiolipin content was determined as described in Materials and Methods. All values are expressed as mean ± SE of 4 separate experiments.

Unsaturated fatty acids of phospholipids are primarily targets during oxidative stress. To investigate whether the loss of cardiolipin in succinate-treated mitochondria could be due to an alteration of its unsaturated fatty acids due to ROS oxidative attack, we measured the degree of mitochondrial lipid peroxidation. This process is accompanied by a rearrangement of the polyunsaturated fatty acid double bonds, leading to the formation of conjugated dienes, which absorb at 233 nm. Thus, lipid peroxidation can be readily assayed by recording the absorbance of extracted membrane lipids at 233 nm (25) . As shown in Fig. 5 , treatment of mitochondria with succinate resulted in a 25% increase in lipid peroxidation compared with untreated mitochondria. This increase was abolished by the addition of buthylhydroxytoluene (BHT), a known free radical scavenger.

View larger version (43K): [in this window] [in a new window]   Figure 5. Absorbance values due to conjugated double bonds of lipid extracts from control and succinate supplemented mitochondria. The conjugated dienes spectra were recorded as described in Materials and Methods. 50 µM BHT was added as indicated. Data represent the absorbance at 233 nm of the lipid extracts from 1 mg of mitochondrial protein. All values are expressed as mean ± SE of 4 separate experiments.

When a comparison was made between the levels of cardiolipin and the amounts of cytochrome c released in mitochondria supplemented with succinate at different times of incubation, a strong linear correlation was observed (see Fig. 6 ). These data provide strong evidence that cardiolipin levels may directly influence cytochrome c release from mitochondria.

View larger version (16K): [in this window] [in a new window]   Figure 6. Release of cytochrome c vs. cardiolipin levels in mitochondria supplemented with succinate at different times of incubation. Mitochondria were incubated at 37°C in the same medium described in the legend of Fig. 3 in the presence of 10 mM succinate. At the indicated times, the incubation was interrupted by centrifugation and the supernatant was withdrawn, filtered, then injected onto the HPLC for determination of the cytochrome c content. Measurements of mitochondrial cardiolipin level were determined as described in Fig. 4 legend and in Materials and Methods. Levels of cardiolipin were compared with values of cytochrome c released from mitochondria. Each value represents the mean ± SE of both the cytochrome c and cardiolipin measurements of 4 independent determinations; r2 = 0.99.

The observed release of cytochrome c from mitochondria supplemented with succinate might involve MPT opening. The results reported in Fig. 7 show that the addition of 10 mM succinate to mitochondria incubated in the same medium used to follow cytochrome c release in the presence of 10 µM EGTA caused no appreciable decrease of light scattering (trace b) compared with control mitochondria (trace a), indicating the absence of mitochondrial swelling and pore opening. By contrast, addition of Ca²⁺ resulted in a full and rapid activation of MPT (trace c).

View larger version (9K): [in this window] [in a new window]   Figure 7. Effect of succinate and Ca2+ on mitochondrial swelling. Mitochondrial swelling was monitored as described in Materials and Methods. Control mitochondria in the absence of succinate (trace a), in the presence of 10 mM succinate (trace b), and in the presence of succinate plus 30 µM Ca 2+ (trace c). Results are typical of 5 independent experiments.

	DISCUSSION

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Mitochondrial dysfunction is a prominent feature of ROS-mediated cell death. A major pathway leading to mitochondrial damage is based on the amplification of mitochondrial and cytosolic ROS production in a variety of inflammatory and ischemia-related conditions. Release of cytochrome c from mitochondria is considered a central event in programmed cell death and appears to be largely mediated by reactive oxygen species. However, the exact mechanism by which ROS induce cytochrome c release from mitochondria and subsequent apoptosis remains unclear.

Mitochondrial electron transport chain is a major intracellular source of ROS (33) . Therefore, the effect of these reactive species should be greatest at the level of mitochondrial constituents, given that they are a highly reactive and short-lived species. Cardiolipin, an inner mitochondrial membrane phospholipid component, appears particularly susceptible to ROS attack either because of its high content of unsaturated fatty acids (90% represented by linoleic acid) or because its location in the IMM, near to the site of ROS production. Recent studies from this laboratory have demonstrated that mitochondrial-mediated ROS production affects the activity of the complexes I, III, and IV of the mitochondrial respiratory chain via oxidative damage of cardiolipin (34 35 36 37) .

In addition to its specific interaction with integral membrane proteins, including anion carriers and complexes of the respiratory chain (38 , 39) , cardiolipin plays an important role in the association of cytochrome c to the IMM (7 8 9) . It would be expected that alterations in the structure and/or in the content of cardiolipin might disturb its interaction with cytochrome c, leading to dissociation of this protein from the IMM, which could be considered an early event in the release of cytochrome from mitochondria and subsequent apoptosis. This raises the intriguing possibility that oxidative modulation of cardiolipin may be involved in the transduction of proapoptotic signaling cascades. This possibility is supported by the finding that cardiolipin is the target for the apoptotic protein tBid, which translocates to mitochondrial membrane in apoptosis (14) .

In a previous work, we reported that mitochondrial-mediated ROS generation induces the dissociation of cytochrome c from submitochondrial particles via peroxidative damage of cardiolipin (18) . In this work, we have explored the role of ROS and cardiolipin in the release of cytochrome c from intact isolated liver mitochondria. The results obtained demonstrate that ROS, produced at the level of the respiratory chain by mitochondria supplemented with succinate, promote the release of cytochrome c from mitochondria. Similar results have been reported by others (16) . This release of cytochrome c is blocked by the addition of ADP or rotenone, which prevent succinate-mediated ROS production, thus indicating a direct involvement of ROS in this effect.

Dissociation of cytochrome c from IMM can be considered an early step in the process of the release of cytochrome c from mitochondria. Unmodified acyl chains of cardiolipin appear to be essential for its interaction with cytochrome c to anchor the protein to the IMM (8 , 9) . Oxidative damage of these acyl chains may alter the interaction between cytochrome c and cardiolipin, leading to the detachment of this protein from the IMM. We found that succinate-supported ROS production leads to a loss of mitochondrial cardiolipin content due to ROS induced cardiolipin peroxidation. An increase in the mitochondrial cardiolipin peroxidation has already been found by different generating ROS systems (40) . The ROS-induced loss of cardiolipin was abolished by the addition of ADP. Moreover, there exists a quantitative correlation between cardiolipin loss and cytochrome c release, thus providing experimental confirmation of the existence of a cause and effect relationship between these two events. Together, these results indicate that mitochondrial-mediated ROS production induces the release of cytochrome c from mitochondria, first liberating this protein from cardiolipin, then releasing cytochrome c through the outer mitochondrial membrane.

A principal mechanism of cytochrome c release could involve ROS-induced promotion of Ca²⁺ dependent MPT opening. In fact, it has been reported that ROS and high intramitochondrial Ca²⁺ may act together to trigger MPT opening (41 42 43) . Permanent MPT opening causes cytochrome c release via matrix swelling and rupture of the mitochondrial membrane. Our results show that ROS-mediated cytochrome c release pathway is independent of the inner membrane components of MPT. ROS-mediated cytochrome c release was observed in mitochondria in the absence of Ca²⁺ (EGTA was used to chelate Ca²⁺) and was insensitive to cyclosporin A, a powerful inhibitor of MPT. Furthermore, no swelling was observed in mitochondria incubated for 10 min in the presence of 10 mM succinate, a condition under which cytochrome c release was observed. These data suggest that the integrity of the mitochondrial inner membrane and matrix space was preserved during ROS-induced cytochrome c release.

ROS-induced disruption of mitochondrial outer membrane could be involved in the release of cytochrome c after treatment of mitochondria with succinate. However, it was recently reported that under experimental conditions similar to those used in our experiments, the outer mitochondrial membrane remains intact (16) . Very recently, a novel mechanism underlying activation of cytochrome c release by superoxide anion has been suggested (17) . By this mechanism, O₂^·– induces cytochrome c release by selective permeabilization of outer mitochondrial membrane, interacting directly with VDAC. A similar mechanism might be involved in the cytochrome c release observed in mitochondria exposed to succinate-mediated ROS production, as suggested by the inhibition of cytochrome c release by DIDS, a compound that has been shown to interact with VDAC (29) .

In conclusion, our data suggest that mitochondrial-mediated ROS production may promote cytochrome c release from mitochondria by a two-step process consisting of the dissociation of this protein from cardiolipin, followed by permeabilization of the outer mitochondrial membrane, probably by interaction with VDAC, thus enabling the release of cytochrome c into the extramitochondrial space. ROS and cardiolipin have come to be recognized to play a central role in the release of cytochrome c from mitochondria and subsequent apoptosis (11 , 16 , 17 , 44) . Our results strongly support this hypothesis and furnish a possible mechanism by which both ROS and cardiolipin may promote the release of cytochrome c from mitochondria by an MPT-independent mechanism. Recently, it has been consistently reported that cardiolipin may cooperate with proapoptotic Bcl-2 family proteins Bax and Bid to permeabilize the outer mitochondrial membrane (45 , 46) .

	ACKNOWLEDGMENTS

 
This work was financially supported by a grant within the National Research Project PRIN "Bioenergetics and Membrane Transport," MURST Italy, 2001–2003.

	FOOTNOTES

 
doi: 10.1096/fj.03-0012com

Received for publication February 12, 2003. Accepted for publication August 5, 2003.

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