Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury.

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N(2)A cells (EC(50) in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca(2+) in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.

The mitochondrial respiratory chain on the inner mitochondrial membrane (IMM) 1 is a major intracellular source of re-active oxygen species (ROS). ROS cause nonspecific damage to lipids, proteins, and DNA, leading to alteration or loss of cellular function. Mitochondria are continuously exposed to ROS and accumulate oxidative damage more rapidly than the rest of the cell, especially because ROS are highly reactive and shortlived (1). Many studies have associated mitochondrial dysfunction caused by ROS with both necrotic and apoptotic cell death (2). The rate of mitochondrial ROS production can be altered by several physiological or pathological conditions. Inhibitors of the respiratory chain such as 3-nitropropionic acid (3NP), an irreversible inhibitor of the complex II enzyme succinate dehydrogenase, tend to increase ROS production (3)(4)(5). The inhibition of this complex seems to be related to neuronal death similar to those occurring in Huntington's disease (6), and antioxidants can attenuate the neurochemical changes and some behavioral disturbances caused by 3NP in animals (5,7). Mitochondrial Ca 2ϩ is another powerful signal for ROS production. Calcium is taken up into mitochondria via a uniporter in the IMM, and elevation of mitochondrial Ca 2ϩ and ROS production is thought to play an important part in cell death associated with ischemia-reperfusion as well as 3NP (4,5).
Increasing evidence suggests that ROS play a key role in promoting cytochrome c release from the mitochondria (8 -11), and cytochrome c in the cytoplasm triggers activation of the caspase cascade that ultimately leads to apoptosis (12,13). The mechanism underlying ROS-mediated cytochrome c release from mitochondria is still not fully understood. Cytochrome c is normally bound to the IMM by an association with cardiolipin (14). It is now believed that cytochrome c release from mitochondria proceeds by a two-step process: dissociation of cytochrome c from cardiolipin in the IMM, followed by release of cytochrome c through the outer mitochondrial membrane (OMM) (15). Cardiolipin is rich in unsaturated fatty acids, and peroxidation of cardiolipin induces the dissociation of cytochrome c from mitochondria into the cytosol (16). However, the mechanism by which cytochrome c is released through the OMM is not clear. One mechanism may involve ROS-induced promotion of Ca 2ϩ -dependent mitochondrial permeability transition (MPT), with swelling of the mitochondrial matrix and rupture of the OMM (17,18). ROS may promote MPT by causing oxidation of thiol groups on the adenine nucleotide translocator (19 -21). This mechanism seems likely in 3NP toxicity and ischemia-reperfusion injury, where increased intracellular Ca 2ϩ and ROS are both present (4,5,22,23). How-ever, there is also evidence showing that cytochrome c can be released through the OMM in an MPT-independent manner (24 -28). It was reported recently that ROS can induce cytochrome c release from mitochondria in the absence of Ca 2ϩ and was insensitive to cyclosporin A (10), suggesting MPT-independent mechanisms. MPT-independent mechanisms may involve the voltage-dependent anion channel on the OMM or an oligomeric form of Bax (15,25,29).
Given the many ways by which cytochrome c may be released through the OMM, the most efficient approach to inhibit ROSinduced cytochrome c release and cell death would be prevention of lipid peroxidation of the IMM. Unfortunately, none of the available antioxidants specifically targets mitochondria, let alone the IMM. In addition, most of the antioxidants are poorly cell-permeable, requiring concentrations in excess of 100 M to prevent oxidative cell death. One approach used to target antioxidants such as coenzyme Q and vitamin E to mitochondria has involved conjugation of these lipid-soluble molecules to lipophilic cations such as triphenylalkylphosphonium ions, which are rapidly taken up into the mitochondrial matrix because of the potential gradient across the IMM (30,31). The introduction of cations into the mitochondrial matrix, however, leads to dissipation of IMM potential, and this was observed in isolated mitochondria with concentrations of triphenylalkylphosphonium ion-conjugated antioxidants greater than 20 M (30, 31). Furthermore, dissipation of the IMM potential would ultimately limit further drug uptake.
We have developed a series of peptide antioxidants that are taken up by mitochondria and concentrate in the IMM. These peptide antioxidants are cell-permeable and are very potent at reducing intracellular ROS and preventing cell death caused by the oxidant t-butylhydroperoxide (tBHP). We have used these IMM-targeted antioxidants to investigate the role of mitochondrially generated ROS in mitochondrial dysfunction in cells exposed to 3NP. To investigate the mechanisms by which these peptide antioxidants protect against mitochondrial dysfunction, we used isolated mitochondria to determine their ability to prevent MPT and cytochrome c release caused by Ca 2ϩ overload and 3NP. In addition, because ROS have been implicated in contractile dysfunction associated with reperfusion of ischemic hearts, we determined the efficacy of these peptide antioxidants in preventing myocardial stunning in an ex vivo perfused heart model. Finally, to prove that the effects of these peptide antioxidants are caused by their ability to scavenge ROS, we designed a peptide analog that lacked antioxidant properties. Our results suggest that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT and rupture of the OMM. These results also confirm a major role for ROS in mitochondrial dysfunction and reperfusion injury and demonstrate the therapeutic potential of these peptide antioxidants in ischemia-reperfusion injury and neurodegeneration.

Measurement of Antioxidant Properties of SS Peptides in Vitro-
The ability of SS peptides to scavenge H 2 O 2 in vitro was determined using luminol chemiluminescence (35). H 2 O 2 (4.4 nmol) was incubated with 1 to 100 M of various peptides in 0.5 ml of phosphate buffer, pH 8.0, for 30 s. Luminol (25 M) and horseradish peroxidase (0.7 IU) were then added to the solution, and chemiluminescence was monitored with an aggregometer (Chronolog, Havertown, PA) for 20 min at 37°C. Antioxidant properties of SS peptides were further established by inhibition of fatty acid peroxidation and low density lipoprotein (LDL) oxidation. Linoleic acid peroxidation was initiated with 2,2Ј-azobis(2-amidinopropane) and the formation of conjugated dienes was monitored spectrophotometrically at 234 nm (36). Freshly prepared human LDL (0.1 mg/ml in phosphate-buffered saline) was oxidized catalytically by the addition of 10 M CuSO 4 , and the formation of conjugated dienes was monitored at 234 nm for 5 h at 37°C (37).
Mitochondrial Preparation-Male CD1 mice were sacrificed by decapitation, and the livers immediately excised and homogenized in ice-cold isolation buffer (10 mM sucrose, 200 mM mannitol, 5 mM HEPES, and 1 mM EGTA, pH 7.4) containing 1 mg/ml fatty acid-free bovine serum albumin. The homogenate was centrifuged for 10 min at 900 ϫ g, and the supernatant was centrifuged again at 13,800 ϫ g for 10 min. The mitochondrial pellets were washed twice, centrifuged at 11,200 ϫ g, and re-suspended in the same buffer (no EGTA). All experiments were conducted in accordance with guidelines approved by the Institution for the Care and Use of Animals at Weill Medical College of Cornell University.
Mitochondrial Uptake Studies-Uptake of SS-19 by isolated mitochondria was examined by fluorescence quenching upon addition of a mitochondrial suspension (0.35 mg) (Hitachi F-4500 fluorescence spectrophotometer; excitation/emission ϭ 320/420 nm). For mitochondrial uptake of [ 3 H]SS-02, mitochondria (0.8 mg) were suspended in buffer (70 mM sucrose, 230 mM mannitol, 3 mM HEPES, 5 mM succinate, 5 mM KH 2 PO 4 , and 0.5 M rotenone, pH 7.4) containing [ 3 H]SS-02 and 1 M SS-02 at room temperature. Uptake was stopped by centrifugation (16,000 ϫ g for 5 min at 4°C), the mitochondrial pellet was washed twice and resuspended in 0.2 ml of 1% SDS/0.2 N NaOH, and radioactivity was determined. Mitochondrial uptake of SS-19 and [ 3 H]SS-02 were also determined in the presence of 1.5 M carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupler that results in mitochondrial depolarization. To determine the localization of the peptide within mitochondria, three cycles of freeze-thaw treatment were used to isolate inner and outer membranes (38). Treatment with 0.2% digitonin was used to disrupt the outer membrane to determine peptide distribution to the IMM and matrix (39).
Cell Culture-Caco-2 cells (American Type Culture Collection, Manassas, VA) and N 2 A cells (provided by Dr. Gunnar Gouras, Department of Neurology, Weill Medical College of Cornell University) were cultured as described previously (40,41). Cell culture supplies were obtained from Invitrogen.
Cellular Uptake and Intracellular Localization of Peptide Antioxidants-Peptide uptake into Caco-2 cells was carried out as described previously (40). Cells (10 6 /well) were incubated with [ 3 H]SS-02 at 37°C for 60 min, and radioactivity was determined in the medium and in cell lysate. To determine intracellular peptide localization, Caco-2 cells were incubated with SS-19 (0.1 M) for 15 min at 37°C, and confocal laser scanning microscopy (CLSM) was carried out with living cells using a C-Apochromat 63ϫ/1.2 W Corr objective (Nikon, Tokyo, Japan) with excitation and emission wavelengths set at 320 and 420 nm, resepctively. To demonstrate localization of SS-19 to mitochondria, Caco-2 cells were incubated with SS-19 and Mitotracker tetramethylrhodamine methyl ester (TMRM; Molecular Probes, Portland, OR; excitation/emission ϭ 550/575 nm) for 30 min at 37°C and then examined by CLSM.
Intracellular Mitochondrial Potential-Caco-2 cells were treated with 3NP (10 mM) in the absence or presence of SS-02 (0.1 M) for 4 h and then incubated with TMRM and examined under CLSM as described above.
Mitochondrial H 2 O 2 Production-0.1 mg of mitochondrial protein was added to 0.5 ml of potassium phosphate buffer (100 mM, pH 8.0) containing 5 mM succinate; 25 M luminol and 0.7 IU of horseradish peroxidase were added, and chemiluminescence was monitored continuously for 20 min at 37°C. The amount of H 2 O 2 produced was determined by area under the curve.
Mitochondrial Oxygen Consumption-Mitochondrial protein (1 mg) was added to 2.0 ml of respiration buffer (70 mM sucrose, 230 mM mannitol, 2 mM HEPES, 5 mM KH 2 PO 4 , 5 mM MgCl 2 , and 0.5 mM EDTA, pH 7.4). Oxygen consumption was measured with a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK). Respiration was measured in the presence of 5 mM succinate, and state 3 respiration was initiated with the addition of 0.35 mM ADP.
Mitochondrial Swelling Assays-Isolated mitochondria (0.1 mg) were added to 0.2 ml of buffer (70 mM sucrose, 230 mM mannitol, 3 mM HEPES, 2 mM Tris-phosphate, 5 mM succinate, and 1 M rotenone) and swelling was measured by decrease in absorbance at 540 nm using a 96-well plate reader (Molecular Devices, Sunnyvale, CA).
Mitochondrial Cytochrome c Release-Isolated mitochondria (0.75 mg/2 ml) were incubated in the absence or presence of SS-02 for 100 s before addition of Ca 2ϩ to induce swelling. Swelling was measured by light scattering at 610 nm. Alamethicin (7 g/ml) was added to induce maximal swelling, and the magnitude of swelling induced by Ca 2ϩ was expressed as a percentage of maximal swelling. After incubation for 400 s, the mitochondrial pellet was collected by centrifugation. Cytochrome c content in the pellet and supernatant was determined using a commercial rat/mouse cytochrome c immunoassay kit (R & D Systems, Minneapolis, MN).
Ischemia-Reperfusion Studies-Details of the isolated perfused guinea pig heart model have been published previously (42). Isolated hearts were perfused continuously with either Krebs-Henseleit solution or Krebs-Henseleit solution containing various SS peptides and allowed to stabilize for 30 min. Contractile force was measured with a small hook inserted into the apex of the left ventricle, and the silk ligature tightly connected to a Grass force-displacement transducer. Global ischemia was then induced by complete interruption of coronary perfusion for 30 min. Reperfusion was carried out for 90 min after ischemia.

Antioxidant Properties of SS Peptides-The antioxidant
properties of SS peptides were demonstrated by their ability to scavenge H 2 O 2 and inhibit the oxidation of linoleic acid and LDL in vitro. The prototype peptide, SS-02, dose-dependently reduced the luminol-derived chemiluminescence produced by H 2 O 2 in the presence of horseradish peroxidase (Fig. 1A). SS-02 also dose-dependently inhibited the oxidation of fatty acids (Fig. 1B) and LDL in vitro (Fig. 1C). The antioxidant activity of SS-02 was not dependent on the specific order of the four amino acids in that SS-31 showed similar antioxidant activity (Fig. 1,  D and E). However, substitution of Dmt 1 by Phe 1 (SS-20) eliminated antioxidant activity (Fig. 1, D and E).
Cellular Uptake of SS-02-To demonstrate that the SS peptides are cell-permeable, we incubated Caco-2 cells with protein (43) and 200 l of media, the intracellular concentration of [ 3 H]SS-02 can be estimated to be 9.8 Ϯ 0.26 times higher than extracellular concentration.
Intracellular Targeting of SS Peptides-The fluorescent analog SS-19 was used to determine cellular uptake and intracellular localization by CLSM. The confocal images showed uptake of SS-19 (blue fluorescence) into Caco-2 cells within 15 min (Fig. 2, left). Fluorescence was detected in the cytoplasm of all cells, but the peptide was entirely excluded from the nucleus. The distribution pattern resembled mitochondrial distribution as shown by Mitotracker TMRM (Fig. 2, middle). The co-localization of SS-19 and TMRM (Fig. 2, right) suggests targeting of SS-19 to mitochondria after cellular uptake.
Mitochondrial Uptake of SS Peptides-Mitochondrial uptake of SS peptides was examined using isolated mouse liver mitochondria. Addition of isolated mitochondria to SS-19 resulted in immediate quenching of the fluorescent signal (Fig. 3A). To ensure that the uptake of SS-19 by mitochondria was not an artifact of the fluorophore, we incubated mitochondria with [ 3 H]SS-02 and determined radioactivity in the mitochondrial pellet. Uptake of [ 3 H]SS-02 by mitochondria was rapid with maximal levels reached by 2 min (Fig. 3B). Radioactivity averaged 67,021 Ϯ 2008 cpm in the mitochondrial pellet, and 128,131 Ϯ 2015 cpm in the supernatant (n ϭ 3). Assuming mitochondrial volume of 1 l/mg protein (44), it can be estimated that [ 3 H]SS-02 accumulates 104.6 Ϯ 1.6-fold in mitochondria.
Pretreatment of mitochondria with FCCP only reduced SS-19 quenching or [ 3 H]SS-02 uptake by ϳ20% (Fig. 3, A and  B), suggesting that only 20% of this cationic peptide was targeted into the mitochondrial matrix in a potential-dependent manner. When mitochondria were incubated with [ 3 H]SS-02 for 5 min and the mitochondrial pellet was subjected to three freeze-thaw cycles, 72% of [ 3 H]SS-02 was retained in the membrane pellet consisting of both IMM and OMM (Fig. 3C). Treatment of the mitochondrial suspension with 1% digitonin to disrupt the OMM allowed us to determine that 85% of the radioactivity was in the mitoplast (IMM and matrix) (Fig. 3C). These results suggest that the peptides are predominantly targeted to the IMM.
SS Peptides Reduce Intracellular ROS and Cell Death Caused by tBHP-To show that SS peptides are effective when applied to whole cells, neuronal N 2 A cells were treated with tBHP (0.5 or 1.0 mM) for 40 min, washed, and then incubated with media containing SS-02 or SS-31, or media alone for 4 h. Incubation with tBHP resulted in dose-dependent increase in intracellular ROS and decrease in cell viability (Fig. 4). Incubation of these cells with either SS-31 or SS-02 dose-dependently reduced intracellular ROS (Fig. 4A)  cell survival (Fig. 4, B and C), with EC 50 in the nanomolar range.
SS-02 Protects against 3NP-induced Mitochondrial Depolarization in Caco-2 Cells-To demonstrate that reduction in mitochondrially generated ROS can protect against mitochondrial dysfunction, we examined the effect of SS-02 on mitochondrial depolarization caused by treatment of cells with 3NP. Caco-2 cells were treated with 10 mM 3NP in the absence or presence of 0.1 M SS-02, and mitochondrial potential was visualized by confocal microscopy using TMRM (Fig. 5). In control cells (left), the mitochondria are clearly visualized as red streaks throughout the cytoplasm. In cells treated with 3NP (middle), the TMRM fluorescence was much reduced, suggesting generalized depolarization. In contrast, concurrent treatment with SS-02 (100 nM) protected against mitochondrial depolarization caused by 3NP (right).
SS Peptides Inhibit ROS Generation by Isolated Mitochondria-Isolated mitochondria were then used to better understand the mechanisms of cytoprotection provided by these peptide antioxidants. SS-31 dose-dependently reduced spontaneous generation of H 2 O 2 in isolated mitochondria (Fig. 6A). Similar results were obtained with SS-02 (Fig. 6B). In addition to reducing spontaneous ROS generation, SS-02 and SS-31 were able to inhibit ROS production induced by antimycin A (Fig.  6B). SS-20, which does not have antioxidant activities, had no effect on spontaneous or induced ROS production by isolated mitochondria (data not shown). did not alter rate of oxygen consumption during state 3 or state 4 respiration (data not shown). Mitochondrial membrane potential, as measured by TMRM, was also not affected by SS-02, SS-19 or SS-31 even when applied at 200 M (Fig. 7A). In contrast, the addition of FCCP caused immediate depolarization, and Ca 2ϩ overload (150 M) resulted in progressive loss of mitochondrial potential, indicative of MPT (Fig. 7A).

Effects of SS Peptides on Mitochondrial Function in Isolated
SS Peptides Protect against MPT in Isolated Mitochondria-Pretreatment of isolated mitochondria with 10 M SS-02 or SS-31 before addition of Ca 2ϩ resulted only in transient depolarization of the mitochondria without eliciting the onset of MPT (Fig. 7, B and C). Pretreatment of mitochondria with SS-02 also dose-dependently delayed the onset of MPT induced by 1 mM 3NP (Fig. 7D). However, SS-20, which cannot reduce mitochondrial ROS production, had no effect on either Ca 2ϩ -or 3NP-induced MPT (data not shown).
SS Peptides Inhibit Mitochondrial Swelling in Isolated Mitochondria-Pretreatment of isolated mitochondria with SS-02 dose-dependently inhibited Ca 2ϩ -induced swelling as measured by decrease in absorbance at 540 nm (Fig. 8A). Mitochondrial swelling was also inhibited by SS-31 but not by SS-20 (Fig. 8B). Ca 2ϩ overload resulted in the translocation of cytochrome c from the mitochondrial pellet to the supernatant (Fig.  8C). The release of mitochondrial cytochrome c was significantly inhibited by SS-02 (Fig. 8C) and SS-31 (data not shown). heart with the antioxidant peptides SS-02 (100 M) or SS-31 (1 nM) significantly improved contractile force after 90 min of reperfusion compared with buffer alone (Fig. 9). In contrast, SS-20 was not able to prevent the contractile dysfunction resulting from reperfusion, providing direct support for a major role for ROS in cardiac reperfusion injury. DISCUSSION These SS peptides (SS-02 and SS-31) are the first antioxidants that selectively target and concentrate in the IMM, thereby enabling scavenging of ROS at the site of production. Using these peptide antioxidants, we were able to show that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP. Our studies with isolated mitochondria also demonstrated that ROS mediate cytochrome c release via MPT and rupture of the OMM. By reducing ROS production, these peptide antioxidants were able to prevent mitochondrial depolarization in cells exposed to 3NP. Finally, these peptide antioxidants were able to prevent myocardial stunning associated with reperfusion in the ischemic heart in an ex vivo model. The inability of SS-20, which does not have antioxidant ability, to prevent Ca 2ϩ -mediated mitochondrial swelling or reperfusion injury confirms that the protective actions of these peptides are mediated via their antioxidant actions. The structural motif of these SS peptides centers on alternating aromatic residues and basic amino acids (aromaticcationic peptides). The antioxidant action of SS peptides can be attributed to the Dmt residue. Tyrosine can scavenge oxyradicals forming relatively unreactive tyrosyl radicals, which can be followed by radical-radical coupling to give dityrosine (45,46), or scavenging by glutathione and/or ascorbate (47,48). Several endogenous tyrosine-containing peptides have been shown to possess antioxidant properties (37,49). We have found that methylation of the phenolic ring, as in Dmt, increases antioxidant potency. Dmt bears much structural similarity to vitamin E; both have the methylated phenol structure. Rearrangement of the amino acid sequence (SS-02 versus SS-31) had no effect on the scavenging properties of the peptides, but substitution of Dmt 1 in SS-02 with Phe 1 (SS-20) resulted in complete loss of antioxidant activity.

SS Peptides Protect against Ischemia/Reperfusion-induced Contractile Dysfunction in the Isolated
Unlike other antioxidants, the SS peptides are water-soluble and readily penetrate cell membranes in a passive manner (40). Given their 3ϩ net charge, they might be expected to target the mitochondrial matrix in a potential-driven manner. Surprisingly, our results revealed that the peptides are primarily associated with the IMM, and this is supported by their lack of effect on mitochondrial potential. By accumulating in the IMM, these peptides are localized to the site of ROS production. Treatment of cells with tBHP causes rapid oxidation of pyridine nucleotides and increased ROS production in mitochondria (17,50). The SS peptides were very potent in reducing intracellular ROS and preventing cell death after tBHP treatment, with EC 50 in the nM range. In contrast, most antioxidants require 100 M to millimolar concentrations to prevent oxidative cell death (50 -53). The triphenylalkylphosphonium ion-conjugated coenzyme Q was able to block H 2 O 2 induction of apoptotic cell death at 1 M, but Ͼ10 M caused cytotoxicity, consistent with the potential of these lipophilic cations to cause mitochondrial depolarization (31). The SS peptides are not toxic to cells even when present at 100 M, and this is consistent with their lack of effect on mitochondrial potential.
Oxidative stress induced by tBHP was shown to enhance mitochondrial Ca 2ϩ , leading to onset of MPT (17). Using iso- FIG. 9. SS peptides prevent reperfusion-associated myocardial stunning in the isolated perfused guinea pig heart. Hearts were subjected to 30 min of global ischemia followed by 90 min of reperfusion. Contractile force before onset of ischemia was 5.8 Ϯ 0.1 g. Contractile force was significantly reduced after 90-min reperfusion with buffer (B). Reperfusion with SS-02 or SS-31 significantly improved contractile force. Reperfusion with SS-20 did not protect against myocardial stunning. lated mitochondria, we showed that SS-02 and SS-31 can inhibit Ca 2ϩ -induced MPT and swelling and reduce cytochrome c release. On the other hand, the peptide analog that does not scavenge ROS (SS-20) was unable to protect against Ca 2ϩinduced mitochondrial swelling. These results support a major role for ROS in Ca 2ϩ -induced MPT and cytochrome c release. A direct interaction of these peptides with the MPT pore, as with cyclosporin A (54,55), seems unlikely because the protective action was not sequence-specific, and SS-31 was as effective as SS-02. Rather, our results support the proposal that ROS may mediate MPT via oxidation of the adenine nucleotide translocator (19 -21). The ability of SS peptides to prevent MPT will minimize MPT-induced ROS accumulation and further reduce oxidative damage on mitochondria (56).
Increased ROS production also plays a role in cell death caused by 3NP, an irreversible inhibitor of complex II of the respiratory chain. The production of ROS seems to be mainly from elevation of mitochondrial Ca 2ϩ , which can then lead to MPT, mitochondrial depolarization, and cell death (4,5). Cyclosporin A, an inhibitor of MPT, can prevent mitochondrial depolarization and neuronal death caused by 3NP (57). We were able to show that SS peptides can prevent mitochondrial depolarization in cells treated with 3NP, and studies in isolated mitochondria confirmed that SS-02 can inhibit MPT induced by 3NP. It was recently reported that some 3NP-induced behavioral abnormalities in mice were attenuated by pre-and posttreatment with vitamin E (5); however, this required rather large doses of vitamin E (20 mg/kg) that had to be given before 3NP exposure and is probably related to the poor bioavailability of the highly lipophilic vitamin E.
Reactive oxygen species and MPT have been postulated to play a major role in cardiac reperfusion injury (22,58). Despite timely reperfusion after transient ischemia, there is often prolonged depression of cardiac contractile function known as myocardial stunning. However, the clinical application of oxygen radical scavengers to the treatment of the stunned myocardium has been disappointing because of the limited ability of most antioxidants to penetrate cell membranes (59). Our results support the free radical theory behind myocardial stunning. Both SS-02 and SS-31 were able to prevent stunning in the ex vivo heart upon reperfusion after global ischemia. In contrast, SS-20, which has no antioxidant activity, was unable to protect the ischemic heart against reperfusion stunning. We had previously reported that SS-02 can prevent myocardial stunning even when administered only during reperfusion (42), consistent with antioxidation as the mechanism of action. The ability of SS-02 to prevent stunning has been confirmed in rats in vivo. 2 Thus SS-02 and SS-31 may benefit patients who have suffered myocardial ischemia and are undergoing reperfusion treatment.
In summary, we have designed cell-permeable peptide antioxidants that target the site of ROS generation and protect mitochondrial function. Our results demonstrate that ROS play a major role in mediating mitochondrial dysfunction induced by tBHP, Ca 2ϩ , and 3NP. These antioxidant peptides may be beneficial in the treatment of aging and diseases associated with oxidative damage such as ischemia-reperfusion injury and neurodegeneration.