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* This work was supported by Multi-Center Program Project Grant DA08924 from the National Institute on Drug Abuse, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ 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)
is a major intracellular source of reactive 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 (
). 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 (
). Mitochondrial Ca2+ is another powerful signal for ROS production. Calcium is taken up into mitochondria via a uniporter in the IMM, and elevation of mitochondrial Ca2+ and ROS production is thought to play an important part in cell death associated with ischemia-reperfusion as well as 3NP (
). 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 (
). 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) (
). Cardiolipin is rich in unsaturated fatty acids, and peroxidation of cardiolipin induces the dissociation of cytochrome c from mitochondria into the cytosol (
). However, the mechanism by which cytochrome c is released through the OMM is not clear. One mechanism may involve ROS-induced promotion of Ca2+-dependent mitochondrial permeability transition (MPT), with swelling of the mitochondrial matrix and rupture of the OMM (
), suggesting MPT-independent mechanisms. MPT-independent mechanisms may involve the voltage-dependent anion channel on the OMM or an oligomeric form of Bax (
Given the many ways by which cytochrome c may be released through the OMM, the most efficient approach to inhibit ROS-induced 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 μm 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 (
). 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 μm (
). 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 Ca2+ 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.
MATERIALS AND METHODS
Chemicals and Reagents—The SS peptides are tetrapeptides with alternating aromatic residues and basic amino acids. SS-02 (Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2′,6′-dimethyltyrosine), SS-20 (Phe-d-Arg-Phe-Lys-NH2), SS-31 (d-Arg-Dmt-Lys-Phe-NH2), and [3H]SS-02 were synthesized as described previously (
). A fluorescent analog (SS-19; Dmt-d-Arg-Phe-atnDap-NH2) containing β-anthraniloyl-l-α,β-diaminopropionic acid in place of the Lys4 residue in SS-02 was prepared for mitochondrial and cellular uptake studies (
). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Measurement of Antioxidant Properties of SS Peptides in Vitro—The ability of SS peptides to scavenge H2O2in vitro was determined using luminol chemiluminescence (
). H2O2 (4.4 nmol) was incubated with 1 to 100 μm of various peptides in 0.5 ml of phosphate buffer, pH 8.0, for 30 s. Luminol (25 μm) 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 2,2′-azobis(2-amidinopropane) and the formation of conjugated dienes was monitored spectrophotometrically at 234 nm (
). Freshly prepared human LDL (0.1 mg/ml in phosphate-buffered saline) was oxidized catalytically by the addition of 10 μm CuSO4, and the formation of conjugated dienes was monitored at 234 nm for 5 h at 37 °C (
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 × g, and the supernatant was centrifuged again at 13,800 × g for 10 min. The mitochondrial pellets were washed twice, centrifuged at 11,200 × 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 = 320/420 nm). For mitochondrial uptake of [3H]SS-02, mitochondria (0.8 mg) were suspended in buffer (70 mm sucrose, 230 mm mannitol, 3 mm HEPES, 5 mm succinate, 5 mm KH2PO4, and 0.5 μm rotenone, pH 7.4) containing [3H]SS-02 and 1 μm SS-02 at room temperature. Uptake was stopped by centrifugation (16,000 × 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 [3H]SS-02 were also determined in the presence of 1.5 μm 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 (
Cell Culture—Caco-2 cells (American Type Culture Collection, Manassas, VA) and N2A cells (provided by Dr. Gunnar Gouras, Department of Neurology, Weill Medical College of Cornell University) were cultured as described previously (
). Cells (106/well) were incubated with [3H]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 μm) for 15 min at 37 °C, and confocal laser scanning microscopy (CLSM) was carried out with living cells using a C-Apochromat 63×/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 = 550/575 nm) for 30 min at 37 °C and then examined by CLSM.
Intracellular ROS and Cell Viability—N2A cells were plated in 96-well plates at a density of 1 × 104/well and allowed to grow for 2 days before treatment with tBHP (0.5 or 1 mm) for 40 min. Cells were washed twice and replaced with medium alone or medium containing varying concentrations of SS-02 or SS-31 for 4 h. Intracellular ROS was measured by 5-(and 6)-carboxy-2′,7′-dichlorohydro-fluorescein diacetate (Molecular Probes). Cell death was assessed by a cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay; Promega, Madison, WI).
Intracellular Mitochondrial Potential—Caco-2 cells were treated with 3NP (10 mm) in the absence or presence of SS-02 (0.1 μm) for 4 h and then incubated with TMRM and examined under CLSM as described above.
Mitochondrial H2O2Production—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 μm luminol and 0.7 IU of horseradish peroxidase were added, and chemiluminescence was monitored continuously for 20 min at 37 °C. The amount of H2O2 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 KH2PO4, 5 mm MgCl2, 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 Membrane Potential—Mitochondrial potential was qualitatively assessed using TMRM fluorescence intensity (excitation/emission = 550/575 nm). Isolated mitochondria (0.3 mg) were added to 2.0 ml of buffer (70 mm sucrose, 230 mm mannitol, 3 mm HEPES, 2 mm Tris-phosphate, 5 mm succinate, and 1 μm rotenone) containing TMRM (0.4–2 μm), and potential was assessed by quenching of the fluorescent signal.
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 μm 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 Ca2+ to induce swelling. Swelling was measured by light scattering at 610 nm. Alamethicin (7 μg/ml) was added to induce maximal swelling, and the magnitude of swelling induced by Ca2+ 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 (
). 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.
RESULTS
Antioxidant Properties of SS Peptides—The antioxidant properties of SS peptides were demonstrated by their ability to scavenge H2O2 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 H2O2 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 Dmt1 by Phe1 (SS-20) eliminated antioxidant activity (Fig. 1, D and E).
Fig. 1In vitro assays showing antioxidant properties of SS peptides.A, SS-02 dose-dependently scavenges H2O2 as measured by luminol chemiluminescence. B, SS-02 dose-dependently inhibits linoleic acid peroxidation. Linoleic acid peroxidation was induced by 2,2′-azobis(2-amidinopropane) and detected by the formation of conjugated dienes measured by absorbance at 234 nm (A234). C, SS-02 dose-dependently inhibits LDL oxidation. Human LDL was oxidized by 10 μm CuSO4, and the formation of conjugated dienes was monitored at A234. D, comparison of different SS peptides (100 μm) in slowing the rate of linoleic acid oxidation. E, comparison of different SS peptides (100 μm) in slowing the rate of LDL oxidation.
Cellular Uptake of SS-02—To demonstrate that the SS peptides are cell-permeable, we incubated Caco-2 cells with [3H]SS-02 at 37 °C for 60 min and measured the amount of radioactivity in cell lysate. [3H]SS-02 was readily taken up into Caco-2 cells. The amount of [3H]SS-02 in cell lysate and media averaged 6152 ± 128 cpm and 229,622 ± 2199, respectively (mean ± S.E.; n = 6). Based on a cell volume of ∼3.3 μl/mg protein (
) and 200 μl of media, the intracellular concentration of [3H]SS-02 can be estimated to be 9.8 ± 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.
Fig. 2Internalization and targeting of fluorescent SS-19 to mitochondria in living cells. Caco-2 cells were incubated with SS-19 (0.1 μm) and TMRM (20 nm)at37 °C for 30 min. CLSM was carried out with excitation/emission set at 320/420 nm for SS-19 and 550/575 nm for TMRM. Left, green-fluorescent SS-19; middle, red-fluorescent TMRM; right, overlay of the two images shows co-localization of SS-19 and TMRM.
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 [3H]SS-02 and determined radioactivity in the mitochondrial pellet. Uptake of [3H]SS-02 by mitochondria was rapid with maximal levels reached by 2 min (Fig. 3B). Radioactivity averaged 67,021 ± 2008 cpm in the mitochondrial pellet, and 128,131 ± 2015 cpm in the supernatant (n = 3). Assuming mitochondrial volume of 1 μl/mg protein (
), it can be estimated that [3H]SS-02 accumulates 104.6 ± 1.6-fold in mitochondria.
Fig. 3Uptake and localization of SS peptides in isolated mouse liver mitochondria.A, mitochondrial uptake of SS-19. Addition of isolated mouse liver mitochondria (0.35 mg/ml) resulted in immediate quenching of SS-19 fluorescence intensity (gray line). Pretreatment of mitochondria with FCCP (1.5 μm) reduced quenching by <20% (black line). B, mitochondrial uptake of SS-02. Isolated mitochondria were incubated with [3H]SS-02 and 1 μm SS-02 at 37 °C for 2 min, and radioactivity was determined in the mitochondrial pellet. Pretreatment of mitochondria with FCCP inhibited SS-02 uptake by ∼20%. Data are shown as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test. C, targeting of SS-02 to mitochondrial membranes. Isolated mitochondria were incubated with [3H]SS-02 at 37 °C for 2 min, and the mitochondrial suspension was subjected to either three cycles of freeze-thaw treatment (F/T) or treatment with 0.2% digitonin (Dig) before determination of radioactivity in the pellet. Radiolabel uptake is expressed as percentage of total radioactivity detected in intact mitochondrial pellet (Int).
Pretreatment of mitochondria with FCCP only reduced SS-19 quenching or [3H]SS-02 uptake by ∼20% (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 [3H]SS-02 for 5 min and the mitochondrial pellet was subjected to three freeze-thaw cycles, 72% of [3H]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 N2A 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) and increased cell survival (Fig. 4, B and C), with EC50 in the nanomolar range.
Fig. 4SS peptides reduce intracellular ROS and prevent cell death caused by tBHP in N2A cells. N2A cells were treated with tBHP (0.5 or 1 mm) for 40 min. Cells were then washed twice and replaced with medium alone or medium containing SS-02 or SS-31 for 4 h. A, effect of SS-31 on intracellular ROS after 0.5 mmtBHP. B, effect of SS-31 on cell survival after 0.5 mmtBHP. C, effect of SS-02 on cell survival after 1 mmtBHP.
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 μm 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).
Fig. 5SS-02 protects against mitochondrial depolarization induced by 3NP in Caco-2 cells. Caco-2 cells were treated with 3NP (10 mm) in the absence or presence of SS-02 (0.1 μm) for 4 h and then incubated with TMRM and examined under CLSM.
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 H2O2 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).
Fig. 6SS peptides inhibit ROS production in isolated mitochondria.A, pretreatment of isolated mouse liver mitochondria with SS-31 dose-dependently inhibited spontaneous generation of H2O2 as measured by luminol chemiluminescence. AUC, area under the curve over 15 min. B, pretreatment with SS-02 inhibited both spontaneous and antimycin-induced H2O2 production. Data are presented as mean ± S.E. (n = 3).
Effects of SS Peptides on Mitochondrial Function in Isolated Mitochondria—The accumulation of SS-02, SS-19, or SS-31 in mitochondria did not alter mitochondrial function. Incubating mouse liver mitochondria with 100 μm SS-02, SS-19, or SS-31 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 μm (Fig. 7A). In contrast, the addition of FCCP caused immediate depolarization, and Ca2+ overload (150 μm) resulted in progressive loss of mitochondrial potential, indicative of MPT (Fig. 7A).
Fig. 7SS peptides protect against MPT induced by Ca2+ and 3NP in isolated mitochondria.A, addition of SS-02 (100 μm) to isolated mitochondria did not alter mitochondrial potential, as measured by TMRM fluorescence. Addition of FCCP (1.5 μm) caused immediate depolarization, whereas Ca2+ (150 μm)resulted in depolarization and progressive onset of MPT. B, pretreatment of isolated mitochondria with 100 μm SS-02 (down arrow) prevented onset of MPT caused by Ca2+ (up arrow). C, pretreatment with 100 μm SS-31 (down arrow) also prevented onset of MPT caused by Ca2+ (up arrow). D, SS-02 dose-dependently delayed the onset of MPT caused by 1 mm 3NP. Arrow indicates addition of buffer or SS-02. Line 1, buffer; line 2, 0.5 μm SS-02; line 3, 5 μm SS-02; line 4, 50 μm SS-02.
SS Peptides Protect against MPT in Isolated Mitochondria— Pretreatment of isolated mitochondria with 10 μm SS-02 or SS-31 before addition of Ca2+ 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 Ca2+- 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 Ca2+-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). Ca2+ 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).
Fig. 8SS peptides inhibit mitochondrial swelling and cytochrome c release in isolated mitochondria.A, pretreatment of isolated mitochondria with SS-02 inhibited mitochondrial swelling induced by 50 μm Ca2+ in a dose-dependent manner. Swelling was measured by absorbance at 540 nm. B, pretreatment of isolated mitochondria with SS-31, but not SS-20, prevented mitochondrial swelling induced by Ca2+. C, SS-02 (100 μm) inhibited Ca2+-induced swelling and release of cytochrome c from isolated mitochondria. Swelling was determined by light scattering at 610 nm, and alamethicin was used to induce maximal swelling. The amount of cytochrome c released was expressed as percentage of total cytochrome c in mitochondria. Data are presented as mean ± S.E. (n = 3).
SS Peptides Protect against Ischemia/Reperfusion-induced Contractile Dysfunction in the Isolated Perfused Heart—Reperfusion of the isolated guinea pig heart after 30 min of global ischemia is associated with progressive loss of contractile force (
). After 90 min of reperfusion, contractile force was 1.3 ± 0.3 g compared with 5.8 ± 0.1 g before ischemia. Perfusion of the heart with the antioxidant peptides SS-02 (100 μm) 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.
Fig. 9SS 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 ± 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.
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 Ca2+-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 (
). 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 Dmt1 in SS-02 with Phe1 (SS-20) resulted in complete loss of antioxidant activity.
Unlike other antioxidants, the SS peptides are water-soluble and readily penetrate cell membranes in a passive manner (
). Given their 3+ 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 (
). The SS peptides were very potent in reducing intracellular ROS and preventing cell death after tBHP treatment, with EC50 in the nm range. In contrast, most antioxidants require 100 μm to millimolar concentrations to prevent oxidative cell death (
). The triphenylalkylphosphonium ion-conjugated coenzyme Q was able to block H2O2 induction of apoptotic cell death at 1 μm, but >10 μm caused cytotoxicity, consistent with the potential of these lipophilic cations to cause mitochondrial depolarization (
). Using isolated mitochondria, we showed that SS-02 and SS-31 can inhibit Ca2+-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 Ca2+-induced mitochondrial swelling. These results support a major role for ROS in Ca2+-induced MPT and cytochrome c release. A direct interaction of these peptides with the MPT pore, as with cyclosporin A (
), 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 (
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 Ca2+, which can then lead to MPT, mitochondrial depolarization, and cell death (
). 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 post-treatment with vitamin E (
); 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 (
). 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 (
). 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 (
W. Song, M. K. Hong, and H. H. Szeto, unpublished results.
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, Ca2+, 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.
Acknowledgments
We thank Guoxiong Luo and Xuanxuan Qian for excellent technical assistance. We thank Dr. Lee Cohen-Gould for invaluable advice with the confocal fluorescent microscopic studies, and Dr. Anatoly Starkov and Dr. Patrick Sullivan for guidance with the mitochondria studies. Human LDL was kindly provided by Dr. Jihong Han (Department of Pathology, Weill Medical College of Cornell University). The N2A cells were provided by Dr. Gunnar Gouras (Department of Neurology, Weill Medical College of Cornell University). The fluorescent microscopic studies were carried out in the Molecular Cytology Core Facility at the Memorial Sloan-Kettering Cancer Center and the Optical Microscopy Core Facility at Weill Medical College of Cornell University.
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