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Am J Physiol Heart Circ Physiol 281: H1422-H1432, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 3, H1422-H1432, September 2001

Knockout mice heterozygous for Sod2 show alterations in cardiac mitochondrial function and apoptosis

Holly Van Remmen1,2,*, Melissa D. Williams1,*, Zhongmao Guo1, Larry Estlack1, Hong Yang1, Elaine J. Carlson4, Charles J. Epstein4, Ting Ting Huang4, and Arlan Richardson1,2,3

1 Department of Physiology, University of Texas Health Science Center at San Antonio; 2 Geriatric Research, Education and Clinical Center, Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, Texas 78284; 3 Sam and Ann Barshop Center for Longevity and Aging Studies and 4 Department of Pediatrics, University of California, San Francisco, California 94143


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heart mitochondria from heterozygous (Sod2-/+) knockout mice have a 50% reduction in manganese superoxide dismutase (MnSOD) activity. The decrease in MnSOD activity was associated with increased mitochondrial oxidative damage as demonstrated by a decrease in the activities of iron sulfhydryl proteins sensitive to oxygen stress (aconitase and reduced nicotinamide adenine dinucleotide-oxidoreductase). Mitochondrial function was altered in the Sod2-/+ mice, as shown by decreased respiration by complex I and an increase in the sensitivity of the permeability transition to induction by calcium and t-butylhydroperoxide. The increased induction of the permeability transition in heart mitochondria from Sod2-/+.mice was associated with increased release of cytochrome c and an increase in DNA fragmentation. Cardiomyocytes isolated from neonatal Sod2-/+ and Sod2-/- mice were more sensitive to cell death than cardiomyocytes from Sod2+/+ mice after t-butylhydroperoxide treatment, and this increased sensitivity was prevented by inhibiting the permeability transition with cyclosporin A. These experiments demonstrate that MnSOD may play an important role in the induction of the mitochondrial pathway of apoptosis in the heart, and this appears to occur primarily through the permeability transition.

permeability transition; oxidative stress


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A COMPLEX ANTIOXIDANT DEFENSE SYSTEM that includes antioxidants, antioxidant enzymes, and a variety of pathways to repair oxidative damage to macromolecules has evolved to protect cells from oxidative stress. The major antioxidant enzymes found in eukaryotes are the superoxide dismutases (SOD), the glutathione peroxidases, and catalase (23). Three different SODs are found in mammals, and all SODs catalyze the dismutation of superoxide anions to hydrogen peroxide (23). Cytosolic copper/zinc SOD (Cu/ZnSOD), is a homodimer coded for by the Sod1 gene, and is the predominant SOD in most cells and tissues (70-80% of cellular SOD activity) (23). Extracellular SOD, a homotetramer, is a minor SOD coded for by the Sod3 gene and is only expressed in significant amounts in a limited number of tissues (lung, kidney, and fat tissue) (51). Manganese SOD (MnSOD), coded for by the Sod2 gene, is also a homotetramer. MnSOD is located in the mitochondrial matrix and contributes 10-20% of the total SOD activity in the cell (23). The presence of MnSOD at the matrix side on the inner mitochondrial membrane allows the ready dissipation of superoxide anions produced as a result of the Q1 semiubiquinone of complex III in the electron transport chain (53). Superoxide anions are charged molecules and thus do not readily cross membranes (20); therefore, if not destroyed, they can directly (or indirectly by formation of other reactive oxygen species) result in oxidative damage to molecules in the mitochondria. Thus the unique cellular location of MnSOD contributes to its ability to play a critical role in protecting the mitochondria from oxidant stress by enzymatically scavenging superoxide anions that are produced as a byproduct of the respiratory chain.

The physiological importance of MnSOD was demonstrated when mice with homozygous null mutations in the Sod2 gene were produced. Mice lacking MnSOD die within 1-18 days from dilated cardiomyopathy or neurodegeneration, depending on the genetic background (30, 38). In contrast, homozygous mutations in the other genes coding for the major antioxidant enzymes; e.g., Sod1, Sod3, and glutathione peroxidase 1 (GPx1), are not lethal, and mice with these null mutations appear normal except for an increased sensitivity to certain types of oxidative stress (5, 12, 27, 28, 57).

A variety of agents that produce a prooxidant environment in the cells have been shown to regulate MnSOD expression at the level of transcription, e.g., ionizing radiation (49), paraquat, (55), thiol-modulating agents (11), and peroxynitrite (32). MnSOD expression is also induced by proinflammatory mediators such as bacterial lipopolysaccharide (8), tumor necrosis factor-alpha (55, 65), interleukin-1 (48, 55), and gamma -interferon (26). Protein kinase activators such as phorbol esters can also induce MnSOD at the level of transcription (63). Thus, in contrast to the other major antioxidant enzymes, e.g., Cu/ZnSOD, catalase, and glutathione peroxidase, the expression of MnSOD is modulated by a variety of physiological and environmental factors, suggesting that MnSOD may play a role in physiological processes other than the antioxidant defense system. This concept is further supported by the reports showing that the overexpression of MnSOD suppresses tumorgenicity in human melanoma cells (7), breast cancer cells (39), and glioma cells (67).

A relationship between MnSOD activity and programmed cell death has also been suggested (16, 33, 43). Programmed cell death or apoptosis is an important cellular mechanism that serves to remove unwanted, damaged, or unnecessary cells. Apoptosis can be initiated by a variety of factors, but in general there are two main pathways by which the process of cell death is executed: an extrinsic pathway initiated by cell surface receptors and an intrinsic pathway involving the mitochondria and the mitochondrial permeability transition. The mitochondrial pathway of apoptosis is induced by factors such as calcium, Bax, and reactive oxygen species (19). These factors can also induce the permeability transition, thus implicating the permeability transition as an important regulator of apoptosis (19). The activation of the mitochondrial membrane permeability transition is believed to result in the release of cytochrome c from the mitochondria and interaction among cytochrome c, Apaf 1, and procaspase 9 in a complex termed the apoptosome, leading to activation of downstream caspases and cell death (37, 40).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and treatments. The Sod2-/+ mice, designated Sod2< tm1> Cje, were originally produced in the CD1 strain of mice (41); however, the mice described in this study have been backcrossed to C57BL/6J mice for 14 generations (B6-Sod2< tm1> Cje). The genotype of the Sod2-/+ mice was determined by PCR analysis as described (41). Female mice were fed ad libitum and maintained under barrier conditions on a 12:12-h dark/light cycle. At 2 to 4 mo of age, the mice were euthanized by cervical dislocation to allow rapid isolation of mitochondria without the interference of drugs, and the hearts were immediately excised and placed on ice. We found that this procedure allowed us to obtain the highest quality mitochondria and most reproducible results for the mitochondrial respiration studies. For measurement of the mitochondrial permeability transition, antioxidant status, and mitochondrial respiration, hearts isolated from two to four mice were pooled before mitochondrial isolation. All procedures involving the mice were in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio and the Audie L. Murphy Veterans Hospital.

Isolation of heart mitochondria. Mitochondria were isolated according to the method of Mela and Seitz (46). The hearts were homogenized in ice-cold homogenization buffer composed of (in mM) 225 mannitol, 75 sucrose, 1 EGTA, and 10 HEPES (pH 7.2) and 0.02% nagarase, with the use of a Potter-Elvehjem glass homogenizer and a Teflon pestle. After homogenization, 0.5% bovine serum albumin was added to the homogenate, and the homogenate was centrifuged at 1,000 g for 5 min to remove cell debris. The resulting supernatant was centrifuged at 10,000 g for 5 min to isolate the mitochondrial pellet. The supernatant was centrifuged again at 100,000 g for 15 min to obtain the cytosolic fraction.

The mitochondrial pellet was resuspended in homogenization buffer without EGTA and centrifuged at 10,000 g for 5 min. This washing procedure was repeated twice and the final mitochondrial pellet was resuspended in homogenization buffer (without EGTA). For enzyme assays, the mitochondria were resuspended in 50 mM Tris · HCl (pH 7.4), 1 mM EDTA, and 0.1% (vol/vol) Triton X-100. Mitochondrial preparations that were used to measure aconitase activity were supplemented with 1 mM sodium citrate to stabilize the iron-sulfur clusters in the enzyme (25, 34). Protein concentrations were determined by using the Bradford assay kit from Bio-Rad (Richmond, CA).

Mitochondrial antioxidant enzyme activities. SOD and glutathione peroxidase activity were measured as described previously (61, 64). SOD activity was measured by using activity gels. Activity gels allow measurement of MnSOD activity distinct from the activity of Cu/ZnSOD based on the difference in electrophoretic mobility. After being stained, the gel was photographed, and the photograph was scanned and quantitated using ImageQuant software. Glutathione peroxidase activity was measured by the coupled reduction of cumene hydroperoxide to the oxidation of NADPH by glutathione reductase. One unit of activity is defined as 1 µmol of NADPH oxidized per minute.

Total glutathione. The concentration of total glutathione in isolated mitochondria was determined by the rate of formation of 5-thio-2-nitrobenzoic acid as described previously (64). The concentration of glutathione was determined by comparison to a standard curve generated by using appropriate concentrations of purified reduced glutathione.

Mitochondrial and cytosolic enzyme activities. Activities of aconitase, fumarase, reduced nicotinamide adenine dinucleotide (NADH):coenzyme Q (CoQ) reductase, and glutamine synthetase were measured as described previously (64). The activity of aconitase was measured in the mitochondrial and cytosolic fractions by the conversion of citrate to alpha -ketoglutarate coupled to the reduction of NADPH. One milliunit of aconitase activity is defined as the amount of enzyme necessary to catalyze the formation of 1 nmol of isocitrate per minute at 37°C. Fumarase and NADH:CoQ reductase activities were measured in the mitochondrial fraction. Fumarase activity was measured by the conversion of fumarate to L-malate. One unit of activity is defined as the production of 1 µmol of fumarate per minute. NADH:CoQ reductase activity was determined by measuring the reduction of CoQ1, an ubiquinone analogue, by NADH. One unit of activity is defined as the oxidation of 1 µmol NADH per minute. Glutamine synthetase activity was measured in the cytosol by the conversion of L-glutamine to gamma -glutamyl hydroxylamate. One unit of activity is defined as the formation of 1 µmol of gamma -glutamyl hydroxylamate per minute.

Mitochondrial respiration. Oxygen consumption was measured using a Gilson oxygraph equipped with a Clark electrode as described by Estabrook (13). Experiments were conducted at 30°C in a 2-ml chamber containing 500 µg of mitochondrial protein in a respiration buffer composed of (in mM) 250 sucrose, 10 KH2PO4, 1 EGTA, and 10 Tris · HCl (pH 7.4), containing different substrates. Respiration rates were measured using substrates that enter the electron transport chain selectively at the following specific complexes: for complex I, glutamate (1.7 mM) and malate (1.7 mM); for complex II, succinate (2.5 mM) with an NADH dehydrogenase inhibitor (5 µg/ml rotenone); and for complex III, duroquinol (500 µM). State 3 respiration was determined by the addition of ADP (375 nM final concentration) to the above reactions. State 4 respiration was defined as oxygen consumption in the presence of adequate substrate but without added ADP. The respiratory control ratio (RCR) was calculated as the ratio of state 3 to state 4 respiration rates. The activity of cytochrome oxidase, which represents the activity of complex IV, was measured with a Clark electrode (24) using 500 µg of mitochondrial protein in the following buffer: 50 mM potassium phosphate (pH 7.4), 40 µM cytochrome c, 12.5 mM ascorbate, 0.63 mM N,N,N',N'tetra-methyl-p-phenylenediamine, and 0.03% Triton X-100.

Mitochondrial permeability transition. The mitochondrial permeability transition is characterized by a sudden increase in the permeability of the mitochondrial inner membrane to small ions and molecules, which can result in the collapse of mitochondrial membrane potential (Psi m). As a consequence of the induction of the permeability transition, matrix components are lost from the mitochondria and the mitochondria swell. The induction of the permeability transition can be measured by following the decrease in mitochondrial absorbance at 540 nm as described by Kristal et al. (36). Mitochondrial protein (1 mg) was resuspended in 1 ml of buffer [215 mM mannitol, 71 mM sucrose, 3 mM HEPES (pH 7.4), and 5 mM succinate] at 25°C and was allowed to equilibrate for 30 s. The following were added to the buffer to induce the transition: 400 µM calcium chloride or 400 µM calcium chloride, pH 7.4, and 75 µM t-butylhydroperoxide (t-BuOOH). The decrease in absorbance was followed until the absorbance value was stabilized. The time in seconds at which one-half of the absorbance (t1/2) is lost was used to measure the induction of the permeability transition, as we have described previously (64).

Cell-free system of apoptosis. Mitochondrial and cytosolic fractions were isolated as described above. Mitochondrial protein (500 µg) isolated from two hearts from wild-type and Sod2-/+ mice were added to 1 ml of cytosol isolated from the heart of the wild-type mice. CaCl2 (400 µM) and t-BuOOH (75 µM) were added to induce the permeability transition. In some experiments, the mitochondria were preincubated in the presence of 2 µM cyclosporin A, an inhibitor of the permeability transition, for 15 min before the addition of the CaCl2 and t-BuOOH. After 5 or 15 min, the mitochondria and cytosol were separated by centrifugation at 16,000 g for 2 min. The supernatant was concentrated with the use of Centricon-3 centrifugal microconcentrators (Amicon; Beverly, MA), which retain molecules >3 kDa. The concentrated supernatants were analyzed by Western blots for the release of cytochrome c from the mitochondria using an antibody purchased from Santa Cruz Biotechnology (Santa Cruz, CA) (16). The intensity of the bands corresponding to cytochrome c was quantitated using ImageQuant software (Molecular Dynamics).

The concentrated supernatant was used to induce DNA fragmentation in wild-type nuclei. Liver nuclei were isolated from wild-type mice essentially as described by Scarlett et al. (56). Forty micrograms of supernatent protein were incubated with equal aliquots of wild-type nuclei, 3 µl of 20 mM dATP, and 6 µl of 20 mM MgCl2 for periods of time varying from 5 min to 1 h. DNA was extracted from the nuclei by adding 500 µl of DNA extraction buffer composed of (in mM) 100 Tris · HCl (pH 8), 5 EDTA, and 0.2 NaCl and 0.2% sodium dodecyl sulfate containing proteinase K (200 µg/ml), and incubated at 37°C overnight. NaCl was added to 1.5 M final concentration to precipitate proteins and the mixture was spun at 1,600 g for 15 min. DNA was isolated by phenol chloroform extraction, washed in cold 70% ethanol, and resuspended in water containing 200 µg/ml RNaseA and incubated at 37°C for 1 h. The extent of DNA fragmentation was visualized using a 2% agarose gel containing ethidium bromide.

Cardiomyocytes. Because of the prenatal lethality of Sod2-/- mice on C57BL/6J (B6) background, it was necessary to cross B6 Sod2-/+ mice to DBA/2J mice to obtain B6D2F2 for deriving cardiomyocytes from Sod2+/+, Sod2-/+, and Sod2-/- mice. In the B6D2F2 background, Sod2-/- mice have an average lifespan of 11 days (30). In contrast, Sod2-/- in the B6 background are resorbed in utero and those that are born survive only ~1.5 days (30). Mouse cardiomyocytes were isolated from 1- to-3-day-old Sod2-/+ and wild-type mouse pups by a modification of the procedure described by Wang et al. (62). Hearts were removed and dissociated at 37°C for 15 min using 0.25% (wt/vol) trypsin in Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+, pH 7.4. Cells released after the first digestion were discarded, and cells from subsequent digestion were added to an equal volume of cold HBSS with Ca2+ and Mg2+ containing 1g/l D-glucose, until all tissue was dissociated (~5×). Cells were obtained as a pellet after centrifugation at 300 g for 8 min and resuspended in Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12) (50/50) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). To exclude nonmuscle cells, the cells were preplated in tissue culture dishes at 37°C with 5% CO2 for 2 h. The suspended cells were then collected and plated at a density of 1.0 × 105 cells/cm2 in the same medium and under the same conditions as above. After 24 h in culture, the medium was discarded and the cells were grown in DMEM/F-12 (50/50), supplemented with 2% fetal bovine serum and 0.1 mM bromodeoxyuridine (to prevent proliferation of nonmyocytes) for 48-72 h before treatment with t-BuOOH. To verify that the cultures were not contaminated with noncardiomyocytes, the cells were stained with Coomassie blue, which stains the cell cytoskeleton, as described by Ulrich et al. (60). This staining method allows discrimination between the cardiomyocytes, which contain myofibrils, and the nonmuscle cells (endothelial cells and fibroblasts), which do not. With the use of this method, >95% of the cells were determined to be cardiomyocytes (data not shown).

Cardiomyocytes were cultured in 6-well plates and treated with 200 µM t-BuOOH for 2 h at 37°C with 5% CO2. In some experiments, cells were pretreated for 20 min with 200 µM manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP), a SOD mimetic, or pretreated for 30 min with 5 µM cyclosporin A-50 µM aristocholic acid to inhibit the permeability transition before the addition of t-BuOOH. Cell viability was determined using neutral red as described by Fauzt et al. (15). After treatment with 200 µM t-BuOOH for 2 h, the cells were washed twice with 1× phosphate-buffered saline (PBS) at 37°C to remove any excess t-BuOOH followed by the addition of 2 ml neutral red medium (50 µg/ml neutral red in DMEM) to the cells. Cell cultures are then incubated for 3 h at 37°C with 5% CO2 to allow the lysosomes of the viable cells to take up the dye. The cells are then washed two times with PBS to eliminate extracellular neutral red. The dye incorporated into the viable cells was eluted from the cells by adding 2 ml of elution medium (50% ethanol-1% acetic acid) into each well, followed by gentle shaking for 10 min. The absorbance of the elution medium was read at 540 nm.

Mitochondrial energization. We measured changes in the Psi m using the dye DiOC6(3), which localizes to the mitochondria as a result of Psi m. The retention of DiOC6(3) (Molecular Probes; Eugene, OR) was measured in cardiomyocytes that had been exposed to 200 µM t-BuOOH for 2 h in the presence or absence of cyclosporin A (52). Cells were pretreated with 100 nM DiOC6(3) during the last 30 min of treatment. At the end of treatment, the cells were pelleted at 700 g for 10 min, the supernatant was removed, and the cells were resuspended in PBS and washed twice. The pellet was lysed in 600 µl of water and homogenized. Retention of DiOC6(3) was measured using a Perkin-Elmer Fluorescence Spectrophotometer at 488-nm excitation and 500-nm emission and the data are expressed as the percentage of dye retained.

Single cell gel electrophoresis (Comet Assay). DNA double-strand breaks were determined using a Comet Assay as described by Olive et al. (50) with slight modifications. Cultured cardiomyocytes (untreated or after 2 h in the presence of 200 µM t-BuOOH) were mixed with 0.5% low-melting agarose and the mixture was layered on a microscope slide. Cells were lysed by immersing the slide in 2.5 M sodium chloride, 0.1 M EDTA, 0.1 M Tris · HCl, pH 10, 10% dimethyl sulfoxide, and 1% Triton X-100 at 4°C for 2 h. The slides were subjected to electrophoresis at 30 V in 1× Tris-borate-EDTA buffer for 10 min. Slides were stained with SYBR green (Molecular Probes; Eugene, OR). The fluorescence images of cells were viewed using a fluorescence microscope (100-W Hg lamp, Nikon) and analyzed using the Komet 4.0 SCG Image Analysis System (Integrated Laboratory System). One hundred cells were analyzed from each slide, and one to three slides were examined for each sample. Cells that possess no or few DNA strand breaks show DNA fluorescence confined to nuclei (Fig. 3A). In cells with DNA strand breaks, a fluorescence tail along the electric field is formed due to small DNA fragments migrating from the nuclei (Fig. 3B). Cells with a tail moment (tail length × fraction of total DNA in the cell) >30 were designated to have significant strand breakage indicative of DNA fragmentation during apoptosis. Electrophoresis was performed under neutral pH, and only cells with DNA double-strand breaks showed tails.

Statistical analysis. Data are expressed as means ± SE. Differences between values obtained from wild-type and Sod2-/+ mice were determined using Student's t-test or two-way analysis of variance using the Tukey-Kramer method for multiple comparisons where appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The characterization of the antioxidant status of mitochondria isolated from hearts of Sod2+/+ and Sod2-/+ mice is shown in Table 1. There were no discernable differences in mitochondrial yield between Sod2+/+ and Sod2-/+ mice, e.g., 1.7 ± 0.2 mg of mitochondrial protein/heart for the Sod2+/+ mice compared with 1.6 ± 0.2 mg of mitochondrial protein/heart for the Sod2-/+ mice (data expressed as means ± SE for 6 animals). The activity of MnSOD was ~50% lower in mitochondria isolated from hearts of Sod2-/+ compared with Sod2+/+ mice. We found no difference in the activities of either glutathione peroxidase or CuZnSOD in mitochondria isolated from hearts of Sod2-/+ and Sod2+/+ mice. We also measured the mitochondrial levels of the antioxidant glutathione. Total mitochondrial glutathione levels were similar in the Sod2+/+ and Sod2-/+ mice (Table 1).

                              
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  		Table 1.   Mitochondrial enzyme activities and glutathione levels

To determine if the lower MnSOD activity in the Sod2-/+ mice resulted in increased oxidative damage, we measured the activities of aconitase and NADH-oxidoreductase because these enzymes have been shown to be sensitive to oxidative stress (18, 47). Table 1 shows that mitochondrial aconitase activity is reduced 35% in mitochondrial extracts from the hearts of Sod2-/+ mice compared with Sod2+/+ mice. The activity of NADH-oxidoreductase using CoQ as the substrate was reduced by 45% in the mitochondria isolated from the hearts of the Sod2-/+ mice compared with Sod2+/+ mice (Table 1). However, the activity of fumarase, a mitochondrial enzyme that is functionally similar to aconitase but insensitive to oxidative damage, was similar in mitochondria isolated from the hearts of Sod2-/+ compared with Sod2+/+ mice.

To determine whether the increase in oxidative stress evident in the mitochondria extended to the cytosolic compartment as well, we measured the activity of aconitase and glutamine synthetase in the cytosol of the hearts from Sod2-/+ and Sod2+/+ mice because these enzymes have been shown to be sensitive to oxidative stress (2, 58). We found no difference in the activity of either cytosolic aconitase or glutamine synthetase in the hearts of Sod2-/+ and Sod2+/+ mice.

Mitochondrial function was compared in mitochondria isolated from the hearts of Sod2-/+ and Sod2+/+ mice by measuring mitochondrial respiration and induction of the permeability transition. The rates of state 3 respiration, state 4 respiration, and the respiratory control ratio (RCR) measured with complex-specific substrates are shown in Table 2. We found a 37% decrease in RCR for complex I by using the substrates glutamate and malate in the heart mitochondrial preparations from Sod2-/+ compared with Sod2+/+ mice. State 3 respiration was also decreased 37% using the substrates glutamate and malate in mitochondria isolated from Sod2-/+ mice. State 4 respiration rates are essentially the same for mitochondria isolated from the hearts of the Sod2-/+ and Sod2+/+ mice, suggesting no differential leakiness in the inner mitochondrial membrane (24). This observation was confirmed by measuring the membrane potential in mitochondria isolated from Sod2-/+ and Sod2+/+ mice using Safranine O as described by Akerman and Wikstrom (1). The membrane potentials for mitochondria isolated from the hearts of Sod2+/+ and Sod2-/+ mice were -155.7 ± 4.2 and -159.4 ± 3.6 mV, respectively (data expressed as means ± SE from 6 experiments pooling mitochondria from 4 hearts).

                              
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  		Table 2.   Oxygen consumption by isolated heart mitochondria

The induction of the permeability transition was also measured in heart mitochondria isolated from the Sod2-/+ and Sod2+/+ mice. Figure 1A shows the data on the induction of the permeability transition by calcium and t-BuOOH in isolated heart mitochondria from Sod2-/+ and Sod2+/+ mice. The t1/2 of induction of the permeability transition by calcium was significantly lower (32%) in mitochondria isolated from the hearts of Sod2-/+ compared with Sod2+/+ mice, indicating that the rate of the induction of the permeability transition is greater for mitochondria isolated from Sod2-/+ mice. The addition of both t-BuOOH and calcium increased the rate of induction of the permeability transition in mitochondria isolated from both Sod2+/+ and Sod2-/+ mice as shown by the decrease in the t1/2. More importantly, the rate of induction of the permeability transition with both calcium and t-BuOOH for mitochondria isolated from hearts of Sod2-/+ mice was greater than that observed with Sod2+/+ mice as shown by the significant decrease (36%) in the t1/2.


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  		Fig. 1.   Induction of the permeability transition and release of apoptogenic factors by isolated heart mitochondria. A: induction, or time in seconds of absorbance loss (t1/2), of the permeability transition in heart mitochondria isolated from Sod2+/+ (solid bars) and Sod2-/+ (open bars) mice incubated with 400 µM calcium or 400 µM calcium and 75 µM t-butylhydroperoxide (t-BuOOH). Each value represents means ± SE of 6 different experiments, in which mitochondria were pooled from 4 animals per experiment. B: laddering of DNA isolated from nuclei treated with extracts of proteins released from wild-type or Sod2-/+ mitochondria following induction of the permeability transition. Data are representative of 4 separate experiments using mitochondria isolated from 2 hearts pooled together from each group for each experiment. C: level of cytochrome c present in the supernatent fraction 5 or 15 min after the induction of the permeability transition in the isolated mitochondria. Representative Western blot (right) from a single experiment measuring cytochrome c in the supernatent is shown. Data are means ± SE for experiments from 5 animals per group. *P < 0.05, difference between wild-type and Sod2+/- mice.

Because the permeability transition may play a role in the mitochondrial pathway of apoptosis (44, 66), we examined whether the increased induction of the permeability transition in mitochondria from the Sod2-/+ mice would also result in an increase in the induction of apoptosis in response to oxidative stress. First, we used a cell-free system, in which heart mitochondria isolated from Sod2-/+ and Sod2+/+ mice were incubated with cytosol from the hearts of Sod2+/+ mice, and the permeability transition was induced by the addition of 400 µM calcium and 75 µM t-BuOOH. The apoptotic activities of the protein extracts released from the mitochondria of Sod2+/+ and Sod2-/+ mice were compared by incubating the extracts with aliquots of liver nuclei isolated from Sod2+/+ mice. As shown in Fig. 1B, the extent of DNA fragmentation is greater in nuclei incubated in the presence of extracts that were obtained from mitochondria from Sod2-/+ mice. No DNA fragmentation was observed when the nuclei were incubated with cytosolic protein extracts from Sod2+/+ mice without the addition of mitochondria or when nuclei were incubated without the addition of extract. The amount of DNA fragmentation was dramatically reduced in the presence of cyclosporin A, an inhibitor of the permeability transition (4, 21). Because cytochrome c release from the mitochondria is a well-established event in the induction of the apoptotic process (42), we also measured the levels of cytochrome c released by the mitochondria into the cytosolic extracts using Western blots (Fig. 1C). The level of cytochrome c present in the cytosolic extracts after the induction of the mitochondrial permeability transition was higher for cytosolic extracts incubated with heart mitochondria isolated from Sod2-/+ mice.

We next wanted to determine if the cells from mice with reduced MnSOD activity were more prone to undergo apoptosis in response to oxidative stress. Cardiomyocytes isolated from Sod2-/+, Sod2-/-, and Sod2+/+ neonatal mice were treated with t-BuOOH, and cell viability was measured after 2 h to determine the extent of cell death induced by this treatment. Some cultures were pretreated with cyclosporin A and aristocholic acid (which prolongs the effect of cyclosporin A) to prevent the induction of the permeability transition before adding t-BuOOH to determine if changes in the viability of the cells to oxidative stress are due to alterations in the permeability transition. The data in Fig. 2A show that cell death was >20% in cardiomyocytes isolated from Sod2-/+ mice and >40% in cells from the Sod2-/- mice compared with <10% for the Sod2+/+ mice, and the loss in cell viability was prevented by cyclosporin A and aristocholic acid. Figure 2A also shows that the addition of the low molecular weight, cell-permeable SOD mimetic, MnTBAP partially protected the cardiomyocytes from t-BuOOH. MnTBAP has been shown to be effective in catalytically degrading superoxide anions both in vivo and in vitro (14). Incubating the cells with MnTBAP decreased significantly the amount of cell death observed in cardiomyocytes isolated in from all three lines of mice after t-BuOOH treatment. In the cardiomyocytes from the Sod2-/+ and Sod2-/- mice, MnTBAP reduced cell death by approximately one-half.


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  		Fig. 2.   Viability of cardiomyocytes after oxidative stress. A: cardiomyocytes isolated from Sod2+/+, Sod2-/+, and Sod2-/- mice were exposed to 200 µM t-BuOOH for 2 h (solid bars), and viability was measured using neutral red. Hatched bars show cell death in response to 200 µM t-BuOOH in cells that were pretreated for 20 min with 200 µM manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP). Open bars show the inhibition of cell death after pretreatment of the cells for 30 min with 5 µM cyclosporin A-50 µM aristocholic acid to inhibit the permeability transition. Data show the means ± SE for cells isolated from 4 to 5 mice per group and were analyzed by two-way analysis of variance. The post hoc test with adjusted multiple comparisons was done using the Tukey-Kramer method. *P < 0.01, cyclosporin A + t-BuOOH vs. t-BuOOH alone; **P < 0.0001 vs. wild-type cells; dagger P < 0.0001 vs. Sod2+/-; dagger dagger P < 0.0001, MnTBAP + t-BuOOH vs. t-BuOOH alone. B: retention of DiOC6(3) in cardiomyocytes from Sod2+/+, Sod2-/+, and Sod2-/- mice after treatment with 200 µM t-BuOOH for 2 h in the presence (open bars) or absence of cyclosporin A (solid bars). Data show means ± SE for experiments from 5 animals per group and were analyzed by two-way analysis of variance. The post hoc test with adjusted multiple comparisons was done by using the Tukey-Kramer method. *P < 0.001, cyclosporin A + t-BuOOH vs. t-BuOOH alone; dagger P < 0.001 vs. wild-type values.

To further demonstrate that the difference in the sensitivity of the cardiomyocytes to t-BuOOH was due to the permeability transition, we also measured Psi m. Induction of the permeability transition would result in a loss of the Psi m as measured by the localization of the fluorescent dye DiOC6(3), which localizes to the mitochondria as a consequence of the Psi m. The data in Fig. 2B show that accumulation of DiOC6(3) in the mitochondria of cardiomyocytes isolated from Sod2-/+ and Sod2-/- mice is significantly reduced after treatment with t-BuOOH compared with cardiomyocytes isolated from Sod2+/+ mice. The loss in the Psi m after t-BuOOH is greater for cardiomyocytes from Sod2-/+ mice compared with Sod2+/+ mice and greatest for cardiomyocytes from Sod2-/- mice, and completely inhibited by cyclosporin A.

To show that the cell death induced by t-BuOOH in Fig. 2 arose from apoptosis, we measured DNA fragmentation in the cardiomyocytes isolated from Sod2+/+, Sod2-/+, and Sod2-/- mice using the comet assay. The data in Fig. 3 show the percentage of cardiomyocytes isolated from Sod2+/+, Sod2-/+, and Sod2-/- mice that have apoptotic nuclei after treatment with t-BuOOH. Incubation of the cardiomyocytes from Sod2-/+ or Sod2-/- mice with t-BuOOH resulted in significant increases in the percentage of cells with apoptotic nuclei. Thus the increased sensitivity of cardiomyocytes from Sod2-/+ and Sod2-/- mice to cell death induced by t-BuOOH observed in Fig. 2 is paralleled by a similar increase in DNA fragmentation, which is indicative of apoptosis.


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  		Fig. 3.   Induction of apoptosis by oxidative stress in cardiomyocytes. Primary cultures of mouse cardiomyocytes isolated from Sod2+/+, Sod2-/+, and Sod2-/- mice were analyzed by the comet assay as described in MATERIALS AND METHODS. Solid bars show percentage of apoptotic cells in the absence of t-BuOOH and open bars show the percentage of apoptotic cells treated for 2 h with 200 µM t-BuOOH. Data are means ± SE for cardiomyocytes isolated from 3 to 4 mice and were analyzed using Student's t-test. *P < 0.05 vs. t-BuOOH alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MnSOD plays a critical role in protecting mitochondria from superoxide anions generated during respiration. In contrast with Sod1 and Sod3, inactivation of Sod2 is lethal in mice. Sod2-/- mice that are generated by targeted disruption of the Sod2 gene usually die within 1-18 days, depending on the genetic background (30, 38, 41). MnSOD is also unique from other antioxidant enzymes in that its expression is modulated by a variety of physiological and environmental factors. Thus MnSOD may play a role in physiological processes other than the antioxidant defense system.

We have studied the effect of reduced MnSOD activity on mitochondrial function using heterozygous MnSOD knockout (Sod2-/+) mice. Sod2-/+ mice are characterized by an ~50% reduction in MnSOD activity in all tissues studied compared with wild-type mice (61). In contrast to the homozygous null mutant, the Sod2-/+ mice are viable and exhibit no apparent abnormal phenotype. However, our initial studies showed increased oxidative damage in mitochondria from the livers of Sod2-/+ mice compared with their wild-type littermates (64). This increased oxidative damage was correlated to reduced mitochondria function, e.g., a decrease in mitochondrial respiration and an increased rate of induction of the permeability transition. The present study with mitochondria isolated from hearts of Sod2-/+ and Sod2+/+ mice confirms our earlier study with liver mitochondria. Mitochondria from hearts of Sod2-/+ mice showed a 50% reduction in MnSOD activity compared with mitochondria isolated from Sod2+/+ mice and have no significant differences in either glutathione peroxidase activity or total glutathione levels. Although CuZn-SOD is primarily a cytosolic enzyme, we found a small but equivalent amount of CuZnSOD activity in heart mitochondria isolated from both Sod2+/+ and Sod2-/+ mice. We did not detect any catalase activity in the mitochondria isolated from either group of mice. This was not surprising because mammalian myocardial catalase has been reported (6) to be located in microperoxisomes and not associated with mitochondria. Thus the reduction in MnSOD activity does not result in any compensatory mechanism that upregulates the other major components of the mitochondrial antioxidant defense system.

We also observed that the activities of aconitase and NADH-oxidoreductase, iron-sulfhydryl proteins in the mitochondria that are sensitive to inactivation by superoxide anions and oxidative stress (17, 18, 47), are significantly lower in mitochondria isolated from the hearts of Sod2-/+ mice compared with Sod2+/+ mice. Aconitase that has been inactivated by superoxide anions is readily reactivated by the addition of a reducing agent and iron (34). We found that reactivation of aconitase resulted in an increase in the mitochondrial aconitase activity in the Sod2-/+ mice to a level similar to that of the Sod2+/+ mice. Therefore, the decrease in aconitase activity in the Sod2-/+ appears to arise from oxidative damage because of increased levels of superoxide anions. We (64) have reported decreased activities of aconitase and NADH-oxidoreductase in liver mitochondria isolated from Sod2-/+ mice. Thus mitochondria from both the heart and liver of Sod2-/+ mice show evidence of increased oxidative damage compared with mitochondria isolated from Sod2+/+ mice. In contrast, the activities of cytosolic aconitase or glutamine synthetase in the hearts of Sod2-/+ and Sod2+/+ mice were not changed, suggesting that the alterations in oxidative stress we observed in the mitochondria were confined primarily to this cellular compartment.

We compared the function of mitochondria isolated from the hearts of Sod2-/+ and Sod2+/+ mice to determine if there was a correlation between increased oxidative damage and mitochondrial function. A decrease in the RCR and state 3 respiration was observed for heart mitochondria isolated from Sod2-/+ mice compared with Sod2+/+ mice for substrates metabolized by complex I. This is consistent with the decrease in NADH-oxidoreductase activity that we observed in heart mitochondria isolated from the Sod2-/+ mice. We previously observed a similar decline in the RCR and state 3 respiration for substrates metabolized by complexes I-III by liver mitochondria isolated from Sod2-/+ mice; however, in the liver, the decrease in the RCR was statistically significant for substrates metabolized by all complexes because the animal-to-animal variation in mitochondrial respiration was lower for mitochondria isolated from the liver compared with mitochondria isolated from the heart (64).

In our intial study with mitochondria isolated from the livers of Sod2-/+ and Sod2+/+ mice, we observed that the rate of induction of the mitochondrial permeability transition by calcium and t-BuOOH was significantly greater for mitochondria isolated from Sod2-/+ mice. In the present study, we observed that the induction of the permeability transition by either calcium or calcium and t-BuOOH was significantly more rapid for mitochondria isolated from the hearts of Sod2-/+ mice as well. The permeability transition has been proposed (68) to be involved in the mitochondrial pathway of apoptosis, and many factors that are known to induce apoptosis, e.g., oxidizing agents, thiol agents, atractyloside, and uncoupling agents that reduce the mitochondrial transmembrane potential, also induce the mitochondrial permeability transition. Therefore, we hypothesized that cells from Sod2-/+ mice would be more prone to the induction of apoptosis by oxidative stress than cells from Sod2+/+ mice. The permeability transition involves a pore that consists of a multiprotein complex located at contact points between the inner and outer mitochondrial membranes and acts as a sensor to detect various types of cellular stress, including oxidative stress (67). Our cell-free studies with isolated heart mitochondria demonstrated that mitochondria isolated from Sod2-/+ mice release more apoptogenic factors when exposed to t-BuOOH than mitochondria isolated from Sod2+/+ mice. In particular, we found that the level of cytochrome c released into the cytosol is greater from mitochondria isolated from the Sod2-/+ mice compared with the Sod2+/+ mice after induction of the permeability transition with t-BuOOH.

We also observed that the cardiomyocytes isolated from Sod2-/+ mice are more sensitive to oxidative stress than cardiomyocytes isolated from Sod2+/+ mice. For example, the percentage of cells that died from a 2-h exposure to t-BuOOH was twice as great for cardiomyocytes from Sod2-/+ mice compared with cardiomyocytes isolated from Sod2+/+ mice. In addition, we observed that cardiomyocytes isolated from Sod2-/- mice, which completely lacked MnSOD activity, were twice as sensitive to treatment with t-BuOOH as cardiomyocytes from the Sod2-/+ mice. With the use of the comet assay, which allows measurement of DNA double-strand breaks in nuclei of individual cells, we found that the increased sensitivity of cardiomyocytes from Sod2-/+ and Sod2-/- mice to cell death induced by t-BuOOH was paralleled by a similar increase in DNA fragmentation, indicating increased apoptosis. Therefore, in cardiomyocyte cultures, we observed an excellent correlation between MnSOD expression and the sensitivity of the cells to apoptosis induced by oxidative stress; in other words, decreased MnSOD expression was correlated to increased induction of apoptosis.

Our data indicate that the increased sensitivity of cardiomyocytes isolated from Sod2-/+ and Sod2-/- mice to apoptosis induced by t-BuOOH is due to alterations in the permeability transition. For example, cell death in our cultures of cardiomyocytes could be prevented by cyclosporin A, which binds to cyclophilin D, a component of the pore, to maintain the pore in a closed state (4, 21). Inhibition of the mitochondrial permeability transition by cyclosporin A has been shown to inhibit apoptotic cell death in a variety of cell types (e.g., hepatocytes, cardiomyocytes, macrophages, and fibroblasts) after treatment with apoptosis-inducing stimuli (29, 31, 35, 59). In addition, we observed an excellent correlation between the increased induction of apoptosis in cultures of cardiomyocytes isolated from Sod2-/- and Sod2-/+ mice and the loss in the Psi m, which is consistent with an increased induction of the permeability transition. The loss in the Psi m after t-BuOOH treatment was completely inhibited by cyclosporin A, which indicates that the increased loss in the Psi m in cardiomyocytes from the Sod2-/+ and Sod2-/- mice was primarily due to increased induction of the permeability transition by t-BuOOH. Thus our studies suggest that MnSOD plays a critical role in the mitochondrial pathway of apoptosis by regulating the sensitivity of the permeability transition pore to oxidative stress.

Our data are consistent with a previous study by Fujimura et al. (16) with Sod2-/+ and Sod2+/+ mice, in which apoptosis was measured in the ischemic and control portions of the brain after focal cerebral ischemia. Twenty-four hours after ischemia, Fujimura et al. (16) observed a greater degree of apoptosis in the brains of the Sod2-/+ mice as measured by increased DNA laddering. They also observed that the increased DNA laddering was correlated to a greater release of cytochrome c from the mitochondria of the ischemic portion of the brain of the Sod2-/+ mice. Therefore, Fujimura et al. (16) suggested that MnSOD prevented apoptosis in brain induced by ischemia-reprefusion by reducing the levels of superoxide anion and preventing release of cytochrome c. In addition, overexpression of MnSOD has been shown to reduce apoptosis. For example, Majima et al. (43) showed that overexpression of MnSOD in a mouse fibrosarcoma cell line protected the cells against cell death induced by alkaline conditions. In addition, Keller et al. (33) reported that apoptosis induced by either iron, amyloid beta -peptide, or nitric oxide-generating agents was reduced in neuronal cell lines from transgenic mice that overexpressed MnSOD. Cyclosporin A prevented the induction of apoptosis in these cells, which is consistent with our study showing a role for the permeability transition pore in the anti-apoptotic action of MnSOD.

The permeability transition involves a nonspecific pore that is thought to consist of a complex of several proteins including the mitochondrial matrix protein cyclophilin D, the inner membrane protein adenine nucleotide translocator (ANT), the voltage-dependent anion channel in the outer mitochondrial membrane and hexokinase-1 (3, 10, 22, 45). In response to apoptogenic factors, including oxidative stress, ANT has been shown to have the ability to form a pore (9, 54). Because ANT is located in the inner mitochondrial membrane, formation of a pore results in permeabilization of the inner membrane and swelling of the mitochondrial matrix. This is believed to lead to the eventual disruption of the outer mitochondrial membrane and release of apoptogenic molecules such as cytochrome c. Once released from the mitochondria, cytochrome c binds to the apoptosome (a complex consisting of Apaf-1, procaspase 9, and dATP), activating caspase 9 and other downstream caspases, leading to apoptosis. Our data indicate that the reduced levels of MnSOD in the mitochondria of the Sod2+/- mice leads to elevated levels of superoxide anion and oxidative stress, which we hypothesize would increase the probability of pore formation by ANT. Therefore, apoptosis would be triggered more readily in the mice deficient in MnSOD because a smaller degree of oxidative stress would be required to induce a critcal level of pore formation and the resulting cascade of apoptotic events. In other words, cells from Sod2-/+ or Sod2-/- mice, which have reduced MnSOD levels, will undergo apoptosis when exposed to a milder oxidative stress compared with Sod2+/+ mice.

In this study, we have examined the effect of reduced MnSOD expression on oxidative damage, mitochondrial function and oxidative stress-induced apoptosis in hearts of Sod2-/+ mice to gain insight into the role of MnSOD in antioxidant defense and apoptosis in the heart. Our data point to MnSOD playing an important role in the regulation of the mitochondrial pathway of apoptosis as well as its well known role in the antioxidant defense system.


    ACKNOWLEDGEMENTS

This work was supported by a Merit Review and a Veterans Integrated Service Network grant from the Department of Veteran Affairs, by an American Heart Association grant, by National Institute on Aging Grants AG-16998 and AG-15908, and by the University of Texas Health Science Center (San Antonio Nathan Shock Aging Center) Grant PO3-AG-13319.


    FOOTNOTES

* H. Van Remmen and M. D. Williams contributed equally to this work.

Address for reprint requests and other correspondence: H. Van Remmen, GRECC (182), Audie Murphy VA Hospital, 7400 Merton Minter Blvd., San Antonio, TX 78229 (E-mail: vanremmen{at}uthscsa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 August 2000; accepted in final form 23 May 2001.


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Am J Physiol Heart Circ Physiol 281(3):H1422-H1432


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