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Am J Physiol Heart Circ Physiol 293: H2726-H2737, 2007. First published August 3, 2007; doi:10.1152/ajpheart.00376.2007
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TNF-{alpha}-induced mitochondrial oxidative stress and cardiac dysfunction: restoration by superoxide dismutase mimetic Tempol

Nithya Mariappan,1 Rajasekaran Namakkal Soorappan,2 Masudul Haque,1 Srinivas Sriramula,1 and Joseph Francis1

1Department of Comparative Biomedical Sciences, Louisiana State University, School of Veterinary Medicine, Baton Rouge, Louisiana; and 2Department of Internal Medicine, Division of Cardiology, University of Utah Health Sciences Center, Salt Lake City, Utah

Submitted 26 March 2007 ; accepted in final form 29 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondria are indispensable for bioenergetics and for the regulation of physiological/signaling events in cellular life. Although TNF-{alpha}-induced oxidative stress and mitochondrial dysfunction are evident in several pathophysiological states, the molecular mechanisms coupled with impaired cardiac function and its potential reversal by drugs such as Tempol or apocyanin have not yet been explored. Here, we hypothesize that TNF-{alpha}-induced oxidative stress compromises cardiac function by altering the mitochondrial redox state and the membrane permeability transition pore (MPTP) opening, thereby causing mitochondrial dysfunction. We measured the redox states in the cytosol and mitochondria of the heart to understand the mechanisms related to the MPTP and the antioxidant defense system. Our studies demonstrate that TNF-{alpha}-induced oxidative stress alters redox homeostasis by impairing the MPTP proteins adenine nucleotide translocator and voltage-dependent anion channel, thereby resulting in the pore opening, causing uncontrolled transport of substances to alter mitochondrial pH, and subsequently leading to dysfunction of mitochondria and attenuated cardiac function. Interestingly, we show that the supplementation of Tempol along with TNF-{alpha} restores mitochondrial and cardiac function.

tumor necrosis factor-{alpha}; redox state; membrane permeability transition pore protein; cardiac function; cytokines

THE POSSIBLE ROLE OF TNF-{alpha}, a potential cytokine (an inflammatory marker and a signaling molecule), in the settings of cardiac hypertrophy and/or dysfunction and end-stage heart failure has been reported in experimental models and human patients (3, 11, 19, 21, 22, 37, 45). Although TNF-{alpha}-induced oxidative stress and mitochondrial dysfunction are evident in several pathophysiological states, its molecular mechanisms and functional significance in target tissue (heart) damage remain unknown. Previous studies have demonstrated the biochemical mechanisms associated with TNF-{alpha} and its direct toxic effect in several disorders, including left ventricle (LV) dysfunction, cardiomyopathy, and pulmonary edema in human patients (26, 33, 36, 42). Additional investigations have used single-bolus or continuous infusions of TNF-{alpha} to examine the dose-dependent effects of TNF-{alpha} on cardiac metabolism, LV dysfunction, and cardiac remodeling in various animal models (4, 5, 18, 24). However, these studies failed to map out the causal relationship with TNF-{alpha}-induced oxidative stress and organelle-dependent mechanisms for the progression of hypertrophy and end-stage heart failure. TNF-{alpha}, besides being a marker of heart failure, could be causally linked to the pathogeneses of both cardiac hypertrophy and cardiomyopathy, because it has been shown to be a potential inducer of oxidative stress under several pathological conditions.

The myocardium is a dynamic tissue that normally generates reactive oxygen species (ROS) as part of its physiological process. The resultant toxic radicals produced during cardiac metabolism are subsequently quenched by intracellular defense mechanisms. An overproduction of oxidants could compromise the defense system, induce pathophysiological signals, and impair the overall function of the cardiac tissue. TNF-{alpha} is reported to stimulate ROS production by several mechanisms, including its direct toxic phenomena and its effects on mitochondrial function (20, 24, 27, 34). It has also been suggested that ROS production via NADPH oxidases is associated with increased levels of angiotensin II and TNF-{alpha} (16, 28, 34). However, the potential implications of an acute infusion of TNF-{alpha} in the progression of LV dysfunction are not clear at the molecular level. Because mitochondria are the potential source for ROS and NADPH oxidases are prime catalysts for the generation of ROS, it is highly critical to understand both the mechanisms linked to mitochondrial function and the role of NADPH oxidases in propagating TNF-{alpha}-induced oxidative stress and cardiac dysfunction.

Considering the well-known facts related to TNF-{alpha}-induced change in the mitochondrial redox state and the associated damage to mitochondria, we hypothesize that TNF-{alpha}-induced oxidative stress could compromise cardiac function by altering the mitochondrial redox state and the membrane permeability transition pore (MPTP) opening and by causing subsequent dysregulation of mitochondrial defense mechanisms. Furthermore, to validate our hypothesis, we have used a gain-of-function strategy by blocking NADPH oxidase with the use of apocyanin, a well-characterized inhibitor of NADPH oxidase, or by scavenging the superoxide pool with the use of Tempol, a potential quencher of ROS. We have also examined whether the actions of NADPH oxidase are inevitable in the context of TNF-{alpha}-induced oxidative stress and its associated mitochondrial dysfunction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Adult male Sprague-Dawley rats weighing 325–350 g were used for the study. Animals were housed in temperature- (23 ± 2°C) and light-controlled (lights on between 7 AM and 7 PM) animal quarters, and rat chow and water were provided ad libitum. All experimental procedures were approved by the Louisiana State University Institutional Animal Care and Use Committee.

Experimental Protocol

Rats were treated with TNF-{alpha} (40 µg /kg ip), TNF-{alpha} + apocyanin (200 µmol/kg po), or TNF -{alpha} + Tempol (300 µmol/kg po), for 5 days. Control animals were injected with 0.9% normal saline. On day 5, LV function was measured by using echocardiography; subsequently, rats were killed and the LV was removed for gene-expression, biochemical, and mitochondrial assays. The structural integrity of the mitochondrial membrane was measured by using a swelling assay, Western blotting, transmission electron microscopy, and antioxidant status.

Echocardiographic Assessment of LV Function

Transthoracic echocardiography was performed as previously described (13). A Toshiba Aplio SSH770 (Toshiba Medical, Tustin, CA) fitted with a PST 65A sector scanner (8-MHz probe) was used, which generates two-dimensional images at a frame rate ranging from 300 to 500 frames/s. LV dimension (LVD) was measured by using m-mode. LV fractional shortening (FS %) was calculated by using the following equation: FS % = [(LVDD – LVESD)/LVEDD] x 100, where LVDD is LV diastolic dimension, LVESD is LV end-systolic dimension, and LVEDD is LV end-diastolic dimension. Tei index was determined from Doppler recordings of LV inflow and outflow as described previously (10).

Blood Pressure in TNF-{alpha}-Induced Oxidative Stress

Blood pressure measurements were obtained for all experimental animals by using the tail-cuff method with the use of a Coda 6 rat tail blood pressure machine (Kent Scientific). Blood pressure was measured once daily for 5 days during the treatment period. Rats were preheated in a chamber at 31°C for 10 min and then placed in plastic restrainers. A cuff with a pneumatic pulse sensor was attached to the tail. Rats were allowed to habituate to this procedure for 3 days before experiments were performed. Blood pressure values were recorded on a MicroSoft Excel spreadsheet and were averaged from at least six consecutive cycles obtained from each rat.

Detection of ROS in the Heart

Superoxide generation was assessed via the conversion of dihydroethidium (DHE) to ethidium. Rats received an intracardiac injection of DHE at a dose of 80 µg/kg body wt. Hearts were harvested, placed into a freezing mold with Tissue-Tek OCT (Sakura Finetek, Torrance, CA), snap frozen with liquid nitrogen, sectioned on a cryostat, and placed on slides. Cryosections (12 µm) were immediately viewed and imaged under epifluorescence with a Zeiss Axiovert 200 microscope by using an ethidium bromide-compatible filter set (Chroma Filters #41006); images were captured with an Olympus Q Capture 5 camera and Q Capture Pro software.

Protein Carbonyl and Superoxide Dismutase Assays

Protein carbonyls were measured as described previously (41). Superoxide dismutase (SOD) levels were measured by using a SOD assay kit (Dojindo Molecular Technologies), following procedures provided by the manufacturer.

Measurement of Serum TNF-{alpha} Levels

TNF-{alpha} concentrations in rat plasma were determined by using a rat TNF-{alpha} immunoassay kit (Biosource), following procedures as described previously (14). Plasma TNF-{alpha} levels were measured on day 5 after intraperitoneal injection of TNF-{alpha}.

Measurement of MPTP Opening in Isolated Cardiac Mitochondria

Heart mitochondria were isolated by a differential centrifugation technique of heart homogenates as described previously (40). Opening of the pore causes mitochondrial swelling, which results in reduction of absorbance at 540 nm (A540). Mitochondrial permeability transition (swelling assay) was monitored as changes at 540 nm at 1-min intervals over 15 min time with 250 µg mitochondrial protein in the swelling buffer, which contains (in mmol/l): 120 KCl (pH 7.4) and 5 KH2PO4. Mitochondrial permeability transition was measured either in the absence or presence of 50 µm Ca2+.

Western Blot Analysis

LV tissue mitochondrial adenine nucleotide translocator (ANT) and anti-voltage-dependent anion channel (VDAC) protein content were determined by Western blot. Goat anti-ANT (1:500) and goat anti-VDAC (1:1000) were used as primary antibodies. The immunoreactive proteins were visualized with rabbit anti-goat IgG (anti-ANT antibody) and goat anti-rabbit IgG (VDAC antibody), all horseradish peroxidase conjugated and used in a dilution of 1:20,000. The band intensities were quantified by using a Konica SRX-101A imaging system and were normalized with GAPDH. Densitometry analysis was performed by using NIH ImageJ software.

Electron Microscopy Studies

Ultrastructural examination of isolated mitochondrial preparations was performed as described before (29) by using electron microscopy. Mitochondrial pellets were immediately fixed in a 2.5% solution of glutaraldehyde. Pellets were then fixed in a 1% solution of osmium tetroxide. The fixed mitochondria were embedded in Epon-Araldite. Ultrathin slices (0.1 µm thick) were obtained, stained with uranyl acetate and lead citrate, and examined in a Joel 100 CX electron microscope at 80 kV, accelerating voltage and film magnification.

Biochemical Assays

For various enzyme assays, heart-tissue samples were homogenized in ice-cold, enzyme-specific homogenization buffer (tissue:buffer ratio, 1:10 wt/vol). The homogenates were centrifuged at 12,000 g at 4°C for 10 min, and the resulting supernatant aliquots were stored at –80°C until use. Protein concentration was determined according to the Bradford method by using BSA as the standard. GSH/GSSG, SOD, and glutathione peroxidase (GPx) concentrations were determined in tissue homogenates and in isolated mitochondria by use of a commercially available kit (Cayman Chemical). All assays were run in triplicate and were averaged to obtain a mean value per sample.

Determination of Catalase Activity

Catalase activity was measured by the method of Beers (1). Briefly, a 0.1-ml tissue homogenate or isolated mitochondria was added to a cuvette containing 1.9 ml of 50 mM phosphate buffer, pH 7.4, and the reaction was started by addition of 1.0 ml of freshly prepared 30 mM H2O2. The change in absorbance per minute at 240 nm was calculated. Measurements were performed in triplicate. Protein concentrations were estimated by the Bradford method. Catalase activity was calculated as units per milligram of protein.

RNA Isolation and Real-Time RT-PCR

Total RNA was extracted from the LV by using TRIzol reagent (Invitrogen) and was reverse transcribed by using oligo(dT) and reverse transcriptase. Expression levels of Nox1, gp91phox, and TNF-{alpha} mRNA were determined by using specific primers (Table 1). GAPDH was used as a housekeeping gene. Real-time RT-PCR (qRT-PCR) was performed in 384-well PCR plates with the use of Bio-Rad PCR Master Mix (iTaq SYBR Green Supermix with ROX) and the ABI Prism 7900 sequence-detection system (Applied Biosystems).


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  		Table 1. Primer sequences for real-time PCR

 
Immunocytochemical Localization of TNF-{alpha} and Nox Subunits

For immunocytochemistry, samples were processed by using standard protocols. The sections were treated with respective primary antibody: Nox1 (1:100 dilution), gp91phox (1:100 dilution), or TNF-{alpha} (1:100 dilution; Santa Cruz Biotechnology) and were incubated overnight at 4°C. Then, sections were washed twice in PBS and were incubated with the secondary antibody, a peroxidase-conjugated IgG antibody, for 30 min. Bound antibodies were detected with a streptavidin-peroxidase complex by using 0.2 mg/ml 3,3'-diaminobenzidine tetrahydrocholride in PBS containing 0.003% hydrogen peroxide. Negative sections were incubated with secondary antibody alone.

Statistical Analysis

Values are expressed as means ± SE for eight animals in each group from each experimental setup. ANOVA followed by Bonferroni's multiple-comparison test was performed by using GraphPad Prism v. 4.00 for Windows, (GraphPad Software, San Diego, CA) to determine differences between groups subjected to repeated measures. Statistical significance was acceptable to a level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Tempol and Apocyanin on LV Structure and Function

Echocardiography revealed that LVD were larger in TNF-{alpha}-treated rats and in rats treated with TNF + apocyanin compared with all other groups (Table 2 ). FS % was decreased in TNF-{alpha}-treated and TNF-{alpha} + apocyanin-treated rats relative to controls. Tei index, an indicator of diastolic dysfunction, was increased in TNF-{alpha}-treated compared with control rats. Treatment with Tempol attenuated changes in these parameters. The changes produced by apocyanin were relatively small and statistically insignificant compared with the control group.


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  		Table 2. Echocardiographic analysis of control and experimental groups

 
Effects of Tempol and Apocyanin on Blood Pressure

Blood pressure measurements were obtained for all experimental animals during the 5-day treatment period. Treatment with TNF-{alpha} and other drugs did not have any effect on blood pressure parameters (Fig. 1).


Figure 1
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  		Fig. 1. Mean arterial pressures for control and experimental groups. No statistically significant differences were observed among mean arterial pressures during 5-day treatment period. TEM, Tempol; APO, apocyanin.

 
Effects of Tempol and Apocyanin on DHE Fluorescence, Indicating Superoxide Levels

Superoxide anions were detected by DHE, and their levels in heart were compared in control and TNF-{alpha}-treated rats. As illustrated by representative images (Fig. 2A), the DHE-labeled nuclei in the hearts of TNF-{alpha}-induced rats contained significantly higher superoxide levels than nuclei of control rats. Treatment of TNF-{alpha}-treated rats with Tempol completely blocked superoxide ions and decreased the DHE signal, suggesting an inhibition of superoxide generation, whereas apocyanin treatment only partially inhibited the formation of superoxide ions. These findings provide evidence that cardiac dysfunction associated with oxidative stress may be largely triggered by TNF-{alpha}, mediated by superoxide ions, and restricted only partially by apocyanin treatment and completely with Tempol treatment.


Figure 2
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  		Fig. 2. A: reactive oxygen species (ROS) generation in hearts of control and treated rats was determined by dihydroethidium (DHE) fluorescence (x20 magnification). Under identical imaging conditions, production of superoxide is significantly elevated in heart tissue of TNF-{alpha}-treated rats compared with control, apocyanin (APO)-treated, or Tempol (TEMP)-treated animals. B: protein carbonyl levels in study groups were determined by ELISA. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF-{alpha} vs. Tempol or apocyanin treatment). C and D: effects of apocyanin and Tempol on myocardial total superoxide dismutase (SOD) enzyme levels and results of accompanying Western blot and densitometric analysis. Note increased SOD levels observed in TNF-{alpha}-treated rats. Tempol or apocyanin treatment attenuated increase in SOD levels. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF-{alpha} vs. Tempol or apocyanin treatment; $P < 0.05, TNF-{alpha} + apocyanin vs. TNF-{alpha} + Tempol).

 
Effects of Apocyanin and Tempol on Cytosolic Protein Carbonyl Content

To assess the degree of protein carbonylization in TNF-{alpha}-induced oxidative stress, protein carbonyl content formation was determined (Fig. 2B). Protein carbonyl content was significantly higher (1.57 ± 0.01 arbitrary units) in the TNF-{alpha}-treated rats compared with the control group (0.099 ± 0.008 arbitrary units). This increase in the protein carbonyl content was significantly attenuated (1.06 ± 0.01 arbitrary units and 1.16 ± 0.01 arbitrary units, respectively) in the groups treated with TNF-{alpha} + Tempol and TNF-{alpha} + apocyanin.

Effects of Apocyanin and Tempol on Cytosolic SOD Levels

The levels of cytosolic SOD in the heart tissues of control and experimental animals are shown in Fig. 2C. Tempol strongly scavenged superoxide radicals and also suppressed hydroxyl radicals, thereby exhibiting significant reductions (7.72 ± 0.05 U/mg protein) in SOD levels compared with TNF-{alpha}-treated rats (14.39 ± 0.12 U/mg protein). Tempol or apocyanin treatment attenuated the increase in SOD levels; these results suggest a difference in the mechanistic actions of apocyanin and Tempol. These results were further confirmed by Western blot and densitometric analysis to quantify SOD levels (Fig. 2D).

Elevated Circulating Levels of TNF-{alpha}

The effects of Tempol and apocyanin on plasma TNF-{alpha} levels in TNF-{alpha}-treated rats are shown in Fig. 3A. The circulating levels of TNF-{alpha} were significantly elevated (33.237 ± 5.29 pg/ml) in TNF-{alpha}-treated rats. In contrast, levels of TNF-{alpha} were significantly lower in Tempol-treated rats (11.004 ± 0.781 pg/ml) compared with controls (12.82 ± 0.18 pg/ml). Treatment with apocyanin attenuated TNF-{alpha} levels, but the level of attenuation was not significant. The tissue levels of TNF-{alpha} in the LV of rats were further confirmed by immunocytochemistry and mRNA expression. Immunocytochemical and mRNA studies also revealed an increase in expression of TNF-{alpha} after treatment with TNF-{alpha} compared with control animals. Treatment with Tempol or apocyanin returned TNF-{alpha} expression to control levels (Fig. 3, B and C).


Figure 3
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  		Fig. 3. Circulating levels of TNF-{alpha}. A: levels of TNF-{alpha} were significantly lower in Tempol-treated rats compared with controls. Treatment with apocyanin attenuated TNF-{alpha} levels, but values were not significantly different from control levels. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF-{alpha} vs. Tempol or apocyanin treatment; $P < 0.05, TNF-{alpha} + apocyanin vs. TNF-{alpha} + Tempol). B: photomicrographs showing immunocytochemical expression of TNF-{alpha} in left ventricle. Note increased expression of TNF-{alpha} in TNF-{alpha}-treated rats compared with control. Tempol attenuated this increased expression. Apocyanin also showed partially attenuated TNF-{alpha} levels in left ventricle. C: effects of apocyanin and Tempol treatment on mRNA expression of TNF-{alpha}. Levels of TNF-{alpha} were elevated in TNF-{alpha}-treated rats compared with control, TNF-{alpha} + Tempol, and TNF-{alpha} + apocyanin treatment groups. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF vs. Tempol or apocyanin treatment).

 
TNF-{alpha}-Mediated Oxidative Stress Induces MPTP Opening Against Calcium Challenge

To determine the effects of Tempol and apocyanin on TNF-{alpha}-mediated oxidative stress, MPTP opening was induced against a 50 µM calcium challenge. When mitochondria are exposed to 50 µM calcium concentrations, especially when accompanied by oxidative stress, they undergo massive swelling. As demonstrated in Fig. 4, A and B, the extent of the swelling was higher in 50 µM Ca2+-loaded mitochondria obtained from TNF-{alpha}-treated rats. The MPTP opened at a faster rate in the in the presence of Ca2+ in TNF-{alpha}- and TNF-{alpha} + apocyanin-treated rats than in the TNF-{alpha} + Tempol-treated group. Mitochondrial swelling was drastically reduced by Tempol but not by apocyanin, thus reinforcing the role played by the MPTP. These results suggest that the protective effect of apocyanin is at the cytosolic level and not at the mitochondrial level. This is in line with the known biological activity of apocyanin as a NADPH oxidase inhibitor that cannot permeate the mitochondrial membrane.


Figure 4
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  		Fig. 4. Effects of apocyanin and Tempol on TNF-{alpha}-induced mitochondrial swelling. A: basal. B: calcium challenge. TNF-{alpha} induced marked membrane permeability transition pore (MPTP) opening; this pore opening was further induced by subsequent additions of Ca2+ to isolated mitochondria. Shown are representative tracings taken from isolated mitochondria from control and experimental rats. MPTP opened at a faster rate in presence of Ca2+ in TNF-{alpha} groups than in TNF-{alpha} + Tempol group.

 
Depletion of ANT Protein in the Heart Mitochondria: Relevance for Mitochondrial Dysfunction

Western-blotting analysis of LV tissue from control and TNF-{alpha}-treated rats was evaluated to assess protein content of ANT and VDAC. Figure 5, A and B, shows treatment-related differences in ANT and VDAC content. TNF-{alpha}-treated rats exhibited a significant decrease in ANT content compared with control and TNF-{alpha} + Tempol-treated groups. Significant differences were also found in VDAC content between the groups. In the TNF-{alpha} +apocyanin-treated group, ANT protein levels were not restored. In the TNF-{alpha} + Tempol-treated group, ANT protein levels were restored to near that of controls. These results further support the assertion that Tempol is protective at the mitochondrial level, whereas apocyanin is only protective at the cytosolic level.


Figure 5
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  		Fig. 5. A: representative blot and densitometric analysis of a left ventricle showing changes in expression of adenine nucleotide translocator (ANT) and voltage-dependent anion channel (VDAC) from isolated mitochondria. TNF-{alpha}-treated rats exhibited a significant decrease in ANT content compared with control, TNF-{alpha} + Tempol-, and TNF-{alpha} +apocyanin-treated groups. B: VDAC protein was also decreased after treatment with TNF-{alpha} compared with controls. Treatment of TNF-{alpha}-treated rats with Tempol or apocyanin prevented VDAC depletion. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF-{alpha} vs. Tempol or apocyanin treatment; $P < 0.05, TNF-{alpha} + apocyanin vs. TNF-{alpha} + Tempol). C: representative electron photomicrographs of mitochondrial ultrastructure. Control shows intact mitochondrial ultrastructure of rat. In TNF-{alpha}-treated group, treatment-induced severe mitochondrial damage is seen. Note mitochondrial swelling accompanied by disruption in membrane integrity. TNF-{alpha} + apocyanin-treated animals showed moderate mitochondrial damage. In the TNF-{alpha} + Tempol group, mitochondria showed slight reductions in damage and preservation of cristae architecture (x100,000).

 
Ultrastructural Details of Mitochondria

The morphological changes of mitochondria isolated from LV heart tissue are shown in Fig. 5C. Normal mitochondria with double membranes were observed in control rats. Cristae were observed to lie parallel from one periphery to the other. Conversely, disorganization and degeneration of mitochondria were observed in TNF-{alpha}-treated rats. Cristae were absent in a few mitochondria, and a few showed dilation. Loss of architecture was also seen, indicating the beginning of degeneration. In contrast, mitochondria from the TNF-{alpha} + Tempol treatment group showed maintenance of structural integrity and preservation of cristae architecture compared with mitochondria from the TNF-{alpha} + apocyanin treatment group. These ultrastructural findings reinforce the overall protective effect of Tempol.

Effects of Apocyanin and Tempol on Antioxidant Parameters

Antioxidant evaluation in tissue.
GSH/GSSG. Glutathione (reduced and oxidized) contents of tissue homogenates and mitochondria were examined in the control and TNF-{alpha}-treated groups (Table 3). Cytosolic GSH and GSSG content in the TNF-{alpha}-treated rats was decreased (17.49 ± 0.49 nmol/mg protein and 12.95 ± 0.33 nmol/mg protein, respectively) compared with its respective control group (26.25 ± 0.41 nmol/mg protein and 16.11 ± 0.2 nmol/mg protein, respectively). Cytosolic GSH and GSSG levels of TNF-{alpha} + Tempol-treated rats were restored to that of control levels (27.86 ± 0.35 nmol/mg protein and 16.43 ± 0.20 nmol/mg protein, respectively). Similar trends were observed in GSH and GSSG content and the GSH/GSSG ratio in mitochondria. These changes were attenuated by treatment with Tempol. The redox ratio was significantly depressed in the TNF-{alpha}-treated groups when compared with respective control groups.


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  		Table 3. Effects of apocyanin and Tempol on levels of reduced glutathione, oxidized glutathione, and redox ratio in cytosol and mitochondria of control and TNF-{alpha}-treated rats

 

GPX. Changes in the activities of cytosolic GPx in the control and experimental groups of rats are shown in Fig. 6A. A significant decrease (78.26 ± 0.95 nmol·min–1·mg protein–1) in GPx activity is observed in TNF-{alpha}-treated rats when compared with control (165.03 ± 1.30 nmol·min–1·mg protein–1). The administration of Tempol or apocyanin to rats treated with TNF-{alpha} restored the GPx activity levels of this group (122.43 ± 1.70 nmol·min–1·mg protein–1) to levels close to those of controls. The activities of antioxidant enzymes such as catalase and GPx were all significantly decreased in the cytosol, and, to our interest, a highly significant decrease was noted in the activity of catalase. Changes seen in GPx levels may be due to an endogenous utilization of catalase and GPx on quenching H2O2 levels that are unregulated when rats are treated with TNF-{alpha}. These results demonstrate that Tempol treatment can completely restore GPx activity.


Figure 6
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  		Fig. 6. Depletion of cytosolic glutathione peroxidase (GPx) and catalase activities (A and B) and mitochondrial GPx and catalase activities (C and D) in TNF-{alpha}-treated animals. TNF-{alpha} treatment depleted antioxidant levels both in cytosol and in mitochondria. Tempol treatment restored cytosolic and mitochondrial enzyme activity. Apocyanin partially restored antioxidant levels in cytosol but not in mitochondria. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF-{alpha} vs. Tempol or apocyanin treatment; $P < 0.05, TNF-{alpha} + apocyanin vs. TNF-{alpha} + Tempol); **P < 0.001.

 

CATALASE. Figure 6B indicates catalase activity from control and treatment groups. Catalase activity in heart homogenates was significantly decreased (17.88 ± 0.49 U·mg–1·min–1) in TNF-{alpha}-treated rats when compared with the control group (29.99 ± 1.05 U·mg–1·min–1) and the TNF-{alpha} + Tempol-treated group (25.72 ± 1.24 U·mg–1·min–1). Treatment of TNF-{alpha}-treated rats with Tempol restored antioxidant enzyme activity, suggesting that Tempol treatment prevented downregulation of catalase. In the apocyanin-treated group, no statistically significant difference was observed in catalase activity.

Antioxidant evaluation in mitochondria. Figure 6, C and D, displays the levels of antioxidant enzymes in isolated mitochondria from the heart. Activities of the mitochondrial enzymes GPx (Fig. 6C) and catalase (Fig. 6D) were significantly decreased in TNF-{alpha}-treated rats (165.36 ± 1.39 nmol·min–1·mg protein–1 and 10.43 ± 0.83 U·min–1·mg protein–1, respectively). Tempol restored the activities of GPx and catalase to levels close to control levels (305.2 ± 1.47 and 17.09 ± 0.4 U·min–1·mg protein–1, respectively). TNF-{alpha}-treated rats treated with apocyanin showed decreased levels of GPx (222.08 ± 2.72 U·min–1·mg protein–1) and catalase (7.89 ± 0.66 U·min–1·mg protein–1) compared with GPx and catalase levels of control rats (305.2 ± 1.48 and 17.08 ± 0.41 U·min–1·mg protein–1, respectively). These results clearly suggest that apocyanin preserves antioxidant levels only at the level of the cytosol and not at the mitochondrial level, whereas Tempol preserves antioxidants at both levels.

Effects of Tempol and Apocyanin on TNF-{alpha}-Induced Increase in gp91phox and Nox1 mRNA and Protein Expression

To further determine whether TNF-{alpha}-induced oxidative stress was accompanied by an increase in the NADPH oxidase subunit gp91phox and its homolog Nox1, levels of these subunits were measured by using real-time RT-PCR for mRNA in heart tissues of all study groups. TNF-{alpha} treatment induced an increase in the mRNA levels of gp91phox (Fig. 7A) and Nox1 (Fig. 8A) in the LV; this increase was prevented by both apocyanin and Tempol. Changes in gp91phox and Nox1 protein expression were further confirmed by immunocytochemistry and Western blot. Immunocytochemistry also revealed an increase in expression of gp91phox and Nox1 in TNF-{alpha}-treated group compared with control (Figs. 7B and 8B); this upregulation was blocked by both Tempol and apocyanin.


Figure 7
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  		Fig. 7. Effects of Tempol and apocyanin treatment on mRNA expression of gp91phox. A: levels of gp91phox were elevated in left ventricles of TNF-{alpha}-treated rats compared with control. Cotreatment with apocyanin or Tempol prevented these increases. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF-{alpha} vs. Tempol or apocyanin treatment; $P < 0.05, TNF-{alpha} + apocyanin vs. TNF-{alpha} + Tempol). B: immunocytochemical localization of gp91phox in left ventricle. Intensities of immunoreactions of gp91phox are notably increased in TNF-{alpha}-treated rats compared with control. Treatment with Tempol completely blocked protein expression for gp91phox. Apocyanin partially blocked this expression (x20).

 

Figure 8
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  		Fig. 8. Effects of Tempol and apocyanin treatment on the mRNA expression of Nox 1. A: levels of Nox1 were elevated in left ventricles of TNF-{alpha}-treated rats compared with control. Cotreatment with apocyanin or Tempol prevented these increases. B: immunocytochemical localization of Nox1 in left ventricle. Intensities of immunoreactions of Nox1 were notably increased in TNF-{alpha}-treated rats compared with control. Treatment with Tempol completely blocked protein expression for Nox1. Apocyanin partially blocked this expression (x20). C: Western blot and densitometric analysis of Nox1, exhibiting an increased level of Nox1 in TNF-{alpha}-treated rats compared with control, apocyanin-treated, and Tempol-treated rats. Values are expressed as means ± SE (*P < 0.05, control vs. TNF-{alpha} treatment; #P < 0.05, TNF-{alpha} vs. Tempol or apocyanin treatment; $P < 0.05 TNF + apocyanin vs. TNF + Tempol).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Redox signals play vital roles in the pathogenesis of acute and chronic stress conditions by mediating the expression patterns of proteins and genes associated with redox balance. Here, we report on TNF-{alpha}-mediated oxidative stress and its possible mechanistic link to the progression of cardiac dysfunction. In the present study, administration of TNF-{alpha} resulted in a marked increase in ROS generation and protein carbonylation in the myocardium of rats. It also decreased myocardial intracellular GSH and GSSG biosynthesis. These observations are suggestive of severe oxidative stress in cardiac tissue, and this stress could intensively affect the role of the heart. Furthermore, phenotypic changes associated with TNF-{alpha}-induced oxidative stress included the suppression of LV function after 5 days of TNF-{alpha} treatment. These interesting findings reveal a stable phenotypic validation in the rat heart due to TNF-{alpha}-induced oxidative stress. We then expanded our experiments to investigate the mechanisms related to 1) TNF-{alpha}-induced oxidative stress and the cardiac defense system and 2) the molecular events coupled with TNF-{alpha}-induced oxidative stress and the NADPH oxidase system.

To elucidate the possible link between TNF-{alpha}-induced production of ROS, oxidative stress, and proinflammatory cytokine expression, we pharmacologically inhibited NADPH oxidase by using apocyanin and scavenged hydrogen peroxide and superoxide anions by using Tempol (a stable radical), which permeates biological membranes and functions as an intracellular scavenger of superoxide ions and other radicals. We found that Tempol prevents mitochondrial damage and subsequent mitochondrial superoxide-scavenging activity that can remove superoxide radicals from both mitochondrial and cytosolic origin to maintain the cellular redox status; this is the mechanism by which Tempol exerts its protective role against TNF-{alpha}-induced oxidative stress.

It is well documented that TNF-{alpha}-induced oxidative stress in rat heart exerts its toxicity by altering the antioxidant defense state in cells (2, 22, 25, 27). However, the underlying mechanisms are still not clear. Administration of TNF-{alpha} to rats significantly decreased GSH content and led to an increased redox (GSH/GSSG) ratio, indicating a direct toxic effect of TNF-{alpha} on the defense state of the heart tissue. This was also reflected at the level of protein oxidation, determined as protein carbonyl content. Interestingly, the effect of TNF-{alpha} is prevented by either Tempol or apocyanin, suggesting that TNF-{alpha}-mediated oxidative stress seems to occur through the generation of excess ROS, which in turn are quenched by a series of reactions that utilize the intracellular GSH pool. These data confirm that Tempol and apocyanin both exert anti-inflammatory properties and clearly demonstrate that these drugs restore the redox state by reducing ROS generation via various Nox isoforms. Together, these observations explain, at least partially, a vital role played by Nox isoforms in the generation of ROS on TNF-{alpha} infusion. Our immunohistochemistry results directly demonstrate evidence for increased translational products of Nox1 and gp91phox in TNF-{alpha}-treated heart tissues and decreased translational products of Nox1 and gp91phox in heart tissues of rats treated with Tempol. Previous reports have revealed an authentic role for various Nox isoforms in the process of ROS generation and antioxidant impairment (11, 15).

Our data demonstrate that increased TNF-{alpha} levels and decreased intracellular GSH levels in the heart tissue are consistent with profound oxidative stress and mitochondrial dysfunction. The coordinated regulation of mitochondrial function always depends on its membrane potential and permeability properties (17, 43, 44). Mitochondria have the unique ability to selectively permit various components to pass through their membranes via MPTP opening (7, 32). To confirm that mitochondrial dysfunction and the hyperactivation of the MPTP are associated with the TNF-{alpha}-altered redox state, we induced mitochondrial swelling after isolating intact mitochondria from control and TNF-{alpha}-treated rat hearts. We have noted that dysregulation of antioxidant mechanisms rapidly leads to altered mitochondrial MPTP opening and MPTP structure; similar observations have been made in mitochondria isolated from cardiomyopathic hearts (35). We hypothesize that the increased mitochondrial swelling due to altered MPTP opening could be related to various MPTP complex proteins, namely cyclophilin-D, ANT, and VDAC (30, 31). The decreased expression of ANT, a protein species seen in our Western blots using isolated mitochondrial protein extracts from TNF-{alpha}-treated rat hearts, indicates disorganization of MPTP and demonstrates its direct consequence on mitochondrial permeability. These observations are further strengthened by ultrastructural studies on isolated mitochondria using electron microscopy. Mitochondrial membrane damage and swelling are evident in rat hearts exhibiting TNF-{alpha}-induced oxidative stress. Our data on antioxidant levels and defense-enzyme activities in both the cytosol and mitochondrial fractions further underscore the parallels between oxidative stress and the expression of VDAC and ANT proteins. The functions of these proteins in the oxidative stress response are unclear, but our results augment the possibility that changes in these proteins have a direct impact on MPTP function. Our future interests include investigating the acute toxic effects of supranormal levels of TNF-{alpha} in heart tissue and the resulting effects on mitochondrial MPTP protein degradation. However, increasing evidence supports the dependence of mitochondrial defense mechanisms on the cytosolic pool of reducing equivalents such as GSH. Furthermore, depletion of these equivalents in the cytosol has direct consequences on the mitochondrial redox state. In the current study, both the cytosolic and mitochondrial redox states are significantly altered because of TNF-{alpha} administration and oxidative stress. An important question concerning the potential role of TNF-{alpha}-induced oxidative stress on the mitochondrial defense state in heart tissue is valid in the context of progression of hypertrophy and cardiac dysfunction. Previous studies have demonstrated that cardiac hypertrophy and dysfunction are believed to be correlated with increased oxidative stress (35). In vitro studies have reported a direct role for TNF-{alpha}-mediated mitochondrial GSH depletion in human umbilical vein endothelial cells via generation of excess ROS and subsequent quenching of antioxidant species (6). Decreased GSH content in TNF-{alpha}-treated rat mitochondria could also account for the limited uptake of GSH from the cytosolic pool, because carrier proteins that facilitate GSH transport are damaged. Importantly, we suspect the decreased total GSH content in this organelle might be the direct result of decreased and damaged mitochondrial number in the TNF-{alpha}-treated group. However, at this point, it is unknown how early an acute infusion of TNF-{alpha} could deplete intracellular GSH and its transport into the mitochondria. It is also unclear how early antioxidant imbalance is reversed by Tempol or apocyanin.

The increase in TNF-{alpha} levels is due to ROS generation, which occurs upstream of NF-{kappa}B activation and acts as an intracellular signal by changing the redox status of the cell. The resulting translocation of NF-{kappa}B into the nucleus is proposed to be proinflammatory, either directly or indirectly leading to a significant increase in TNF-{alpha} production as well as inducing the inflammatory response (12, 23, 38). Hence, TNF-{alpha}-treated animals exhibit enhanced production of TNF-{alpha}, whereas Tempol-treated animals exhibit decreased TNF-{alpha} levels. This decrease may be a result of both the attenuated effects of superoxide anions and the formation of hydroxyl radicals, both of which attenuate the cytotoxic effects of hydrogen peroxide. Reductions in NF-{kappa}B activity by Tempol administration have been demonstrated previously (9). However, the exact mechanisms by which Tempol suppresses NF-{kappa}B activation in inflammation are not known. Our gain-of-function studies using supplementation of Tempol or apocyanin to the TNF-{alpha}-treated rat reveal significant insights. Although multiple intracellular sources for oxygen free radical formation exist (e.g., xanthine oxidase, peroxynitrite), our results support the idea that the major enzyme activated by TNF-{alpha} during oxidative stress is NADPH oxidase. Furthermore, superoxide is charged and may not easily diffuse across cell membranes (39). Tempol is a stable radical that permeates the biological membrane and functions as an intracellular scavenger of superoxide anions and other radicals. Our results demonstrate that Tempol prevents mitochondrial damage by its superoxide-scavenging activity and thereby results in maintenance of intracellular and intraorganelle redox status. Our results are well in line with the observed moderate cardiac function in Tempol-supplemented rats. Our results also indicate that the pathway for TNF-{alpha}-induced oxidative stress is mediated by the activation of NADPH oxidase and the subsequent production of superoxide. Administration of apocyanin, a specific NADPH inhibitor that prevents interactions between the NADPH oxidase subunits Nox1 and gp91phox, to TNF-{alpha}-treated animals partially eliminated the TNF-{alpha}-induced oxidative stress-mediated cellular damage by reducing the generation of ROS. Thus Tempol treatment, in conjunction with TNF-{alpha} administration, could minimize myocardial oxidative stress by either scavenging ROS or by decreasing ROS generation. Tempol can easily permeate the mitochondrial membrane; thus quenching of ROS is significantly reflected in both the cytosol and mitochondria of Tempol-treated rats. Conversely, the action of apocyanin is only seen at the cytosolic level, because it cannot effectively cross the mitochondrial membrane (8).

In summary, we demonstrate that TNF-{alpha} administration induces mitochondrial dysfunction by depleting cytosolic and mitochondrial antioxidants, resulting in altered cardiac function. Furthermore, Tempol, a SOD mimetic, can mitigate oxidative stress, improve mitochondrial structural integrity, and restore intracellular and intraorganelle redox status, thus restoring cardiac function, whereas apocyanin does not restore TNF-{alpha}-induced mitochondrial oxidative stress.


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These studies were supported by a grant from the National Heart Lung and Blood Institute (1-RO1-HL-80544–01), an American Heart Association Scientist Development Grant (AHA 0330191N), and Louisiana Board of Regents LEQSF (2005-2007) RD-A-06 for J. Francis.


   ACKNOWLEDGMENTS
 
We thank Sherry Ring for sectioning tissue samples and Julie Millard for help with immunohistochemistry.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Francis, LSU School of Veterinary Medicine, Comparative Biomedical Sciences, Louisiana State Univ., Baton Rouge, LA 70803 (e-mail: jfrancis{at}lsu.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.


   REFERENCES
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  1. Beers RF Jr, Sizer IW. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195: 133–140, 1952.[Free Full Text]
  2. Bhattacharyya S, Mandal D, Sen GS, Pal S, Banerjee S, Lahiry L, Finke JH, Tannenbaum CS, Das T, Sa G. Tumor-induced oxidative stress perturbs nuclear factor-kappaB activity-augmenting tumor necrosis factor-alpha-mediated T-cell death: protection by curcumin. Cancer Res 67: 362–370, 2007.[Abstract/Free Full Text]
  3. Boesten LS, Zadelaar AS, van Nieuwkoop A, Gijbels MJ, de Winther MP, Havekes LM, van Vlijmen BJ. Tumor necrosis factor-alpha promotes atherosclerotic lesion progression in APOE*3-Leiden transgenic mice. Cardiovasc Res 66: 179–185, 2005.[Abstract/Free Full Text]
  4. Bozkurt B, Kribbs SB, Clubb FJ Jr, Michael LH, Didenko VV, Hornsby PJ, Seta Y, Oral H, Spinale FG, Mann DL. Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats. Circulation 97: 1382–1391, 1998.[Abstract/Free Full Text]
  5. Bradham WS, Bozkurt B, Gunasinghe H, Mann D, Spinale FG. Tumor necrosis factor-alpha and myocardial remodeling in progression of heart failure: a current perspective. Cardiovasc Res 53: 822–830, 2002.[Abstract/Free Full Text]
  6. Chen KH, Reece LM, Leary JF. Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction. Free Radic Biol Med 27: 100–109, 1999.[CrossRef][Web of Science][Medline]
  7. Correa F, Soto V, Zazueta C. Mitochondrial permeability transition relevance for apoptotic triggering in the post-ischemic heart. Int J Biochem Cell Biol 39: 787–798, 2007.[CrossRef][Web of Science][Medline]
  8. Cuzzocrea S, McDonald MC, Mazzon E, Dugo L, Lepore V, Fonti MT, Ciccolo A, Terranova ML, Caputi AP, Thiemermann C. Tempol, a membrane-permeable radical scavenger, reduces dinitrobenzene sulfonic acid-induced colitis. Eur J Pharmacol 406: 127–137, 2000.[CrossRef][Web of Science][Medline]
  9. Cuzzocrea S, Pisano B, Dugo L, Ianaro A, Patel NS, Caputi AP, Thiemermann C. Tempol reduces the activation of nuclear factor-kappaB in acute inflammation. Free Radic Res 38: 813–819, 2004.[CrossRef][Web of Science][Medline]
  10. Dujardin KS, Tei C, Yeo TC, Hodge DO, Rossi A, Seward JB. Prognostic value of a Doppler index combining systolic and diastolic performance in idiopathic-dilated cardiomyopathy. Am J Cardiol 82: 1071–1076, 1998.[CrossRef][Web of Science][Medline]
  11. Fernandez-Velasco M, Ruiz-Hurtado G, Hurtado O, Moro MA, Delgado C. TNF-{alpha} down-regulates transient outward potassium current (Ito) in rat ventricular myocytes through iNOS over-expression and oxidant species generation. Am J Physiol Heart Circ Physiol 293: H238–H245, 2007.[Abstract/Free Full Text]
  12. Francis J, Chu Y, Johnson AK, Weiss RM, Felder RB. Acute myocardial infarction induces hypothalamic cytokine synthesis. Am J Physiol Heart Circ Physiol 286: H2264–H2271, 2004.[Abstract/Free Full Text]
  13. Francis J, Wei SG, Weiss RM, Felder RB. Brain angiotensin-converting enzyme activity and autonomic regulation in heart failure. Am J Physiol Heart Circ Physiol 287: H2138–H2146, 2004.[Abstract/Free Full Text]
  14. Francis J, Zhang ZH, Weiss RM, Felder RB. Neural regulation of the proinflammatory cytokine response to acute myocardial infarction. Am J Physiol Heart Circ Physiol 287: H791–H797, 2004.[Abstract/Free Full Text]
  15. Frantz S, Brandes RP, Hu K, Rammelt K, Wolf J, Scheuermann H, Ertl G, Bauersachs J. Left ventricular remodeling after myocardial infarction in mice with targeted deletion of the NADPH oxidase subunit gp91PHOX. Basic Res Cardiol 101: 127–132, 2006.[CrossRef][Web of Science][Medline]
  16. Fu YC, Chi CS, Yin SC, Hwang B, Chiu YT, Hsu SL. Norepinephrine induces apoptosis in neonatal rat cardiomyocytes through a reactive oxygen species-TNF alpha-caspase signaling pathway. Cardiovasc Res 62: 558–567, 2004.[Abstract/Free Full Text]
  17. Gateau-Roesch O, Argaud L, Ovize M. Mitochondrial permeability transition pore and postconditioning. Cardiovasc Res 70: 264–273, 2006.[Abstract/Free Full Text]
  18. Guggilam A, Haque M, Kerut EK, McIiwain E, Lucchesi PA, Sehgal I, Francis J. TNF-{alpha} blockade decreases oxidative stress in the paraventricular nucleus and attenuates sympathoexcitation in heart failure rats. Am J Physiol Heart Circ Physiol 293: H599–H609, 2007.[Abstract/Free Full Text]
  19. Irwin MW, Mak S, Mann DL, Qu R, Penninger JM, Yan A, Dawood F, Wen WH, Shou Z, Liu P. Tissue expression and immunolocalization of tumor necrosis factor-alpha in postinfarction dysfunctional myocardium. Circulation 99: 1492–1498, 1999.[Abstract/Free Full Text]
  20. Jamaluddin M, Wang S, Boldogh I, Tian B, Brasier AR. TNF-alpha-induced NF-kappaB/RelA Ser(276) phosphorylation and enhanceosome formation is mediated by an ROS-dependent PKAc pathway. Cell Signal 19: 1419–1433, 2007.[CrossRef][Web of Science][Medline]
  21. Kang YM, Zhang ZH, Johnson RF, Yu Y, Beltz T, Johnson AK, Weiss RM, Felder RB. Novel effect of mineralocorticoid receptor antagonism to reduce proinflammatory cytokines and hypothalamic activation in rats with ischemia-induced heart failure. Circ Res 99: 758–766, 2006.[Abstract/Free Full Text]
  22. Kaur K, Sharma AK, Dhingra S, Singal PK. Interplay of TNF-alpha and IL-10 in regulating oxidative stress in isolated adult cardiac myocytes. J Mol Cell Cardiol 41: 1023–1030, 2006.[CrossRef][Web of Science][Medline]
  23. Kawano S, Kubota T, Monden Y, Kawamura N, Tsutsui H, Takeshita A, Sunagawa K. Blockade of NF-kappaB ameliorates myocardial hypertrophy in response to chronic infusion of angiotensin II. Cardiovasc Res 67: 689–698, 2005.[Abstract/Free Full Text]
  24. Kimura H, Shintani-Ishida K, Nakajima M, Liu S, Matsumoto K, Yoshida K. Ischemic preconditioning or p38 MAP kinase inhibition attenuates myocardial TNF alpha production and mitochondria damage in brief myocardial ischemia. Life Sci 78: 1901–1910, 2006.[CrossRef][Web of Science][Medline]
  25. Koike N, Takamura T, Kaneko S. Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-alpha stimulation, and effects of a phosphodiesterase inhibitor. Life Sci 80: 1721–1728, 2007.[CrossRef][Web of Science][Medline]
  26. Liakopoulos OJ, Teucher N, Muhlfeld C, Middel P, Heusch G, Schoendube FA, Dorge H. Prevention of TNFalpha-associated myocardial dysfunction resulting from cardiopulmonary bypass and cardioplegic arrest by glucocorticoid treatment. Eur J Cardiothorac Surg 30: 263–270, 2006.[Abstract/Free Full Text]
  27. Moe GW, Marin-Garcia J, Konig A, Goldenthal M, Lu X, Feng Q. In vivo TNF-alpha inhibition ameliorates cardiac mitochondrial dysfunction, oxidative stress, and apoptosis in experimental heart failure. Am J Physiol Heart Circ Physiol 287: H1813–H1820, 2004.[Abstract/Free Full Text]
  28. Nakamura K, Fushimi K, Kouchi H, Mihara K, Miyazaki M, Ohe T, Namba M. Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-alpha and angiotensin II. Circulation 98: 794–799, 1998.[Abstract/Free Full Text]
  29. Nithya M, Suguna L, Rose C. The effect of nerve growth factor on the early responses during the process of wound healing. Biochim Biophys Acta 1620: 25–31, 2003.[Medline]
  30. Oliveira PJ, Wallace KB. Depletion of adenine nucleotide translocator protein in heart mitochondria from doxorubicin-treated rats–relevance for mitochondrial dysfunction. Toxicology 220: 160–168, 2006.[CrossRef][Web of Science][Medline]
  31. Portman MA, Xiao Y, Qian K, Tucker RL, Parish SM, Ning XH. Thyroid hormone coordinates respiratory control maturation and adenine nucleotide translocator expression in heart in vivo. Circulation 102: 1323–1329, 2000.[Abstract/Free Full Text]
  32. Rodriguez-Enriquez S, He L, Lemasters JJ. Role of mitochondrial permeability transition pores in mitochondrial autophagy. Int J Biochem Cell Biol 36: 2463–2472, 2004.[CrossRef][Web of Science][Medline]
  33. Somasundaram P, Sung BH, Mousa SA, Wilson MF. Acute congestive heart failure and tumor necrosis factor alpha. Am J Geriatr Cardiol 9: 76–80, 2000.[Medline]
  34. Suematsu N, Tsutsui H, Wen J, Kang D, Ikeuchi M, Ide T, Hayashidani S, Shiomi T, Kubota T, Hamasaki N, Takeshita A. Oxidative stress mediates tumor necrosis factor-alpha-induced mitochondrial DNA damage and dysfunction in cardiac myocytes. Circulation 107: 1418–1423, 2003.[Abstract/Free Full Text]
  35. Sun M, Chen M, Dawood F, Zurawska U, Li JY, Parker T, Kassiri Z, Kirshenbaum LA, Arnold M, Khokha R, Liu PP. Tumor necrosis factor-alpha mediates cardiac remodeling and ventricular dysfunction after pressure overload state. Circulation 115: 1398–1407, 2007.[Abstract/Free Full Text]
  36. Sun M, Dawood F, Wen WH, Chen M, Dixon I, Kirshenbaum LA, Liu PP. Excessive tumor necrosis factor activation after infarction contributes to susceptibility of myocardial rupture and left ventricular dysfunction. Circulation 110: 3221–3228, 2004.[Abstract/Free Full Text]
  37. Torre-Amione G. Immune activation in chronic heart failure. Am J Cardiol 95: 3C–8C, 2005.[Web of Science][Medline]
  38. Trescher K, Bernecker O, Fellner B, Gyongyosi M, Schafer R, Aharinejad S, DeMartin R, Wolner E, Podesser BK. Inflammation and postinfarct remodeling: overexpression of IkappaB prevents ventricular dilation via increasing TIMP levels. Cardiovasc Res 69: 746–754, 2006.[Abstract/Free Full Text]
  39. Xie H, Ray PE, Short BL. NF-kappaB activation plays a role in superoxide-mediated cerebral endothelial dysfunction after hypoxia/reoxygenation. Stroke 36: 1047–1052, 2005.[Abstract/Free Full Text]
  40. Yan LJ, Rajasekaran NS, Sathyanarayanan S, Benjamin IJ. Mouse HSF1 disruption perturbs redox state and increases mitochondrial oxidative stress in kidney. Antioxid Redox Signal 7: 465–471, 2005.[CrossRef][Web of Science][Medline]
  41. Yan LJ, Sohal RS. Current protocols in protein sciences. New York: John Wiley & Sons, 2000.
  42. Zhang M, Xu YJ, Saini HK, Turan B, Liu PP, Dhalla NS. Pentoxifylline attenuates cardiac dysfunction and reduces TNF-alpha level in ischemic-reperfused heart. Am J Physiol Heart Circ Physiol 289: H832–H839, 2005.[Abstract/Free Full Text]
  43. Zhu WZ, Xie Y, Chen L, Yang HT, Zhou ZN. Intermittent high altitude hypoxia inhibits opening of mitochondrial permeability transition pores against reperfusion injury. J Mol Cell Cardiol 40: 96–106, 2006.[CrossRef][Web of Science][Medline]
  44. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta 1757: 509–517, 2006.[Medline]
  45. Zymek P, Nah DY, Bujak M, Ren G, Koerting A, Leucker T, Huebener P, Taffet G, Entman M, Frangogiannis NG. Interleukin-10 is not a critical regulator of infarct healing and left ventricular remodeling. Cardiovasc Res 74: 313–322, 2006.[Web of Science][Medline]



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