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Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis
Free Radical Biology & Medicine 40 (2006) 127 – 137 www.elsevier.com/locate/freeradbiomed
Original Contribution
Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis i Gerald S. Supinski *, Leigh A. Callahan Pulmonary and Critical Care Division, Department of Medicine, 1120 15th St BBR-5513, Medical College of Georgia, Augusta, GA 30912, USA Received 15 March 2005; revised 2 September 2005; accepted 9 September 2005 Available online 17 October 2005
Abstract Free radical-mediated mitochondrial dysfunction may play a role in the genesis of sepsis-induced multiorgan failure. Several cellular defenses protect against free radicals, including heme oxygenase. No previous study has determined if measures that increase heme oxygenase levels reduce mitochondrial dysfunction following endotoxin. The purpose of the present study was to determine if mitochondrial dysfunction following endotoxin (LPS) administration can be attenuated by administration of hemin, a pharmacological inducer of heme oxygenase. Blood pressure, heart rate, cardiac and diaphragm mitochondrial function, plasma nitrite/nitrate levels, and tissue markers of free radical generation were compared among rats given saline, LPS, hemin, or a combination of hemin and LPS. Endotoxin (LPS) administration produced large reductions in mitochondrial function (e.g., ATP production rate decreased in both tissues, P < 0.001). Administration of hemin increased tissue heme oxygenase levels, ablated LPS-induced alterations in mitochondrial function, attenuated LPS-induced increases in plasma nitrite/nitrate levels, and prevented LPS-mediated increases in tissue markers of free radical generation. These data indicate that tissue heme oxygenase levels modulate the degree of LPS-induced mitochondrial dysfunction. Measures that increase heme oxygenase levels may provide a means of reducing sepsis-induced mitochondrial dysfunction and tissue injury. D 2005 Elsevier Inc. All rights reserved. Keywords: Free radicals; Mitochondria; Endotoxin; Sepsis; Heme oxygenase
Introduction Increasing evidence indicates that mitochondria play an important role in modulating the development of manifestations of septic shock in patients with bacterial infection [1– 4]. This work suggests that sepsis-induced mitochondrial alterations may deleteriously influence organ function in three ways. First, sepsis has been shown to elicit significant reductions in mitochondrial ATP generation rates [4]. This impairment may increase cellular reliance on anaerobic pathways of ATP generation during periods of enhanced tissue ATP utilization, leading to intracellular lactate formation and acidosis. Secondly, mitochondrial generation of oxygen-derived free radical species (e.g., superAbbreviations: LPS, lipopolysaccaride; MOPS, 4-morpholinepropanesulfonic acid; FCCP, carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone; RCR, respiratory control ratios; DNP, 2,4-dinitrophenylhydrazine; SDS-PAGE, sodium dodecyl sulfate – polyacrylamide gel electrophoresis; HO1, heme oxygenase 1; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PEG-SOD, polyethylene glycol – superoxide dismutase. i Supported by NHLBI 69821 and 63698. * Corresponding author. Fax: +1 706 721 3069. E-mail address: [email protected] (G.S. Supinski). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.09.025
oxide, hydrogen peroxide) may increase in sepsis [5]. These potentially toxic species can react with multiple cellular constituents, altering the function of critical protein receptors, enzymes, and structural elements. Finally, mitochondrial damage can result in release of cytochrome c, which, in turn, can elicit caspase activation and initiate cell death by activation of proapoptotic pathways [6]. Sepsis-induced organ damage can result from the effects of one or more of these mitochondrially driven alterations in cell function. Currently, no therapies are available that are specifically designed to prevent mitochondrial dysfunction in sepsis. In theory, there are several ways in which this goal may be accomplished. One approach is to administer pharmacological agents that block the cellular signaling pathways responsible for inducing infection-related mitochondrial dysfunction. A second approach is to administer agents that directly detoxify metabolic products (e.g., free radicals) that play an important role in damaging mitochondria in sepsis. A third approach is to administer agents that upregulate endogenous intracellular defenses against cellular toxins. One such agent is hemin, which upregulates cellular levels of heme oxygenase [7]. Heme oxygenase is an important cellular defense against free radical-
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induced tissue damage; this protein catalyzes formation of biliverdin and carbon monoxide, both of which exert significant anti-inflammatory effects. No previous study, however, has examined the effect of administration of hemin on mitochondrial dysfunction in sepsis or a sepsis-like state. In the current study we used endotoxin (LPS) administration to induce a sepsis-like inflammatory state and tested the hypothesis that hemin administration can ameliorate LPS-induced alterations in mitochondrial function. Comparison was made of mitochondrial respiration for tissue samples isolated from saline-injected control rats, rats given hemin alone, rats given LPS, and rats given both LPS and hemin. We expected LPS administration to reduce mitochondrial ATP generation; our hypothesis would be supported if hemin administration prevented this LPS-induced reduction in mitochondrial respiration. Some of the results of these studies have been previously reported in the form of an abstract [8].
mesoporphyrin IX (5 AM/kg, intraperitoneally, with 50 mg/kg hemin, intraperitoneally), an inhibitor of heme oxygenase, affected the response to hemin administration (n = 4). These animals were given hemin and chromium mesoporphyrin IX at time zero and LPS beginning at 24 h after time zero (8 mg/kg/d) and were sacrificed at 72 h into the protocol. All animals in all experiments were also given saline to prevent dehydration (60 ml/kg/day subcutaneously). At the end of all protocols, animals were deeply anesthetized with 60 mg/ kg pentobarbital, the abdomen was opened, the aorta was perfused with isolation buffer (60 ml), and the heart and diaphragms were removed. In the animals used for assessment of plasma nitrite/nitrate levels, cardiac puncture was used to remove blood prior to aortic perfusion. For those experiments in which blood pressure and heart rate were monitored, animals had implantation (under pentobarbital anesthesia) of telemetry monitors attached to an intraarterial probe 10– 14 days prior to implementation of hemin and LPS injections.
Methods Assessment of mitochondrial oxidative phosphorylation Experimental protocol Studies used adult male rats (Harlan, Indianapolis, IN) weighing between 225 and 350 g. Rats were housed in the Animal Resource Center. Food and water were allowed ad lib. All protocols were approved by the Institutional Animal Care and Use Committee. Five groups of studies were performed. In the first group, we determined the time course of effect of endotoxin (LPS) on mitochondrial function by assessing mitochondrial respiration (diaphragm and cardiac) for samples from control animals (n = 3) and from animals at 24, 48, 72, and 96 h after LPS administration (intraperitoneally, 8 mg/kg for the first 2 days, n = 2, 3, 2, and 2, respectively, for 24, 48, 72, and 96-h groups). In a second group of studies, we determined if hemin administration would augment cardiac and diaphragm heme oxygenase 1 (HO1) levels by using Western blots to assess heme oxygenase levels 24 h after administration of saline (0.5 ml intraperitoneally) or hemin (intraperitoneally, 50 mg/kg) (n = 3/group). In a third group of experiments, we examined blood pressure, heart rate, plasma nitrite/nitrate levels, and tissue (diaphragm and heart) heme oxygenase levels (HO1) in groups of salineinjected control animals (n = 3), animals given hemin alone (50 mg/kg) (n = 3), animals given LPS alone (8 mg/kg/day) (n = 3), and animals given both hemin and LPS (n = 3). Hemin or control solution was given at time zero to all animals, LPS or saline given at 24 h after time zero, and animals sacrificed at 72 h (i.e., at 48 h after initiation of LPS administration). In a fourth set of experiments, we compared cardiac and diaphragm mitochondrial function for groups of saline-injected control animals (n = 6), animals given hemin alone (50 mg/kg) (n = 5), animals given LPS alone (8 mg/kg/day) (n = 6), and animals given both hemin and LPS (n = 5). Hemin or control solution was given at time zero to all animals, LPS or saline given at 24 h after time zero, and animals sacrificed at 72 h. In a fifth group of studies, we determined the specificity of the effect of hemin by determining if administration of chromium
Mitochondrial oxygen consumption was assessed using a Clark-type electrode on mitochondrial isolates [9,10]. Following removal from the animal, tissues were rinsed in cold isolation buffer (180 mM KCl, 5 mM MOPS, 2 mM EGTA, pH 7.25, at 4-C), blotted dry, weighed, and placed in fresh isolation buffer. After mincing finely with scissors, muscle pieces were homogenized using a Polytron homogenizer set at 1/2 speed. The homogenate was filtered and then centrifuged at 600 g for 7.5 min at 4-C. The resulting supernatant was centrifuged at 5000 g for 10 min at 4-C, and the isolated mitochondrial pellet gently resuspended in isolation buffer to yield a final mitochondrial protein concentration of 10 – 20 mg/ ml (Bio-Rad protein assay, Hercules, CA). State 3 and state 4 mitochondrial oxygen consumption was measured for mitochondrial suspensions using established procedures [9,10]. Mitochondria were diluted to a protein concentration of 0.5 mg/ml in buffer (120 mM KCl, 5 mM KH2PO4, 5 mM MOPS, 1 mM EDTA, 10 mM pyruvate, 2.5 mM malate, pH 7.25) inside a chamber containing a Clark-type oxygen electrode (Instech, Plymouth Meeting, PA). ADP (0.5 mM) was added to initiate state 3. For uncoupled respiration, 300 AM FCCP (carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone) was used instead of ADP. Respiratory control ratios (RCR) were calculated as the ratio of state 3 to state 4 respiration. The ADP/O ratio, an index of the coupling of electron flow to oxidative phosphorylation, was calculated as the amount of ADP consumed (in moles) per atom of oxygen utilized during state 3 respiration. Assessment of protein carbonyl levels Protein carbonyl content was measured as previously described [11]. For protein carbonyl content, frozen muscle samples were homogenized in 0.05 M K2HPO4, 5 mM EDTA, pH 7.4. Duplicate homogenate aliquots for each tissue specimen were precipitated with trichloroacetic acid and centrifuged; one
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pellet from each pair was resuspended using 2 N HCl containing 0.1% DNP (2,4-dinitrophenylhydrazine) and the second pellet of the pair was resuspended in HCl without DNP. Samples were then incubated at room temperature for 60 min. Sample proteins were precipitated with trichloroacetic acid. Precipitates were washed three times with an ethanol– ethyl acetate mixture (1:1), and dissolved in 6 M guanidine-HCl. Carbonyl levels were calculated from the absorbance (370 nm) of the DNP-derived sample, minus the absorbance of the nonderived sample, normalized for protein level.
3% BSA, 0.05% Tween 20. The membranes were then incubated overnight at 4-C with an anti-HO1 antibody (Stressgen Biotechnologies, Victoria, BC, Canada) diluted 1 Ag/ml in PBS-BSA. Subsequently, membranes were incubated with anti-mouse HRP-conjugated IgG. Antibody binding to proteins was detected using chemiluminescence (NEN Life Science Products, Boston, MA). Gel densitometry was performed using a Microtek scanner (Carson, CA) and UNSCAN-IT software (Silk Scientific, Orem, UT) to determine the density of HO1 bands.
Measurement of lipid peroxidation
Assessment of plasma nitrite/nitrate concentration
Lipid peroxidation was assessed by measuring malondialdehyde and 4-hydroxyalkenal using a technique that is more specific than the usual thiobarbituric acid method [12]. For these assays, muscle samples were washed in ice-cold 20 mM Tris buffer (pH 7.4), blotted, minced in buffer, and homogenized. After centrifugation (3000 g for 10 min) to remove debris, cell supernatant was collected and added (200 Al) to Nmethyl-2-phenylindole in acetonitrile (10.3 mM in 650 Al). Methanesulfonic (15.4 mM) was then added (150 Al) and this mixture was incubated at 45-C for 40 min. Samples were subsequently cooled on ice, absorbance measured at 586 nm, and lipid peroxidation content determined by comparison to a standard curve generated using known concentrations of malondialdehyde and 4-hydroxynonenol.
For experiments in which blood was removed by ventricular puncture, plasma was stored at 80-C and subsequently analyzed for nitrite plus nitrate levels using a Molecular Probes (Eugene, OR) Griess reagent kit per the manufacturer’s instructions. Nitrate was converted to nitrite prior to use of Griess reagent by incubating plasma aliquots with nitrate reductase (0.1 U/ml), 100 AM NADPH, and 10 AM FAD for 10 min at room temperature. Excess NADPH was eliminated by subsequently adding lactate dehydrogenase 10 U/ml and 10 mM sodium pyruvate for a 10-min, room temperature incubation. Samples were then mixed with Griess reagent and nitrite was assessed spectrophotometrically at 548 nm. Nitrite standards (1– 100 AM) were used to calibrate the assay. Statistical analysis
Western blot analysis of heme oxygenase protein levels SDS-PAGE techniques were used to assess tissue levels of heme oxygenase (HO1) protein content [13]. For each determination, sample protein suspensions were loaded onto tandem 12% Tris glycine polyacrylamide gels (60 Ag of protein per lane) and proteins separated by electrophoresis (Novex Minicell II, Carlsbad, CA). Proteins from one gel were visualized with Coomassie blue and their approximate molecular weights determined using scan software (SigmaScan Gel, Chicago, IL) and known standard molecular weight markers. Mitochondrial proteins from the second SDS-PAGE gel were transferred to polyvinylidene fluoride membranes and assessed using Western blotting. In brief, after electroblotting, membranes were washed several times in PBS and then blocked for 1.5 h at room temperature in freshly prepared PBS containing
ANOVA testing (Sigma-Stat Software) was used to compare parameters (state 3, state 4, ATP generation, ADP/O, FCCP respiration) across experimental groups. Post hoc testing (Tukeys) was used to determine differences between individual groups following ANOVA. For comparison of HO1 protein levels, unpaired t tests were used. A P value of less than 0.05 was taken as indicating statistical significance. Data are presented as mean T 1 SE (standard error). Results Time course of LPS-induced mitochondrial dysfunction We first assessed the time course of alterations in cardiac and diaphragm mitochondrial respiration following LPS adminis-
Table 1 Time course of LPS-induced mitochondrial alterations Control
24 h LPS
48 h LPS
72 h LPS
96 h LPS
Diaphragm State 3 rate (natom O/min/mg) State 4 rate (natom O/min/mg) ADP/O
172 T 13 46 T 2 3.15 T 0.03
168 T 6 58 T 5 3.23 T 0.07
90 T 3* 42 T 2 2.56 T 0.14*
87 T 8* 37 T 3 3.07 T 0.01
120 T 11* 46 T 5 3.11 T 0.04
Heart State 3 rate (natom O/min/mg) State 4 rate (natom O/min/mg) ADP/O
403 T 13 70 T 7 3.29 T 0.01
311 T 29 46 T 3 3.46 T 0.05
237 T 8* 51 T 4 2.83 T 0.12*
275 T 19* 43 T 2 3.29 T 0.06
274 T 8* 44 T 7 3.22 T 0.04
* Statistically different from control.
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tration; state 3 respiration rates are presented in Table 1. For both heart and diaphragm, there was a small change in state 3 rates over the first 24 h following the initiation of LPS administration. There was, however, a substantial reduction in state 3 respiration rates by 48 h for both cardiac and diaphragm mitochondria ( P < 0.001 for both comparisons). In addition, the ADP/O ratio at 48 h after LPS administration was significantly lower than for mitochondria from control animals ( P < 0.005 for both cardiac and diaphragm mitochondrial comparisons, Table 1). State 3 respiration rates remained reduced at 48 and 72-h time points ( P < 0.01 for comparison to controls). Mean arterial pressure and heart rate determinations Rats with implanted telemetry monitors were used to determine the effects of LPS and hemin on blood pressure and heart rate. Neither LPS nor hemin administration appeared to have substantial effects on mean arterial pressure, as shown in Fig. 1. On the other hand, LPS treatment did increase rat heart rates, as shown in Fig. 2. For example, heart rate averaged 505 T 11 at 24 h after LPS administration while averaging only 351 T 15 at the same time point for saline-treated control animals ( P < 0.002). Heart rates at the 24-h time point for hemin + LPS and hemin alone treated animals were both significantly lower than heart rates for the LPS group ( P < 0.035), indicating that hemin administration blunted the LPSinduced tachycardia. Plasma nitrite/nitrate concentrations Plasma nitrite/nitrate levels are a general indicator of nitric oxide generation. We found that plasma nitrite/nitrate increased in response to LPS administration (see Fig. 3). Prior
Fig. 2. Heart rate for control, hemin, LPS, and LPS + hemin-treated animals over time after injection of LPS or saline. LPS (endotoxin)-treated animals demonstrated a significant increase in heart rate, with heart rate significantly greater for this group at both 24- and 36-h time points than for saline-treated control animals ( P < 0.002 and P < 0.01, respectively). Heart rates for hemintreated animals fell between control and LPS-treated groups, and were significantly lower than LPS group at the 24-h time point ( P < 0.035).
administration of hemin largely prevented this LPS-induced increase, with nitrite/nitrate levels for the hemin + LPS-treated animals similar to controls and significantly lower than levels for the LPS alone treated group ( P < 0.03). Heme oxygenase concentrations We also sought to determine the effect of our regimen of hemin administration on cardiac and diaphragm heme oxyge-
Fig. 1. Mean arterial pressure for control, hemin, LPS, and LPS + hemin-treated animals over time from the initiation of LPS (endotoxin) injections or timematched saline control injections. Pressures were not substantially different across the experimental groups and LPS administration did not induce significant reductions in mean arterial pressure.
Fig. 3. Plasma levels of nitrite + nitrate for control, hemin, LPS, and LPS + hemin-treated animals. Levels for LPS-treated animals were significantly higher than for controls ( P < 0.03). Hemin administration prevented this LPSinduced increase ( P < 0.03 for comparison of LPS to LPS + hemin groups).
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nase levels. Hemin administration induced large increases in heme oxygenase levels within 24 h in both cardiac and diaphragm tissue, as shown in Fig. 4. On average, hemin administration increased HO1 levels by 44% in cardiac tissue and 126% in diaphragm tissue at 24 h after hemin administration ( P < 0.03 and P < 0.002, respectively). We also measured HO1 levels at the time point at which mitochondrial functional assessments were made (i.e., at 72 h after hemin administration and 48 h after beginning administration of LPS or saline) and these data are presented in Fig. 5. We observed increases in both diaphragm and cardiac HO1 levels, compared to saline-treated controls, in animals receiving either hemin or a combination of hemin plus LPS ( P < 0.05 for comparison of diaphragm levels between control and hemin groups, P < 0.02 for comparison of diaphragm levels for control and hemin + LPS, P < 0.001 for comparison of cardiac levels for control and hemin groups, P < 0.02 for comparison of cardiac levels for control and hemin + LPS). LPS alone also induced a significant increase in diaphragm HO1 levels ( P < 0.02). There was a trend for LPS alone to also increase cardiac HO1 levels, but this latter comparison did not achieve statistical significance. We also found that chromium mesoporphyrin IX administration did not block the effect of hemin to increase HO1 levels. Specifically, diaphragms and hearts from animals given a combination of chromium mesoporphyrin IX, hemin, and endotoxin had HO1 levels significantly greater than controls and equal to or greater than HO1 levels for animals given either hemin, LPS, or hemin + LPS (see Fig. 6).
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Effect of hemin administration on mitochondrial function We next determined the effect of hemin administration on LPS-induced mitochondrial dysfunction. Animals were first given hemin or saline control solution; 24 h later, animals were started on saline injections or injections of LPS. Because administration of endotoxin alone had its biggest effect on mitochondrial function at 48 h, we sacrificed animals at 48 h following initiation of LPS administration (i.e., 72 h after hemin administration). We found that hemin administration almost completely ablated LPS-induced diaphragm and cardiac mitochondrial abnormalities (data presented in Table 2, Figs. 7 – 10). For example, hemin prevented LPS-induced reductions in state 3 rates (Fig. 7, P < 0.001 for comparison of state 3 rates for diaphragm or cardiac mitochondrial samples from LPS treated to samples from LPS + hemin). Hemin, given in the absence of endotoxin, had no effect on state 3 rates, arguing that hemin’s effects in LPS-treated animals were not due to a due to a nonspecific effect to increase mitochondrial respiration rates. LPS also reduced the respiratory control ratio (Table 2), decreased FCCP-stimulated respiratory rates (Table 2), reduced ATP generation rates (Fig. 9), and decreased the ADP/O ratio (Fig. 10). Hemin largely prevented each of these LPS-induced reductions in mitochondrial function (see Table 2 and legends for Figs. 7 – 10). On the other hand, LPS did not significantly alter state 4 respiration rates in either diaphragm or cardiac tissues (Fig. 8). Hemin administration somewhat reduced state
Fig. 4. Heme oxygenase 1 protein levels as assessed by Western blotting. These blots compare diaphragm and heart HO1 levels for saline-treated control animals and animals sacrificed 24 h after hemin administration. HO1 levels increased significantly after hemin administration ( P < 0.002 for diaphragm and P < 0.03 for heart samples).
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Fig. 5. Heme oxygenase 1 protein levels assessed at 48 h after LPS administration (or time-matched saline injections). Comparison is made of HO1 levels for salinetreated controls, animals given hemin alone (i.e., 72 h prior to sacrifice), animals given LPS (48 h earlier), and animals given both hemin and LPS (at 72 and 48 h, respectively, prior to sacrifice). For diaphragm samples, HO1 levels were higher than control following hemin, LPS, or the combination of hemin + LPS ( P < 0.05, P < 0.02, and P < 0.02, respectively). For heart samples, both hemin ( P < 0.001) and hemin + LPS ( P < 0.02) significantly increased HO1 but LPS alone did not increase HO1 levels.
4 rates for cardiac samples (Fig. 8, P < 0.001), but did not significantly alter diaphragm state 4 rates. Hemin, however, had no effect on any other parameter of mitochondrial function in non-LPS-treated animals. Protein carbonyl and lipid peroxidation levels Protein carbonyl levels for diaphragm samples were increased in response to LPS administration and this increase was blocked by concomitant administration of hemin (Fig. 11, P < 0.03 for comparison of carbonyl levels for control and LPS groups, with values for the hemin + LPS group similar to controls). On the other hand, cardiac mitochondrial protein carbonyl levels were not statistically different across the four experimental groups (Fig. 11). Mitochondrial lipid peroxidation levels for cardiac samples did, however, increase in response to LPS administration (Fig. 12) and this increase was suppressed by hemin administration ( P < 0.03). Effect of chromium mesoporphyrin IX administration Chromium mesoporphyrin IX administration inhibits heme oxygenase. We therefore determined the effect of coadministration of hemin and chromium mesoporphyrin IX on the
response to LPS administration in a final group of experiments. This group of animals was initially given both hemin and chromium mesoporphyrin IX; 24 h later, LPS administration was initiated (8 mg/kg/day), and at 48 h after endotoxin administration, animals were sacrificed. In this group of studies, cardiac and diaphragm state 3 respiration rates were similar to those observed for animals given LPS alone and lower ( P < 0.001) than for animals given hemin prior to LPS administration (see Table 3). This group of experiments demonstrates that the protective effect of hemin administration is prevented if heme oxygenase activity is blocked. Discussion In these experiments we found that tissue heme oxygenase concentrations rose dramatically in response to hemin administration, in keeping with previous reports of the effects of this agent to induce tissue heme oxygenase concentrations and activity [7,13]. More importantly, we found that LPS administration evoked large reductions in state 3 respiration and ATP formation rates for cardiac and diaphragm mitochondrial samples from LPS-treated animals in the absence of hemin administration, while LPS administration to hemin-treated animals resulted in virtually no mitochondrial dysfunction.
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Fig. 6. Heme oxygenase 1 protein levels compared among control animals, hemin-treated animals (72 h previously), and animals given chromium mesoporphyrin IX, hemin, and LPS. The combination of chromium mesoporphyrin plus hemin plus LPS resulted in significant increases in HO1 levels for both diaphragm ( P < 0.02) and cardiac ( P < 0.001) tissues when compared to levels in control animals.
Indices of tissue protein and lipid oxidation (i.e., diaphragm protein carbonyl levels and cardiac lipid peroxidation levels) increased in animals given LPS without hemin, but not in animals given both LPS and hemin. Mitochondrial dysfunction in sepsis Multiorgan system failure is a leading cause of death in sepsis, and the pathogenesis of this syndrome is currently under intense investigation [14,15]. Some have argued that this syndrome is largely a consequence of alterations in oxygen delivery due to sepsis-induced microvascular disturbances that limit delivery of oxygen to tissue beds [16]. This concept has been challenged, however, since several reports indicate that
tissue oxygen levels can be well preserved at time points coinciding with the development of significant sepsis-induced tissue injury [1– 4,17]. In addition, administration of cytokines to isolated tissues and cells leads to cellular injury even when oxygen levels remain high [18]. These reports have lead to a renewed interest in the potential role of mitochondrial dysfunction as a mechanism of cell injury in sepsis [1– 4]. Sepsisinduced reductions in mitochondrial function could easily account for the lactate production and intracellular acidosis commonly associated with this syndrome. In addition, mitochondrial generation of toxic free radicals could damage multiple cellular structures while release of cytochrome c from damaged mitochondrial could trigger activation of caspase pathways and induce apoptosis in cells [5,6]. A combination
Table 2 Effect of hemin on endotoxin-induced alterations in RCR and FCCP respiration rate Control
Hemin
LPS
LPS + hemin
Diaphragm RCR FCCP rate (natom O/min/mg)
3.71 T 0.13 197 T 10
3.65 T 0.13 209 T 19
2.16 T 0.07* 84 T 24*
2.72 T 0.12 181 T 27
Heart RCR FCCP rate (natom O/min/mg)
6.38 T 0.18 400 T 22
6.19 T 0.23 379 T 29
5.28 T 0.19* 223 T 22*
6.13 T 0.11 377 T 29
* Statistically different from control.
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Fig. 7. State 3 respiration rates for diaphragm and cardiac mitochondria isolated from (right to left) control, hemin, LPS, and LPS + hemin groups of animals. LPS elicited significant reductions in state 3 respiration for both diaphragm ( P < 0.001) and cardiac ( P < 0.001) samples. Hemin administration prevented LPSinduced reductions in state 3 respiration ( P < 0.001 for diaphragm and P < 0.001 for heart sample comparisons of LPS to hemin + LPS).
Fig. 9. ATP production rates for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. LPS evoked a large reduction in ATP production rates ( P < 0.001 for both diaphragm and cardiac samples). Hemin administration blocked the effects of LPS ( P < 0.001 for comparison of LPS to LPS + hemin for both diaphragm and cardiac samples).
of these factors could be a major contributor to the multiorgan tissue failure syndrome in sepsis, affecting renal, liver, muscle, cardiac, intestinal, and cerebral function. The current study used endotoxin (LPS) to produce a systemic inflammatory state and examined the effects of LPS on cardiac and skeletal muscle, since the most consistent evidence for mitochondrial dysfunction has been observed in these latter two organs [19 –21] and both tissue types are easily amenable to mitochondrial isolation and measurement of
respiration parameters. The effect of LPS on cardiac mitochondrial function has been the subject of several recent studies. For example, Trumbeckaite et al. found that LPS induced marked reductions in cardiac mitochondrial state 3 respiration [19]. The effect of LPS on mitochondrial function in the diaphragm, the primary muscle of the ventilatory pump, has also been the subject of several recent studies [20,22,23]. One such study employed the NADH oxidase assay (in which mitochondria are permeabilized and respiration rates assayed
Fig. 8. State 4 respiration rates for control, hemin, LPS, and hemin + LPStreated groups. LPS did not significantly alter state 4 levels. On the other hand, hemin reduced state 4 levels for cardiac samples ( P < 0.001), but had no significant effect on diaphragm sample state 4 rates.
Fig. 10. ADP/O for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. LPS slightly reduced the ADP/O ratio ( P < 0.001 in diaphragm and P < 0.02 in cardiac tissue). Hemin administration prevented this effect of LPS ( P < 0.04 for both diaphragm and cardiac samples regarding comparison of LPS to LPS + hemin groups).
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Table 3 Effect of chromium mesoporphyrin IX (CrMSPIX) on the response to hemin Control
LPS
LPS + hemin
LPS + hemin + CrMSPIX
Diaphragm State 3 rate 194 T 11 96 T 5* 166 T 13 64 T 4* (natom O/min/mg) State 4 rates 53 T 2 44 T 2* 60 T 3 40 T 2* (natom O/min/mg) ADP/O 3.13 T 0.01 2.69 T 0.09* 2.99 T 0.02 2.53 T 0.04* Heart State 3 rate 420 T 10 263 T 9* 402 T 11 224 T 11* (natom O/min/mg) State 4 rate 69 T 2 50 T 1* 66 T 2 56 T 3* (natom O/min/mg) ADP/O 3.29 T 0.01 3.05 T 0.09* 3.22 T 0.03 2.96 T 0.04* * Statistically different from control.
Fig. 11. Protein carbonyls for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. Protein carbonyl levels for diaphragm samples from LPS animals were significantly greater than levels for controls and significantly lower than for samples from animals treated with both LPS and hemin ( P < 0.03). Protein carbonyl levels for cardiac samples were not significantly different across the four groups.
following direct administration of exogenous NADH to the electron transport chain) to demonstrate that the effects of sepsis on diaphragm mitochondrial function can be primarily attributed to alterations in electron transport chain function rather than alterations in NADH generation and/or transmitochondrial transport [23].
The effects of LPS on mitochondrial function observed in the current study are consistent with these previous reports. We found that mitochondrial state 3 and ATP generation rates were reduced in both cardiac and diaphragm muscles. We also found that respiration of cardiac and diaphragm mitochondrial samples from endotoxin-treated animals were lower than controls following administration of large doses of a chemical uncoupling agent (i.e., FCCP), paralleling LPS-induced reductions in state 3 respiration rates. This latter finding argues that the LPS-induced defect is not related to alterations in ATP synthase (if this were the problem FCCP-induced respiration would be similar for control and sepsis samples) but, rather, is due to a defect at a more proximal portion of the electron transport chain (i.e., before complex IV). Previous studies have also provided important clues as to the process by which LPS/sepsis induces alterations in electron transport chain activity. In the diaphragm and liver, LPS and other models of sepsis evoke a significant increase in mitochondrial free radical generation [5,24]. Moreover, administration of free radical scavengers (i.e., PEG-SOD) has been shown to prevent LPS-induced mitochondrial dysfunction, arguing that free radicals either directly (by reacting with and damaging electron transport chain components) or indirectly (e.g., by activating signaling pathways) are critically important in mediating LPS-induced alterations in mitochondrial function [20]. Heme oxygenase and sepsis
Fig. 12. Lipid peroxide levels for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. Lipid peroxidation was increased for cardiac mitochondrial samples from the LPS-treated animal group ( P < 0.03) and hemin administration prevented this LPS-induced increase ( P < 0.03). Levels were not significantly different for diaphragm samples from the four groups of animals.
Heme oxygenase is an important cellular defense against oxidant-mediated tissue damage [25,26]. This enzyme acts on heme to generate biliverdin (an antioxidant) and carbon monoxide. Recent work suggests that both of these products have cell protective effects. Biliverdin directly scavenges free radicals and free radical products, and has been shown in vitro to prevent damage to cell constituents incubated with oxidants [25,26]. Biliverdin can pass across cell membranes, and can thereby theoretically protect intracellular organelles, such as the mitochondria, even when the biliverdin is generated in the cytosol. Carbon monoxide also has anti-inflammatory effects,
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and has been shown to protect organs (e.g., the lung) in models of oxidant tissue injury [27]. Several reports have used hemin to induce increases in tissue levels of heme oxygenase [7,13]. In one of these reports, hemin administration was found to subsequently blunt the effects of sepsis to reduce diaphragm-force-generating capacity [7]. The present study extends these previous reports by demonstrating that hemin administration upregulates heme oxygenase in both the heart and the diaphragm and prevents LPS-induced alterations in protein oxidation and lipid peroxidation in both tissues. More importantly, this is the first study to demonstrate that hemin administration can preserve mitochondrial function in these organs in a model of systemic inflammation. The fact that hemin prevented alterations in tissue levels of oxidant damage in parallel with an effect to prevent mitochondrial function argues that these two phenomena are linked. It seems likely that the antioxidant effects associated with heme oxygenase upregulation are responsible for both the protective effect of hemin administration on mitochondrial function and its action to prevent protein oxidation/lipid peroxidation.
[10]
Implications
[11]
The present study was limited to the examination of the effects of LPS on diaphragm and heart, but similar patterns of mitochondrial dysfunction have been reported in other organs in other models of sepsis [28]. As a result, upregulation of cellular antioxidant concentrations represents a potential therapeutic approach to preventing sepsis-induced mitochondrial dysfunction throughout the body. Use of standard antioxidants (e.g., vitamin E, N-acetylcysteine) to achieve this goal has substantial limitations, because traditional antioxidants achieve relatively low concentrations in tissue sites (e.g., mitochondria) requiring the highest antioxidant efficacy. On the other hand, pharmacological upregulation of heme oxygenase may represent a strategy that would permit potent and relatively specific inhibition of free radicals within mitochondria. Moreover, heme oxygenase upregulation could theoretically prevent intracellular damage to parenchymal cells in sepsis without preventing cytokine-mediated activation of neutrophils and other elements important for host defense. While the present study presents the potential benefits of using hemin to accomplish upregulation of HO1, liver toxicity with this agent has been reported [29]. In addition, the present data suggests that this particular agent requires a significant time period (24 h) before beneficial effects can be observed. As a result, other agents which upregulate heme oxygenase more rapidly and with fewer potential side effects will be needed before the full potential of this therapeutic strategy (i.e., of upregulating heme oxygenase) can be realized.
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mechanism contributing to organ dysfunction in sepsis. Crit. Care Clin. 17(1):219 – 237; 2001. Singer, M.; Brealey, D. Mitochondrial dysfunction in sepsis. Biochem. Soc. Symp. 66:149 – 166; 1999. Brealey, D.; Brand, M.; Hargreaves, I.; Heales, S.; Land, J.; Smolenski, R.; Davies, N. A.; Cooper, C. E.; Singer, M. Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360(9328):219 – 223; 2002. Nethery, D.; Callahan, L. A.; Stofan, D.; Mattera, R.; DiMarco, A.; Supinski, G. PLA(2) dependence of diaphragm mitochondrial formation of reactive oxygen species. J. Appl. Physiol. 89(1):72 – 80; 2000. Lang-Rollin, I. C.; Rideout, H. J.; Noticewala, M.; Stefanis, L. Mechanisms of caspase-independent neuronal death: energy depletion and free radical generation. J. Neurosci. 23(35):11015 – 11025; 2003. Taille, C.; Foresti, R.; Lanone, S.; Zedda, C.; Green, C.; Aubier, M.; Motterlini, R.; Boczkowski, J. Protective role of heme oxygenases against endotoxin-induced diaphragmatic dysfunction in rats. Am. J. Respir. Crit. Care Med. 63(3 Pt 1):753 – 761; 2001. Callahan, L. A.; Supinski, G. S. Hemin administration prevents sepsis induced mitochondrial alterations. Am. J. Respir. Crit. Care Med. 167:A555; 2003. Lucas, D. T.; Szweda, L. I. Cardiac reperfusion injury: aging, lipid peroxidation, and mitochondrial dysfunction. Proc. Natl. Acad. Sci. USA 95(2):510 – 514; 1998. Humphries, K. M.; Yoo, Y.; Szweda, L. I. Inhibition of NADH-linked mitochondrial respiration by 4-hydroxy-2-nonenal. Biochemistry 37(2):527 – 552; 1998. Reznick, A. Z.; Packer, L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 233:357 – 363; 1994. Meagher, E. A.; FitzGerald, G. A. Indices of lipid peroxidation in vivo: strengths and limitations. Free Radic. Biol. Med. 28(12):1745 – 1750; 2000. Barreiro, E.; Comtois, A. S.; Mohammed, S.; Lands, L. C.; Hussain, S. N. Role of heme oxygenases in sepsis-induced diaphragmatic contractile dysfunction and oxidative stress. Am. J. Physiol. Lung Cell. Mol. Physiol. 283(2):L476 – L484; 2002. Sharma, S.; Kumar, A. Septic shock, multiple organ failure, and acute respiratory distress syndrome. Curr. Opin. Pulm. Med. 9(3):199 – 209; 2003. Van Amersfoort, E. S.; Van Berkel, T. J.; Kuiper, J. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin. Microbiol. Rev. 16(3):379 – 414; 2003. Ince, C.; Sinaasappel, M. Microcirculatory oxygenation and shunting in sepsis and shock. Crit. Care Med. 27:1369 – 1377; 1999. Hotchkiss, R. S.; Rust, R. S.; Dence, C. S.; Wasserman, T. H.; Song, S.-K.; Hwang, D.-R.; Karl, I. E.; Welch, M. J. Evaluation of the role of cellular hypoxia in sepsis by the hypoxic marker [18F]fluoroisonidazole. Am. J. Physiol. 261:R965 – R972; 1991. Zell, R.; Geck, P.; Werden, K.; Boekstegers, P. TNF-alpha and IL-1alpha inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: evidence for primary impairment of mitochondrial function. Mol. Cell. Biochem. 177:61 – 67; 1997. Trumbeckaite, S.; Poalka, J. R.; Neuhof, C.; Zierz, S.; Gellerich, F. N. Different sensitivity of rabbit heart and skeletal muscle to endotoxininduced impairment of mitochondrial function. Eur. J. Biochem. 268:1422 – 1429; 2001. Callahan, L. A.; Stofan, D.; Szweda, L.; Nethery, D.; Supinski, G. S. Free radicals alter maximal diaphragmatic oxygen consumption in endotoxin-induced sepsis. Free Radic. Biol. Med. 30:129 – 138; 2001. Boczkowski, J.; Lisdero, C. L.; Lanone, S.; Samb, A.; Carreras, C.; Boveris, A.; Aubier, M.; Poderoso, J. Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia. FASEB J. 13:1637 – 1647; 1999. Supinski, G. S.; Stofan, D.; Callahan, L. A.; Nethery, D.; DiMarco, A.; Szweda, L. Endotoxin-induced sepsis elicits free radical mediated alterations in diaphragm mitochondrial proteins. FASEB J. 14:A198; 2000.
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Original Contribution
Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis i Gerald S. Supinski *, Leigh A. Callahan Pulmonary and Critical Care Division, Department of Medicine, 1120 15th St BBR-5513, Medical College of Georgia, Augusta, GA 30912, USA Received 15 March 2005; revised 2 September 2005; accepted 9 September 2005 Available online 17 October 2005
Abstract Free radical-mediated mitochondrial dysfunction may play a role in the genesis of sepsis-induced multiorgan failure. Several cellular defenses protect against free radicals, including heme oxygenase. No previous study has determined if measures that increase heme oxygenase levels reduce mitochondrial dysfunction following endotoxin. The purpose of the present study was to determine if mitochondrial dysfunction following endotoxin (LPS) administration can be attenuated by administration of hemin, a pharmacological inducer of heme oxygenase. Blood pressure, heart rate, cardiac and diaphragm mitochondrial function, plasma nitrite/nitrate levels, and tissue markers of free radical generation were compared among rats given saline, LPS, hemin, or a combination of hemin and LPS. Endotoxin (LPS) administration produced large reductions in mitochondrial function (e.g., ATP production rate decreased in both tissues, P < 0.001). Administration of hemin increased tissue heme oxygenase levels, ablated LPS-induced alterations in mitochondrial function, attenuated LPS-induced increases in plasma nitrite/nitrate levels, and prevented LPS-mediated increases in tissue markers of free radical generation. These data indicate that tissue heme oxygenase levels modulate the degree of LPS-induced mitochondrial dysfunction. Measures that increase heme oxygenase levels may provide a means of reducing sepsis-induced mitochondrial dysfunction and tissue injury. D 2005 Elsevier Inc. All rights reserved. Keywords: Free radicals; Mitochondria; Endotoxin; Sepsis; Heme oxygenase
Introduction Increasing evidence indicates that mitochondria play an important role in modulating the development of manifestations of septic shock in patients with bacterial infection [1– 4]. This work suggests that sepsis-induced mitochondrial alterations may deleteriously influence organ function in three ways. First, sepsis has been shown to elicit significant reductions in mitochondrial ATP generation rates [4]. This impairment may increase cellular reliance on anaerobic pathways of ATP generation during periods of enhanced tissue ATP utilization, leading to intracellular lactate formation and acidosis. Secondly, mitochondrial generation of oxygen-derived free radical species (e.g., superAbbreviations: LPS, lipopolysaccaride; MOPS, 4-morpholinepropanesulfonic acid; FCCP, carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone; RCR, respiratory control ratios; DNP, 2,4-dinitrophenylhydrazine; SDS-PAGE, sodium dodecyl sulfate – polyacrylamide gel electrophoresis; HO1, heme oxygenase 1; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PEG-SOD, polyethylene glycol – superoxide dismutase. i Supported by NHLBI 69821 and 63698. * Corresponding author. Fax: +1 706 721 3069. E-mail address: [email protected] (G.S. Supinski). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.09.025
oxide, hydrogen peroxide) may increase in sepsis [5]. These potentially toxic species can react with multiple cellular constituents, altering the function of critical protein receptors, enzymes, and structural elements. Finally, mitochondrial damage can result in release of cytochrome c, which, in turn, can elicit caspase activation and initiate cell death by activation of proapoptotic pathways [6]. Sepsis-induced organ damage can result from the effects of one or more of these mitochondrially driven alterations in cell function. Currently, no therapies are available that are specifically designed to prevent mitochondrial dysfunction in sepsis. In theory, there are several ways in which this goal may be accomplished. One approach is to administer pharmacological agents that block the cellular signaling pathways responsible for inducing infection-related mitochondrial dysfunction. A second approach is to administer agents that directly detoxify metabolic products (e.g., free radicals) that play an important role in damaging mitochondria in sepsis. A third approach is to administer agents that upregulate endogenous intracellular defenses against cellular toxins. One such agent is hemin, which upregulates cellular levels of heme oxygenase [7]. Heme oxygenase is an important cellular defense against free radical-
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induced tissue damage; this protein catalyzes formation of biliverdin and carbon monoxide, both of which exert significant anti-inflammatory effects. No previous study, however, has examined the effect of administration of hemin on mitochondrial dysfunction in sepsis or a sepsis-like state. In the current study we used endotoxin (LPS) administration to induce a sepsis-like inflammatory state and tested the hypothesis that hemin administration can ameliorate LPS-induced alterations in mitochondrial function. Comparison was made of mitochondrial respiration for tissue samples isolated from saline-injected control rats, rats given hemin alone, rats given LPS, and rats given both LPS and hemin. We expected LPS administration to reduce mitochondrial ATP generation; our hypothesis would be supported if hemin administration prevented this LPS-induced reduction in mitochondrial respiration. Some of the results of these studies have been previously reported in the form of an abstract [8].
mesoporphyrin IX (5 AM/kg, intraperitoneally, with 50 mg/kg hemin, intraperitoneally), an inhibitor of heme oxygenase, affected the response to hemin administration (n = 4). These animals were given hemin and chromium mesoporphyrin IX at time zero and LPS beginning at 24 h after time zero (8 mg/kg/d) and were sacrificed at 72 h into the protocol. All animals in all experiments were also given saline to prevent dehydration (60 ml/kg/day subcutaneously). At the end of all protocols, animals were deeply anesthetized with 60 mg/ kg pentobarbital, the abdomen was opened, the aorta was perfused with isolation buffer (60 ml), and the heart and diaphragms were removed. In the animals used for assessment of plasma nitrite/nitrate levels, cardiac puncture was used to remove blood prior to aortic perfusion. For those experiments in which blood pressure and heart rate were monitored, animals had implantation (under pentobarbital anesthesia) of telemetry monitors attached to an intraarterial probe 10– 14 days prior to implementation of hemin and LPS injections.
Methods Assessment of mitochondrial oxidative phosphorylation Experimental protocol Studies used adult male rats (Harlan, Indianapolis, IN) weighing between 225 and 350 g. Rats were housed in the Animal Resource Center. Food and water were allowed ad lib. All protocols were approved by the Institutional Animal Care and Use Committee. Five groups of studies were performed. In the first group, we determined the time course of effect of endotoxin (LPS) on mitochondrial function by assessing mitochondrial respiration (diaphragm and cardiac) for samples from control animals (n = 3) and from animals at 24, 48, 72, and 96 h after LPS administration (intraperitoneally, 8 mg/kg for the first 2 days, n = 2, 3, 2, and 2, respectively, for 24, 48, 72, and 96-h groups). In a second group of studies, we determined if hemin administration would augment cardiac and diaphragm heme oxygenase 1 (HO1) levels by using Western blots to assess heme oxygenase levels 24 h after administration of saline (0.5 ml intraperitoneally) or hemin (intraperitoneally, 50 mg/kg) (n = 3/group). In a third group of experiments, we examined blood pressure, heart rate, plasma nitrite/nitrate levels, and tissue (diaphragm and heart) heme oxygenase levels (HO1) in groups of salineinjected control animals (n = 3), animals given hemin alone (50 mg/kg) (n = 3), animals given LPS alone (8 mg/kg/day) (n = 3), and animals given both hemin and LPS (n = 3). Hemin or control solution was given at time zero to all animals, LPS or saline given at 24 h after time zero, and animals sacrificed at 72 h (i.e., at 48 h after initiation of LPS administration). In a fourth set of experiments, we compared cardiac and diaphragm mitochondrial function for groups of saline-injected control animals (n = 6), animals given hemin alone (50 mg/kg) (n = 5), animals given LPS alone (8 mg/kg/day) (n = 6), and animals given both hemin and LPS (n = 5). Hemin or control solution was given at time zero to all animals, LPS or saline given at 24 h after time zero, and animals sacrificed at 72 h. In a fifth group of studies, we determined the specificity of the effect of hemin by determining if administration of chromium
Mitochondrial oxygen consumption was assessed using a Clark-type electrode on mitochondrial isolates [9,10]. Following removal from the animal, tissues were rinsed in cold isolation buffer (180 mM KCl, 5 mM MOPS, 2 mM EGTA, pH 7.25, at 4-C), blotted dry, weighed, and placed in fresh isolation buffer. After mincing finely with scissors, muscle pieces were homogenized using a Polytron homogenizer set at 1/2 speed. The homogenate was filtered and then centrifuged at 600 g for 7.5 min at 4-C. The resulting supernatant was centrifuged at 5000 g for 10 min at 4-C, and the isolated mitochondrial pellet gently resuspended in isolation buffer to yield a final mitochondrial protein concentration of 10 – 20 mg/ ml (Bio-Rad protein assay, Hercules, CA). State 3 and state 4 mitochondrial oxygen consumption was measured for mitochondrial suspensions using established procedures [9,10]. Mitochondria were diluted to a protein concentration of 0.5 mg/ml in buffer (120 mM KCl, 5 mM KH2PO4, 5 mM MOPS, 1 mM EDTA, 10 mM pyruvate, 2.5 mM malate, pH 7.25) inside a chamber containing a Clark-type oxygen electrode (Instech, Plymouth Meeting, PA). ADP (0.5 mM) was added to initiate state 3. For uncoupled respiration, 300 AM FCCP (carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone) was used instead of ADP. Respiratory control ratios (RCR) were calculated as the ratio of state 3 to state 4 respiration. The ADP/O ratio, an index of the coupling of electron flow to oxidative phosphorylation, was calculated as the amount of ADP consumed (in moles) per atom of oxygen utilized during state 3 respiration. Assessment of protein carbonyl levels Protein carbonyl content was measured as previously described [11]. For protein carbonyl content, frozen muscle samples were homogenized in 0.05 M K2HPO4, 5 mM EDTA, pH 7.4. Duplicate homogenate aliquots for each tissue specimen were precipitated with trichloroacetic acid and centrifuged; one
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pellet from each pair was resuspended using 2 N HCl containing 0.1% DNP (2,4-dinitrophenylhydrazine) and the second pellet of the pair was resuspended in HCl without DNP. Samples were then incubated at room temperature for 60 min. Sample proteins were precipitated with trichloroacetic acid. Precipitates were washed three times with an ethanol– ethyl acetate mixture (1:1), and dissolved in 6 M guanidine-HCl. Carbonyl levels were calculated from the absorbance (370 nm) of the DNP-derived sample, minus the absorbance of the nonderived sample, normalized for protein level.
3% BSA, 0.05% Tween 20. The membranes were then incubated overnight at 4-C with an anti-HO1 antibody (Stressgen Biotechnologies, Victoria, BC, Canada) diluted 1 Ag/ml in PBS-BSA. Subsequently, membranes were incubated with anti-mouse HRP-conjugated IgG. Antibody binding to proteins was detected using chemiluminescence (NEN Life Science Products, Boston, MA). Gel densitometry was performed using a Microtek scanner (Carson, CA) and UNSCAN-IT software (Silk Scientific, Orem, UT) to determine the density of HO1 bands.
Measurement of lipid peroxidation
Assessment of plasma nitrite/nitrate concentration
Lipid peroxidation was assessed by measuring malondialdehyde and 4-hydroxyalkenal using a technique that is more specific than the usual thiobarbituric acid method [12]. For these assays, muscle samples were washed in ice-cold 20 mM Tris buffer (pH 7.4), blotted, minced in buffer, and homogenized. After centrifugation (3000 g for 10 min) to remove debris, cell supernatant was collected and added (200 Al) to Nmethyl-2-phenylindole in acetonitrile (10.3 mM in 650 Al). Methanesulfonic (15.4 mM) was then added (150 Al) and this mixture was incubated at 45-C for 40 min. Samples were subsequently cooled on ice, absorbance measured at 586 nm, and lipid peroxidation content determined by comparison to a standard curve generated using known concentrations of malondialdehyde and 4-hydroxynonenol.
For experiments in which blood was removed by ventricular puncture, plasma was stored at 80-C and subsequently analyzed for nitrite plus nitrate levels using a Molecular Probes (Eugene, OR) Griess reagent kit per the manufacturer’s instructions. Nitrate was converted to nitrite prior to use of Griess reagent by incubating plasma aliquots with nitrate reductase (0.1 U/ml), 100 AM NADPH, and 10 AM FAD for 10 min at room temperature. Excess NADPH was eliminated by subsequently adding lactate dehydrogenase 10 U/ml and 10 mM sodium pyruvate for a 10-min, room temperature incubation. Samples were then mixed with Griess reagent and nitrite was assessed spectrophotometrically at 548 nm. Nitrite standards (1– 100 AM) were used to calibrate the assay. Statistical analysis
Western blot analysis of heme oxygenase protein levels SDS-PAGE techniques were used to assess tissue levels of heme oxygenase (HO1) protein content [13]. For each determination, sample protein suspensions were loaded onto tandem 12% Tris glycine polyacrylamide gels (60 Ag of protein per lane) and proteins separated by electrophoresis (Novex Minicell II, Carlsbad, CA). Proteins from one gel were visualized with Coomassie blue and their approximate molecular weights determined using scan software (SigmaScan Gel, Chicago, IL) and known standard molecular weight markers. Mitochondrial proteins from the second SDS-PAGE gel were transferred to polyvinylidene fluoride membranes and assessed using Western blotting. In brief, after electroblotting, membranes were washed several times in PBS and then blocked for 1.5 h at room temperature in freshly prepared PBS containing
ANOVA testing (Sigma-Stat Software) was used to compare parameters (state 3, state 4, ATP generation, ADP/O, FCCP respiration) across experimental groups. Post hoc testing (Tukeys) was used to determine differences between individual groups following ANOVA. For comparison of HO1 protein levels, unpaired t tests were used. A P value of less than 0.05 was taken as indicating statistical significance. Data are presented as mean T 1 SE (standard error). Results Time course of LPS-induced mitochondrial dysfunction We first assessed the time course of alterations in cardiac and diaphragm mitochondrial respiration following LPS adminis-
Table 1 Time course of LPS-induced mitochondrial alterations Control
24 h LPS
48 h LPS
72 h LPS
96 h LPS
Diaphragm State 3 rate (natom O/min/mg) State 4 rate (natom O/min/mg) ADP/O
172 T 13 46 T 2 3.15 T 0.03
168 T 6 58 T 5 3.23 T 0.07
90 T 3* 42 T 2 2.56 T 0.14*
87 T 8* 37 T 3 3.07 T 0.01
120 T 11* 46 T 5 3.11 T 0.04
Heart State 3 rate (natom O/min/mg) State 4 rate (natom O/min/mg) ADP/O
403 T 13 70 T 7 3.29 T 0.01
311 T 29 46 T 3 3.46 T 0.05
237 T 8* 51 T 4 2.83 T 0.12*
275 T 19* 43 T 2 3.29 T 0.06
274 T 8* 44 T 7 3.22 T 0.04
* Statistically different from control.
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tration; state 3 respiration rates are presented in Table 1. For both heart and diaphragm, there was a small change in state 3 rates over the first 24 h following the initiation of LPS administration. There was, however, a substantial reduction in state 3 respiration rates by 48 h for both cardiac and diaphragm mitochondria ( P < 0.001 for both comparisons). In addition, the ADP/O ratio at 48 h after LPS administration was significantly lower than for mitochondria from control animals ( P < 0.005 for both cardiac and diaphragm mitochondrial comparisons, Table 1). State 3 respiration rates remained reduced at 48 and 72-h time points ( P < 0.01 for comparison to controls). Mean arterial pressure and heart rate determinations Rats with implanted telemetry monitors were used to determine the effects of LPS and hemin on blood pressure and heart rate. Neither LPS nor hemin administration appeared to have substantial effects on mean arterial pressure, as shown in Fig. 1. On the other hand, LPS treatment did increase rat heart rates, as shown in Fig. 2. For example, heart rate averaged 505 T 11 at 24 h after LPS administration while averaging only 351 T 15 at the same time point for saline-treated control animals ( P < 0.002). Heart rates at the 24-h time point for hemin + LPS and hemin alone treated animals were both significantly lower than heart rates for the LPS group ( P < 0.035), indicating that hemin administration blunted the LPSinduced tachycardia. Plasma nitrite/nitrate concentrations Plasma nitrite/nitrate levels are a general indicator of nitric oxide generation. We found that plasma nitrite/nitrate increased in response to LPS administration (see Fig. 3). Prior
Fig. 2. Heart rate for control, hemin, LPS, and LPS + hemin-treated animals over time after injection of LPS or saline. LPS (endotoxin)-treated animals demonstrated a significant increase in heart rate, with heart rate significantly greater for this group at both 24- and 36-h time points than for saline-treated control animals ( P < 0.002 and P < 0.01, respectively). Heart rates for hemintreated animals fell between control and LPS-treated groups, and were significantly lower than LPS group at the 24-h time point ( P < 0.035).
administration of hemin largely prevented this LPS-induced increase, with nitrite/nitrate levels for the hemin + LPS-treated animals similar to controls and significantly lower than levels for the LPS alone treated group ( P < 0.03). Heme oxygenase concentrations We also sought to determine the effect of our regimen of hemin administration on cardiac and diaphragm heme oxyge-
Fig. 1. Mean arterial pressure for control, hemin, LPS, and LPS + hemin-treated animals over time from the initiation of LPS (endotoxin) injections or timematched saline control injections. Pressures were not substantially different across the experimental groups and LPS administration did not induce significant reductions in mean arterial pressure.
Fig. 3. Plasma levels of nitrite + nitrate for control, hemin, LPS, and LPS + hemin-treated animals. Levels for LPS-treated animals were significantly higher than for controls ( P < 0.03). Hemin administration prevented this LPSinduced increase ( P < 0.03 for comparison of LPS to LPS + hemin groups).
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nase levels. Hemin administration induced large increases in heme oxygenase levels within 24 h in both cardiac and diaphragm tissue, as shown in Fig. 4. On average, hemin administration increased HO1 levels by 44% in cardiac tissue and 126% in diaphragm tissue at 24 h after hemin administration ( P < 0.03 and P < 0.002, respectively). We also measured HO1 levels at the time point at which mitochondrial functional assessments were made (i.e., at 72 h after hemin administration and 48 h after beginning administration of LPS or saline) and these data are presented in Fig. 5. We observed increases in both diaphragm and cardiac HO1 levels, compared to saline-treated controls, in animals receiving either hemin or a combination of hemin plus LPS ( P < 0.05 for comparison of diaphragm levels between control and hemin groups, P < 0.02 for comparison of diaphragm levels for control and hemin + LPS, P < 0.001 for comparison of cardiac levels for control and hemin groups, P < 0.02 for comparison of cardiac levels for control and hemin + LPS). LPS alone also induced a significant increase in diaphragm HO1 levels ( P < 0.02). There was a trend for LPS alone to also increase cardiac HO1 levels, but this latter comparison did not achieve statistical significance. We also found that chromium mesoporphyrin IX administration did not block the effect of hemin to increase HO1 levels. Specifically, diaphragms and hearts from animals given a combination of chromium mesoporphyrin IX, hemin, and endotoxin had HO1 levels significantly greater than controls and equal to or greater than HO1 levels for animals given either hemin, LPS, or hemin + LPS (see Fig. 6).
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Effect of hemin administration on mitochondrial function We next determined the effect of hemin administration on LPS-induced mitochondrial dysfunction. Animals were first given hemin or saline control solution; 24 h later, animals were started on saline injections or injections of LPS. Because administration of endotoxin alone had its biggest effect on mitochondrial function at 48 h, we sacrificed animals at 48 h following initiation of LPS administration (i.e., 72 h after hemin administration). We found that hemin administration almost completely ablated LPS-induced diaphragm and cardiac mitochondrial abnormalities (data presented in Table 2, Figs. 7 – 10). For example, hemin prevented LPS-induced reductions in state 3 rates (Fig. 7, P < 0.001 for comparison of state 3 rates for diaphragm or cardiac mitochondrial samples from LPS treated to samples from LPS + hemin). Hemin, given in the absence of endotoxin, had no effect on state 3 rates, arguing that hemin’s effects in LPS-treated animals were not due to a due to a nonspecific effect to increase mitochondrial respiration rates. LPS also reduced the respiratory control ratio (Table 2), decreased FCCP-stimulated respiratory rates (Table 2), reduced ATP generation rates (Fig. 9), and decreased the ADP/O ratio (Fig. 10). Hemin largely prevented each of these LPS-induced reductions in mitochondrial function (see Table 2 and legends for Figs. 7 – 10). On the other hand, LPS did not significantly alter state 4 respiration rates in either diaphragm or cardiac tissues (Fig. 8). Hemin administration somewhat reduced state
Fig. 4. Heme oxygenase 1 protein levels as assessed by Western blotting. These blots compare diaphragm and heart HO1 levels for saline-treated control animals and animals sacrificed 24 h after hemin administration. HO1 levels increased significantly after hemin administration ( P < 0.002 for diaphragm and P < 0.03 for heart samples).
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Fig. 5. Heme oxygenase 1 protein levels assessed at 48 h after LPS administration (or time-matched saline injections). Comparison is made of HO1 levels for salinetreated controls, animals given hemin alone (i.e., 72 h prior to sacrifice), animals given LPS (48 h earlier), and animals given both hemin and LPS (at 72 and 48 h, respectively, prior to sacrifice). For diaphragm samples, HO1 levels were higher than control following hemin, LPS, or the combination of hemin + LPS ( P < 0.05, P < 0.02, and P < 0.02, respectively). For heart samples, both hemin ( P < 0.001) and hemin + LPS ( P < 0.02) significantly increased HO1 but LPS alone did not increase HO1 levels.
4 rates for cardiac samples (Fig. 8, P < 0.001), but did not significantly alter diaphragm state 4 rates. Hemin, however, had no effect on any other parameter of mitochondrial function in non-LPS-treated animals. Protein carbonyl and lipid peroxidation levels Protein carbonyl levels for diaphragm samples were increased in response to LPS administration and this increase was blocked by concomitant administration of hemin (Fig. 11, P < 0.03 for comparison of carbonyl levels for control and LPS groups, with values for the hemin + LPS group similar to controls). On the other hand, cardiac mitochondrial protein carbonyl levels were not statistically different across the four experimental groups (Fig. 11). Mitochondrial lipid peroxidation levels for cardiac samples did, however, increase in response to LPS administration (Fig. 12) and this increase was suppressed by hemin administration ( P < 0.03). Effect of chromium mesoporphyrin IX administration Chromium mesoporphyrin IX administration inhibits heme oxygenase. We therefore determined the effect of coadministration of hemin and chromium mesoporphyrin IX on the
response to LPS administration in a final group of experiments. This group of animals was initially given both hemin and chromium mesoporphyrin IX; 24 h later, LPS administration was initiated (8 mg/kg/day), and at 48 h after endotoxin administration, animals were sacrificed. In this group of studies, cardiac and diaphragm state 3 respiration rates were similar to those observed for animals given LPS alone and lower ( P < 0.001) than for animals given hemin prior to LPS administration (see Table 3). This group of experiments demonstrates that the protective effect of hemin administration is prevented if heme oxygenase activity is blocked. Discussion In these experiments we found that tissue heme oxygenase concentrations rose dramatically in response to hemin administration, in keeping with previous reports of the effects of this agent to induce tissue heme oxygenase concentrations and activity [7,13]. More importantly, we found that LPS administration evoked large reductions in state 3 respiration and ATP formation rates for cardiac and diaphragm mitochondrial samples from LPS-treated animals in the absence of hemin administration, while LPS administration to hemin-treated animals resulted in virtually no mitochondrial dysfunction.
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Fig. 6. Heme oxygenase 1 protein levels compared among control animals, hemin-treated animals (72 h previously), and animals given chromium mesoporphyrin IX, hemin, and LPS. The combination of chromium mesoporphyrin plus hemin plus LPS resulted in significant increases in HO1 levels for both diaphragm ( P < 0.02) and cardiac ( P < 0.001) tissues when compared to levels in control animals.
Indices of tissue protein and lipid oxidation (i.e., diaphragm protein carbonyl levels and cardiac lipid peroxidation levels) increased in animals given LPS without hemin, but not in animals given both LPS and hemin. Mitochondrial dysfunction in sepsis Multiorgan system failure is a leading cause of death in sepsis, and the pathogenesis of this syndrome is currently under intense investigation [14,15]. Some have argued that this syndrome is largely a consequence of alterations in oxygen delivery due to sepsis-induced microvascular disturbances that limit delivery of oxygen to tissue beds [16]. This concept has been challenged, however, since several reports indicate that
tissue oxygen levels can be well preserved at time points coinciding with the development of significant sepsis-induced tissue injury [1– 4,17]. In addition, administration of cytokines to isolated tissues and cells leads to cellular injury even when oxygen levels remain high [18]. These reports have lead to a renewed interest in the potential role of mitochondrial dysfunction as a mechanism of cell injury in sepsis [1– 4]. Sepsisinduced reductions in mitochondrial function could easily account for the lactate production and intracellular acidosis commonly associated with this syndrome. In addition, mitochondrial generation of toxic free radicals could damage multiple cellular structures while release of cytochrome c from damaged mitochondrial could trigger activation of caspase pathways and induce apoptosis in cells [5,6]. A combination
Table 2 Effect of hemin on endotoxin-induced alterations in RCR and FCCP respiration rate Control
Hemin
LPS
LPS + hemin
Diaphragm RCR FCCP rate (natom O/min/mg)
3.71 T 0.13 197 T 10
3.65 T 0.13 209 T 19
2.16 T 0.07* 84 T 24*
2.72 T 0.12 181 T 27
Heart RCR FCCP rate (natom O/min/mg)
6.38 T 0.18 400 T 22
6.19 T 0.23 379 T 29
5.28 T 0.19* 223 T 22*
6.13 T 0.11 377 T 29
* Statistically different from control.
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Fig. 7. State 3 respiration rates for diaphragm and cardiac mitochondria isolated from (right to left) control, hemin, LPS, and LPS + hemin groups of animals. LPS elicited significant reductions in state 3 respiration for both diaphragm ( P < 0.001) and cardiac ( P < 0.001) samples. Hemin administration prevented LPSinduced reductions in state 3 respiration ( P < 0.001 for diaphragm and P < 0.001 for heart sample comparisons of LPS to hemin + LPS).
Fig. 9. ATP production rates for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. LPS evoked a large reduction in ATP production rates ( P < 0.001 for both diaphragm and cardiac samples). Hemin administration blocked the effects of LPS ( P < 0.001 for comparison of LPS to LPS + hemin for both diaphragm and cardiac samples).
of these factors could be a major contributor to the multiorgan tissue failure syndrome in sepsis, affecting renal, liver, muscle, cardiac, intestinal, and cerebral function. The current study used endotoxin (LPS) to produce a systemic inflammatory state and examined the effects of LPS on cardiac and skeletal muscle, since the most consistent evidence for mitochondrial dysfunction has been observed in these latter two organs [19 –21] and both tissue types are easily amenable to mitochondrial isolation and measurement of
respiration parameters. The effect of LPS on cardiac mitochondrial function has been the subject of several recent studies. For example, Trumbeckaite et al. found that LPS induced marked reductions in cardiac mitochondrial state 3 respiration [19]. The effect of LPS on mitochondrial function in the diaphragm, the primary muscle of the ventilatory pump, has also been the subject of several recent studies [20,22,23]. One such study employed the NADH oxidase assay (in which mitochondria are permeabilized and respiration rates assayed
Fig. 8. State 4 respiration rates for control, hemin, LPS, and hemin + LPStreated groups. LPS did not significantly alter state 4 levels. On the other hand, hemin reduced state 4 levels for cardiac samples ( P < 0.001), but had no significant effect on diaphragm sample state 4 rates.
Fig. 10. ADP/O for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. LPS slightly reduced the ADP/O ratio ( P < 0.001 in diaphragm and P < 0.02 in cardiac tissue). Hemin administration prevented this effect of LPS ( P < 0.04 for both diaphragm and cardiac samples regarding comparison of LPS to LPS + hemin groups).
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Table 3 Effect of chromium mesoporphyrin IX (CrMSPIX) on the response to hemin Control
LPS
LPS + hemin
LPS + hemin + CrMSPIX
Diaphragm State 3 rate 194 T 11 96 T 5* 166 T 13 64 T 4* (natom O/min/mg) State 4 rates 53 T 2 44 T 2* 60 T 3 40 T 2* (natom O/min/mg) ADP/O 3.13 T 0.01 2.69 T 0.09* 2.99 T 0.02 2.53 T 0.04* Heart State 3 rate 420 T 10 263 T 9* 402 T 11 224 T 11* (natom O/min/mg) State 4 rate 69 T 2 50 T 1* 66 T 2 56 T 3* (natom O/min/mg) ADP/O 3.29 T 0.01 3.05 T 0.09* 3.22 T 0.03 2.96 T 0.04* * Statistically different from control.
Fig. 11. Protein carbonyls for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. Protein carbonyl levels for diaphragm samples from LPS animals were significantly greater than levels for controls and significantly lower than for samples from animals treated with both LPS and hemin ( P < 0.03). Protein carbonyl levels for cardiac samples were not significantly different across the four groups.
following direct administration of exogenous NADH to the electron transport chain) to demonstrate that the effects of sepsis on diaphragm mitochondrial function can be primarily attributed to alterations in electron transport chain function rather than alterations in NADH generation and/or transmitochondrial transport [23].
The effects of LPS on mitochondrial function observed in the current study are consistent with these previous reports. We found that mitochondrial state 3 and ATP generation rates were reduced in both cardiac and diaphragm muscles. We also found that respiration of cardiac and diaphragm mitochondrial samples from endotoxin-treated animals were lower than controls following administration of large doses of a chemical uncoupling agent (i.e., FCCP), paralleling LPS-induced reductions in state 3 respiration rates. This latter finding argues that the LPS-induced defect is not related to alterations in ATP synthase (if this were the problem FCCP-induced respiration would be similar for control and sepsis samples) but, rather, is due to a defect at a more proximal portion of the electron transport chain (i.e., before complex IV). Previous studies have also provided important clues as to the process by which LPS/sepsis induces alterations in electron transport chain activity. In the diaphragm and liver, LPS and other models of sepsis evoke a significant increase in mitochondrial free radical generation [5,24]. Moreover, administration of free radical scavengers (i.e., PEG-SOD) has been shown to prevent LPS-induced mitochondrial dysfunction, arguing that free radicals either directly (by reacting with and damaging electron transport chain components) or indirectly (e.g., by activating signaling pathways) are critically important in mediating LPS-induced alterations in mitochondrial function [20]. Heme oxygenase and sepsis
Fig. 12. Lipid peroxide levels for diaphragm and cardiac mitochondria from control, hemin, LPS, and LPS + hemin groups. Lipid peroxidation was increased for cardiac mitochondrial samples from the LPS-treated animal group ( P < 0.03) and hemin administration prevented this LPS-induced increase ( P < 0.03). Levels were not significantly different for diaphragm samples from the four groups of animals.
Heme oxygenase is an important cellular defense against oxidant-mediated tissue damage [25,26]. This enzyme acts on heme to generate biliverdin (an antioxidant) and carbon monoxide. Recent work suggests that both of these products have cell protective effects. Biliverdin directly scavenges free radicals and free radical products, and has been shown in vitro to prevent damage to cell constituents incubated with oxidants [25,26]. Biliverdin can pass across cell membranes, and can thereby theoretically protect intracellular organelles, such as the mitochondria, even when the biliverdin is generated in the cytosol. Carbon monoxide also has anti-inflammatory effects,
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and has been shown to protect organs (e.g., the lung) in models of oxidant tissue injury [27]. Several reports have used hemin to induce increases in tissue levels of heme oxygenase [7,13]. In one of these reports, hemin administration was found to subsequently blunt the effects of sepsis to reduce diaphragm-force-generating capacity [7]. The present study extends these previous reports by demonstrating that hemin administration upregulates heme oxygenase in both the heart and the diaphragm and prevents LPS-induced alterations in protein oxidation and lipid peroxidation in both tissues. More importantly, this is the first study to demonstrate that hemin administration can preserve mitochondrial function in these organs in a model of systemic inflammation. The fact that hemin prevented alterations in tissue levels of oxidant damage in parallel with an effect to prevent mitochondrial function argues that these two phenomena are linked. It seems likely that the antioxidant effects associated with heme oxygenase upregulation are responsible for both the protective effect of hemin administration on mitochondrial function and its action to prevent protein oxidation/lipid peroxidation.
[10]
Implications
[11]
The present study was limited to the examination of the effects of LPS on diaphragm and heart, but similar patterns of mitochondrial dysfunction have been reported in other organs in other models of sepsis [28]. As a result, upregulation of cellular antioxidant concentrations represents a potential therapeutic approach to preventing sepsis-induced mitochondrial dysfunction throughout the body. Use of standard antioxidants (e.g., vitamin E, N-acetylcysteine) to achieve this goal has substantial limitations, because traditional antioxidants achieve relatively low concentrations in tissue sites (e.g., mitochondria) requiring the highest antioxidant efficacy. On the other hand, pharmacological upregulation of heme oxygenase may represent a strategy that would permit potent and relatively specific inhibition of free radicals within mitochondria. Moreover, heme oxygenase upregulation could theoretically prevent intracellular damage to parenchymal cells in sepsis without preventing cytokine-mediated activation of neutrophils and other elements important for host defense. While the present study presents the potential benefits of using hemin to accomplish upregulation of HO1, liver toxicity with this agent has been reported [29]. In addition, the present data suggests that this particular agent requires a significant time period (24 h) before beneficial effects can be observed. As a result, other agents which upregulate heme oxygenase more rapidly and with fewer potential side effects will be needed before the full potential of this therapeutic strategy (i.e., of upregulating heme oxygenase) can be realized.
[3] [4]
[5]
[6]
[7]
[8]
[9]
[12]
[13]
[14]
[15]
[16] [17]
[18]
[19]
[20]
[21]
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