Published ahead of print on June 30, 2005, doi:10.1164/rccm.200410-1344OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200410-1344OC
Sepsis Induces Diaphragm Electron Transport Chain Dysfunction and Protein DepletionDivision of Pulmonary and Critical Care Medicine, Department of Medicine, Medical College of Georgia, Augusta, Georgia Correspondence and requests for reprints should be addressed to Leigh A. Callahan, MD, 1120 15th St, Room BBR-5513, Medical College of Georgia, Augusta GA 30912-3135. E-mail: lcallahan{at}mail.mcg.edu
Rationale: Sepsis significantly alters skeletal muscle mitochondrial function, but the mechanisms responsible for this abnormality are unknown. Objectives: We postulated that endotoxin elicits specific changes in electron transport chain proteins that produce derangements in mitochondrial function. To examine this issue, we compared the effects of endotoxin-induced sepsis on mitochondrial ATP (adenosine triphosphate) formation and electron transport chain protein composition. Methods and Measurements: Diaphragm mitochondrial oxygen consumption and mitochondrial nicotinamide adenine dinucleotide, reduced form, oxidase assays were measured in control rats (n = 13) and rats given endotoxin (8 mg/kg/d) for 12 (n = 14), 24 (n = 14), 36 (n = 14), and 48 h (n = 13). Electron transport chain subunits from Complexes I, III, IV, and V were isolated using Blue Native polyacrylamide gel electrophoresis techniques. Main Results: Endotoxin administration: 1) elicited large reductions in mitochondrial oxygen consumption (e.g., 201 ± 3.9 SE natoms O/min/mg for controls and 101 ± 4.5 SE natoms O/minutes/mg after 48 h endotoxin, p < 0.001), in nicotinamide adenine dinucleotide, reduced form, oxidase activity (p < 0.002), and in uncoupled respiration (p < 0.001) and 2) induced selective reductions in two subunits of Complex I, three subunits of Complex III, one subunit of Complex IV, and one subunit of Complex V. The time course of depletion of protein subunits mirrored alterations in oxygen consumption. Conclusions: Our data indicate that endotoxin selectively depletes critical components of the electron transport chain that diminishes electron flow, reduces proton pumping and decreases ATP formation.
Key Words: Blue-Native PAGE • diaphragm • endotoxin • mitochondria • sepsis • skeletal muscle Recent work suggests that mitochondrial dysfunction may play a central role in producing the biochemical abnormalities that are characteristic of the sepsis syndrome (1–5). For example, studies by Crouser and colleagues show that alterations in tissue oxygen consumption during the development of sepsis correlate with histologic evidence of mitochondrial damage, but have no relationship to levels of oxygen delivery (5). This has led to the assertion that alterations in mitochondrial metabolism may be the major cause of abnormalities in oxygen extraction, oxygen consumption, lactate generation, and organ dysfunction in the sepsis syndrome (2–5). Skeletal muscle comprises approximately 50% of body mass. As a result, abnormalities in tissue oxygen utilization in this large organ account for a significant portion of the metabolic derangements that are associated with the induction of sepsis (4, 6, 7). In keeping with this concept, one recent study found severe reductions in skeletal muscle mitochondrial function were present in many critically ill patients with sepsis, with poor mitochondrial function a predictor of substantially increased mortality (4). Despite these important observations, there is much that remains unknown regarding alterations in mitochondrial function during the development of sepsis or after endotoxin administration. Previous studies have only described sepsis and endotoxin-induced changes in mitochondrial function in gross terms (e.g., overall reductions in oxygen consumption). In theory, sepsis or endotoxin administration could alter mitochondrial oxygen consumption by affecting any one of several cellular pathways (the Krebs cycle, the adenine nucleotide translocase, the electron transport chain, or the adenosine triphosphate [ATP] synthase) by any one of several different biophysical mechanisms (i.e., sidegroup modification of pathway protein constituents, proteolytic degradation of pathway components, disruption of organelle membrane integrity, dissegregation of mulitprotein complexes, activation of specific signaling pathways). The purpose of the present study, therefore, was to test the specific hypothesis that endotoxin administration evokes a depletion of specific electron transport chain complex subunit proteins and that this protein depletion, in turn, causes a severe reduction in the ability of the electron transport chain to accept electrons from nicotinamide adenine dinucleotide, reduced form (NADH). Studies were performed using control rats and rats sacrificed at several time points after endotoxin administration. Our hypothesis was tested by isolating diaphragm mitochondria from control and endotoxin treated animals and making the following comparisons: (1) mitochondrial oxygen consumption, (2) electron transport chain function, and (3) electron transport chain subunit protein composition (8). Some of the results of these studies have previously been presented in abstract form (9).
Animal Preparation Studies were performed in adult male rats (Harlan) weighing between 250 and 350 g in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. Animals were housed in the Animal Resource Center with food and water allowed ad libitum. We studied groups of control animals (n = 13) and animals killed at either 12, 24, 36, or 48 h after endotoxin administration (n = 14, 14, 14, and 13, respectively). Endotoxin (Escherichia coli lipopolysaccharide; Sigma Chemicals, St. Louis, MO) was administered intraperitoneally as 8 mg/kg/d (i.e., a single dose in animals studied at 24 h, two doses for animals studied at 48 h). Control animals were injected with 1 ml saline/d as placebo. All animals received 60 ml/kg/d saline subcutaneously to prevent dehydration. At the time of sacrifice, animals were anesthetized with pentobarbital (50 mg/kg intraperitoneally), the abdomen was opened, and the abdominal aorta was isolated, cannulated, and flushed with mitochondrial isolation buffer (see online supplement for composition). Excised diaphragms were used for immediate mitochondrial isolation or frozen and stored for Blue Native polyacrylamide gel electrophoresis (BN-PAGE) determination.
Diaphragm Mitochondrial Isolation and Measurements of Mitochondrial Oxygen Consumption
NADH Oxidase Assay
Mitochondrial Protein Assessment Using BN-PAGE
Statistical Analysis All data are expressed as mean ± SEM. Statistics were performed using SigmaStat (SPSS Inc.; Chicago, IL). Comparison of parameters across experimental groups was accomplished using analysis of variance; the Student-Neuman-Keuls post hoc test was used to determine significance for individual experimental conditions. A p value < 0.05 was taken to indicate statistical significance. Additional detail on the statistical analysis is provided in an online supplement.
Diaphragm Oxygen Consumption after Endotoxin Administration We observed no significant alterations in any assessed mitochondrial parameters up to 12 h after endotoxin administration. At later time points, however, we observed significant reductions in State 3 respiration rates and in the respiratory control ratio (Table 1 and Figure 1A). Endotoxin administration had no appreciable effect on State 4 respiration rates or on the ADP/oxygen ratio, suggesting that sepsis did not result in "uncoupling" of oxidative phosphorylation (Table 1). Administration of a chemical uncoupling agent (DNP) stimulated respiration to values similar to State 3 respiration rates for samples from both control and endotoxin-treated animals. As a result, respiration rates with addition of the chemical uncoupler DNP were also lower for the endotoxin-treated group as compared with control animals (Table 1).
NADH Oxidase Assay after Endotoxin Administration NADH oxidase assays were performed to exclude the possibility that the lower respiration rates observed for endotoxin treated animals were solely because of depletion of cellular NAD/NADH stores. We found that permeabilization of mitochondria and direct addition of NADH to mitochondria resulted in markedly lower stimulation of oxygen consumption for samples from endotoxin-treated animals when compared with controls (Figure 1B). The time course of endotoxin-induced reductions in mitochondrial NADH oxidase activity paralleled reductions in State 3 respiration (Figure 1B), with no reduction in NADH oxidase activity at 12 h after endotoxin, larger reductions between 12 and 36 h, and maximal reductions at 48 h.
Analysis of Mitochondrial Complex Protein Subunits by BN-PAGE
A representative second-dimension gel comparing Complex I diaphragm mitochondrial samples from a control and an animal treated with endotoxin for 48 h is shown in Figure 3A. Endotoxin resulted in a marked reduction in Complex I NADH dehydrogenase 24 kD subunit and Complex I PDSW subunit protein bands, when compared with control. For comparison, endotoxin did not appear to appreciably alter the level of NADH dehydrogenase 30 kD subunit protein. Group mean densitometry data for Complex I protein subunits is presented in Figure 3B. At 48 h after endotoxin, NADH dehydrogenase 24 kD and PDSW subunit levels fell to 55 ± 5% and 58 ± 7% of control levels (p < 0.05 for both comparisons). In contrast, NADH dehydrogenase 30 kD levels were not significantly different from control values at 48 h.
A representative gel comparing Complex III samples from a control and endotoxin-treated animal is shown in Figure 4A, whereas group mean data are shown in Figure 4. Endotoxin administration evoked significant reductions in three Complex III subunits, including cytochrome b, cytochrome c1, and the Rieske Iron Sulfur Complex protein, which fell to 45 ± 9, 56 ± 6, and 67 ± 4% of control levels, respectively, after 48 h of endotoxin administration (p < 0.05 for each). For comparison, Core I protein level was not altered by endotoxin administration.
Representative and mean data for Complex IV gels are presented in Figure 5. Endotoxin administration evoked a large reduction in the Cox III subunit of complex IV (p < 0.05 for protein level of Cox III at 48 h as compared with control). In addition, endotoxin elicited a large increase in Cox IV subunit levels by 48 h (p < 0.05). This represents the only subunit protein observed to increase in response to endotoxin administration. As a loading control, we also present protein levels for Cox I, which was unaffected by endotoxin administration.
Representative and mean data for Complex V gels are presented in Figure 6. Endotoxin produced a significant reduction in subunit b, which decreased to 40 ± 10% of control at 48 h after endotoxin administration (p < 0.05). For comparison, endotoxin administration had no effect on the  subunit of Complex V.
Note that the time course of the reductions in the content of the seven band found to decrease after endotoxin administration roughly paralleled the alterations we observed in mitochondrial State 3 respiration rates and NADH oxidase activity, with little or no reduction in band intensity at 12 h, and with large reductions in protein content between 36 and 48 h (Figures 3–6).
The major findings of the current study are: (1) diaphragm State 3 mitochondrial oxygen consumption is markedly reduced after endotoxin administration; (2) endotoxin also reduces NADH oxidase activity for diaphragm mitochondrial isolates, suggesting that diaphragm State 3 respiration rates are decreased because of reductions in electron flow through the electron transport chain; and (3) endotoxin produces selective depletion of seven diaphragm electron transport chain proteins, including two subunits of Complex I, three subunits of Complex III, one subunit of Complex IV, and one subunit of Complex V.
Mitochondrial Oxygen Consumption in Sepsis One of the organs whose function is severely compromised in bacterial sepsis and after endotoxin administration is the diaphragm (16). For example, in the dog animal model of endotoxin-induced sepsis, respiratory failure from diaphragmatic dysfunction is the proximate cause of early death (16). In keeping with this observation, diaphragms excised from endotoxin-treated animals generate forces in vitro that are typically a fraction of achieved by specimens from control animals (17, 18). Several abnormalities have been reported to contribute to the development of sepsis and endotoxin-induced diaphragmatic failure, including alterations in diaphragm mitochondrial function (6, 7, 19, 20). Recent studies have reported severe reductions in mitochondrial ATP generation capacity and an abnormally heightened production of free radical species (reactive oxygen species) by diaphragm mitochondrial isolates from endotoxin treated animals (21). Both phenomena may affect diaphragm force generation (22, 23). Although these previous studies demonstrated significant alterations in diaphragm mitochondrial respiration after endotoxin administration, they were limited in scope (6, 7). No previous study performed specific assays to determine if electron transport chain NADH utilization was impaired and none have identified specific electron transport chain protein abnormalities. The present study extends these previous observations. We used a combination of the NADH oxidase assay, conventional assessments of State 3–4 respiration, and assessment of respiration rates in response to addition of a large dose of an exogenous chemical uncoupler to define the type of mitochondrial abnormality responsible for endotoxin-induced alterations in mitochondrial respiration. For the NADH oxidase assay, we permeabilized mitochondria, permitting direct delivery of NADH to the electron transport chain. If the major endotoxin-induced alteration in mitochondrial metabolism was an alteration at "upstream" metabolic sites (e.g., altered activity of Krebs cycle enzymes, impaired NADH transport, inadequate NAD reserves), then isolates from control and endotoxin treated animals should have had identical NADH assay results. Instead, we found that NADH oxidase rates were significantly lower for samples from endotoxin-treated animals, indicating that the endotoxin-induced respiratory defect lies within or distal to the electron transport chain. We also found that respiratory rates in response to administration of DNP, a potent chemical uncoupler, were lower for samples from endotoxin-treated animals than for samples from controls. These data further limit the site of the observed defect, ruling out the possibility that endotoxin-induced alterations are due to changes in ATP synthase or transport of ATP or ADP into and out of mitochondria. That ADP/oxygen ratios were not affected by endotoxin indicates this stress did not alter respiration by causing mitochondrial uncoupling. As a result, our data indicate that endotoxin administration alters diaphragm mitochondrial function and oxygen consumption primarily by reducing electron flow through Complexes I, III, and IV.
Diaphragm Mitochondrial Electron Transport Chain Complex Alterations in Sepsis It is important to consider the potential functional consequences resulting from loss of the particular subunits found to be depleted by endotoxin in this study. Recent work indicates that the NADH dehydrogenase 24-kD subunit of Complex I is critically important for Complex I function (25). Experimental deletion of this protein compromises electron transfer function and impairs assembly of the complex (25). The other subunit in Complex I found to be depleted in this study, PDSW (also known as TYKY), is homologous to a protein present in lower organisms that has been shown to play a critical role in maintaining the connection between the membranous domain and the peripheral domain of Complex I (26–29). As a result, depletion of these two particular proteins would be expected to have significant effects on Complex I assembly and electron transport. The three Complex III proteins found to be depleted in the current study (i.e., cytochrome b, cytochrome c1, and the Rieske iron–sulfur complex protein) compose the operational center of Complex III. During normal function, electrons are first transferred to cytochrome b and then via the iron sulfur complex to cytochrome c1. Cytochrome c1 then acts as an electron donor to cytochrome c and to components of Complex IV (i.e., Cox subunits) (30). Transfer of electrons through the Q centers in cytochrome b generates an electrogenic potential that drives protons across the mitochondrial membrane, producing the mitochondrial transmembrane hydrogen ion electrochemical gradient (30). As a result, depletion of these particular protein subunits would be expected to have major effects on electron transport chain function, impairing electron flow and maintenance of transmembrane potential gradient. The potential effect of depletion of the other subunits noted in Table 2 is less clear. Little is known regarding the Cox III subunit of complex IV, but it is thought that this protein must play some role in complex function or structure because it is highly conserved across species. It has been speculated that this subunit may play a role in proton pumping, but the consequence of loss of this subunit is not known (31, 32). Subunit b of complex V is a structural component of the "stalk" region of this complex connecting the catalytic and membrane portions of ATP synthase (33, 34). The effects produced by depletion of this particular subunit have not been examined. It is worth noting that cytochrome b and Cox III subunits are mitochondrially encoded, whereas the other subunits found to be depleted are encoded by nuclear DNA. In addition, many other nuclear and mitochondrially encoded proteins did not appear to be depleted by our BN-PAGE analysis, so it is difficult to argue that the pattern of subunit depletion observed is just on the basis of reduced translation of proteins from either mitochondrial or nuclear gene pools. On the other hand, five of the depleted proteins contain metal ion complexes (i.e., NADH dehydrogenase 24 kD subunit, cytochrome b, cytochrome c1, Rieske iron–sulfur complex protein, and Cox III) (35–37). In addition, PDSW is intimately bound to two Fe-S complexes in its normal configuration and is highly susceptible to modification by reactive oxygen species (26–29). As a result, one possible explanation for the pattern of protein depletion observed in the present study is that metal centers result in increased susceptibility of proteins to oxidative modification as the result of metal-catalyzed reactions (i.e., the Fenton reaction) using superoxide as a substrate. Such a possibility is in keeping with previous research indicating that mitochondrial superoxide generation is increased after endotoxin and that administration of free radical scavengers preserves mitochondrial function in this condition (6, 7). Yet another possibility is that the seven proteins found to be depleted were selectively targeted for proteolytic degradation by a proteosomal-like process. Mechanisms of mitochondrial protein degradation are not the same as for cytosolic proteins, however, and there is no current evidence that E3 ligases play a role in modulating mitochondrial protein degradation.
Limitations and Potential Implications Another limitation is that our assessment was confined to analysis of diaphragm muscle mitochondria. It is conceivable, however, that endotoxin or bacterial sepsis may also evoke electron transport chain protein depletion in limb skeletal muscle, heart, and other organs. If so, the phenomenon examined in the present study may represent a fairly generalized process responsible for widespread sepsis induced alterations in mitochondrial function. Therapeutic interventions that can prevent these abnormalities may provide a means of reducing sepsis-mediated organ failure.
This article has an online supplement, which is accessible from this issue's table of contents at www.ats.journals.org Conflict of Interest Statement: Neither of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form October 11, 2004; accepted in final form June 28, 2005
This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
METHODS
