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Pulmonary Division, Department of Medicine, Case Western Reserve University, and MetroHealth Medical Center, Cleveland, Ohio 44109
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Recent work indicates that free radicals mediate sepsis-induced diaphragmatic dysfunction. These previous experiments have not, however, established the source of the responsible free radical species. In theory, this phenomenon could be explained if one postulates that sepsis elicits an upregulation of contraction-related free radical formation in muscle. The purpose of the present study was to test this hypothesis by examination of the effect of sepsis on contraction-related free radical generation [i.e., formation of reactive oxygen species (ROS)] by the diaphragm. Rats were killed 18 h after injection with either saline or endotoxin. In vitro hemidiaphragms were then prepared, and ROS generation during electrically induced contractions (20-Hz trains delivered for 10 min) was assessed by measurement of the conversion of hydroethidine to ethidium. ROS generation was negligible in noncontracting diaphragms from both saline- and endotoxin-treated groups (2.0 ± 0.6 and 2.8 ± 1.0 ng ethidium/mg tissue, respectively), but it was marked in contracting diaphragms from saline-treated animals (19.0 ± 2.8 ng/mg tissue) and even more pronounced (30.0 ± 2.8 ng/mg tissue) in diaphragms from septic animals (P < 0.01). This enhanced free radical generation occurred despite the fact that the force-time integral (i.e., the area under the curve of force vs. time) for control diaphragms was higher than that for the septic group. In additional studies, in which we altered the stimulation paradigm in control muscles to achieve a force-time integral similar to that achieved in septic muscles, an even greater difference between control and septic muscle ROS formation was observed. These data indicate that ROS formation during contraction is markedly enhanced in diaphragms from endotoxin-treated septic animals. We speculate that ROS generated in this fashion plays a central role in producing sepsis-related skeletal muscle dysfunction.
free radicals; skeletal muscle; respiratory muscles
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IN PREVIOUS PUBLICATIONS, we and others have suggested that sepsis-induced muscle dysfunction is secondary, in large part, to the actions of oxygen-derived free radicals (14, 16, 17, 22). Specifically, these past studies found evidence of both contractile dysfunction and lipid peroxidation in the respiratory muscles of septic animals (14, 16, 17, 22). Moreover, these studies demonstrated that administration of free radical scavengers to septic animals largely prevents sepsis-related lipid peroxidation and contractile alterations (14, 16, 22).
In this past work, free radical generation within or by septic muscles was not directly demonstrated, and the cellular mechanisms responsible for heightened formation of free radicals in the respiratory muscles in sepsis were not explored. In other pathological and physiological conditions, however, a number of cellular processes have been identified that appear to induce free radical formation in muscle. For example, limb ischemia, followed by reperfusion, is known to elicit free radical-mediated muscle injury (15). It is conceivable that free radical formation in skeletal muscle during sepsis also represents a form of ischemia-induced injury that arises as a consequence of sepsis-related microvascular blood flow dysregulation. Sepsis is also associated with extravasation of white blood cells into the parenchyma of a number of organs (e.g., lung), and it is possible that infiltration of white blood cells provides a source of free radicals in the muscles of septic animals. Finally, it is known that the respiratory muscles normally generate small quantities of free radicals during contraction, and it is conceivable that this contraction-related formation of free radicals in muscle is upregulated in septic muscle (11a). The purpose of the present study was to examine the possible contribution of the latter process to free radical formation in muscle during sepsis, that is, to determine whether contraction-induced formation of free radicals is heightened in muscles taken from septic animals compared with control animals' muscles that were contracting in response to an identical electrical stimulation paradigm. Studies were performed by using rat diaphragms harvested from control animals, saline-injected animals, or animals made septic by injection of endotoxin. Generation of reactive oxygen species (ROS) within the diaphragm was assessed by using a modification of a recently described assay, in which hydroethidine is infused into tissue and ethidium, the reaction product of ROS (e.g., superoxide, peroxynitrite, hydrogen peroxide) with hydroethidine, is measured (1, 5).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Care
Studies were performed by using 42 adult male Sprague-Dawley rats. These animals were housed in the Animal Resource Center of Case Western Reserve University. As per American Laboratory Animal Care guidelines, rats were examined daily by veterinarians. Food and water were allowed to the animals ad libitum before they were used in experiments.Experimental Preparation
To accomplish the goals of the present study, an arterially perfused, isolated diaphragm muscle preparation, specially developed by our laboratory, was employed (18). Compared with conventional preparations, this experimental model offers several advantages that are especially useful for this particular experiment. First, this preparation permits direct infusion of the fluorescent indicator used in this study (i.e., hydroethidine) into the diaphragm vascular bed and thereby facilitates the achievement of uniform penetration of this agent into diaphragm myocytes. Second, because a large portion of the hemidiaphragm is used in this preparation, sufficient quantities of muscle are available to perform the ethidium assay required in these experiments.At the time of study, rats were anesthetized with halothane and were
then decapitated. The thoracic aorta was cannulated by using a 16-gauge
catheter, and perfusion was initiated [Krebs-Henseleit solution
(in mM): 135 NaCl, 5 KCl, 11.1 dextrose, 2.5 CaCl2, 1 MgSO4, 14.9 NaHCO
3, and 1 NaHPO4, and 50 U/l insulin, 95%
O2-5%
CO2, pH 7.40]. After
the abdomen was opened, the distal aorta was ligated below the exit of
the phrenic arteries, and the heart, lungs, and liver were removed. The
left hemidiaphragm was then isolated. Perfusion from the left
intercostal and phrenic arteries was left intact. At this point, the
right hemidiaphragm was removed, flash frozen in liquid nitrogen, and
stored at 
70°C (to be used as described in
Ethidium Analysis). The left
hemidiaphragm (which was still attached to the lower nine ribs, a
section of the spinal column, and the aorta) was submerged en bloc in
an organ bath that contained Krebs-Henseleit solution, at room
temperature, with 50 mg/l curare added. Once the preparation was
submerged, arterial perfusion was discontinued, and the aortic catheter
was capped. The preparation was then secured to a support in the base of the organ bath by using stainless steel pins. The edges of the
hemidiaphragm were then trimmed away, leaving a large central portion
(~300 mg) of the left costal diaphragm intact. Two ties were placed
in the central tendon of the muscle and connected to a rigid steel rod
suspended from a Grass FT10 force transducer (Grass Instruments,
Quincy, MA) mounted above the organ bath. The transducer was attached
to a transducer positioner (Radnoti Glass, Monrovia, CA) that allowed
adjustment of diaphragm muscle length. Platinum-mesh field electrodes
were positioned on each side of the left costal diaphragm so that the
distance between each electrode and the muscle was ~5 mm. Electrical
cables connected these electrodes to an isolated current-output stage
(Biomedical Technology, Cleveland, OH) that was attached, in turn, to a
Grass S48 stimulator (Grass Instruments).
Ethidium Analysis
The present study employed a recently described fluorescent assay to examine ROS generation in the diaphragm (1, 5). This assay makes use of the fact that hydroethidine reacts with several free radical species (i.e., superoxide, peroxynitrite, hydrogen peroxide, and the hydroxyl radical) to form ethidium. This latter compound fluoresces and can be detected spectrofluorometrically (excitation wavelength, 465 nm; emission wavelength, 585 nm). When this assay is employed, tissue preparations are infused with hydroethidine, and ethidium formation is assessed spectrofluorometrically. Previous investigations have used this assay to measure superoxide formation in the lung after ischemia-reperfusion and to measure superoxide generation in phorbol 12-myristate 13-acetate-activated neutrophils (1, 5). Utilization of this assay to measure generation of free radicals in contracting respiratory muscles represents a further extension of this technique. Because muscle contraction results in widespread alterations to intracellular metabolism, we thought it prudent to conduct a number of preliminary studies to determine 1) whether common cellular constituents (e.g., enzymes, ions) directly interfere with the fluoroscopic assessment of ethidium or hydroethidine concentrations, 2) whether variation in common experimental variables (temperature, storage conditions, etc.) affect ethidium formation, and 3) whether the two most important endogenous free radical scavengers (glutathione and vitamin E) affect the reaction of hydroethidine with free radicals to form ethidium.When examining the effect of common cellular constituents on ethidium
and hydroethidine measurements, we found
1) incubation of hydroethidine and
ethidium in solutions of varying pH and calcium concentration (i.e., a
pH range of 5-8 and calcium concentrations from 0 to 5 mM were
examined) does not substantially alter the fluorescent signals
obtained; 2) incubation in solutions
containing Fe2+, transferrin,
Cu2+/Cu3+,
Zn2+, or lactate dehydrogenase for
30 min did not substantially alter ethidium determinations;
3) incubation in calpain for 30 min
had no significant effect on hydroethidine or ethidium fluorescent signals; and 4) addition of high
concentrations of free phosphate (i.e., either
HPO+4 or
HPO4 had no effect on
ethidium signals, but the addition increased the measured fluorescence
of hydroethidine by eightfold.
We also examined a variety of experimental variables that may affect
our use of this assay and found the following:
1) muscle tissue samples may be
stored for extended periods (up to 2 wk) at 70°C without any
alteration in recovered hydroethidine or ethidium concentrations,
2) there is a small degree of
autooxidation of hydroethidine if this compound is kept in
Krebs-Henseleit solution at 37°C for protracted periods (i.e., a
10% oxidation over 2 h at 37°C) or if it is exposed to fluorescent
lights for long periods, 3) there is
essentially no autoxidation of hydroethidine to ethidium during
incubation at room temperatures in a darkened room for >2 h, and
4) repeated measurement (up to 15 determinations in a spectrofluorophotometer) did not alter
hydroethidine or ethidium concentrations in a sample. The latter result
indicates that this level of exposure to light is insufficient to
promote substantial photobleaching.
It is possible that antioxidant levels in the muscles of rats may rise
or fall during the course of development of sepsis. It is also
theoretically possible for endogenous antioxidants to react with free
radicals before hydroethidine has the chance to do so, thus blunting
formation of ethidium and compromising the ability of our assay to
detect free radical formation. To try to estimate the magnitude of the
potential interference of endogenous antioxidants, we assessed the
formation of ethidium over time by a mixture of hydroethidine and a
superoxide-generating solution (hypoxanthine-xanthine oxidase) in the
presence and absence of two antioxidants [glutathione and Trolox
(a water-soluble analog of vitamin E)]. Figure
1 displays the oxidation of hydroethidine by the hypoxanthine-xanthine oxidase superoxide-generating system alone, with the addition of 1,000 nmol/ml glutathione, and with the
addition of 100 µM Trolox. Neither glutathione nor Trolox affected
the rate or the magnitude of ethidium formation over 5 min. Because of
these results, it seems unlikely that pathophysiologically relevant in
vivo fluctuations of endogenous antioxidants in muscle should have a
significant impact on the reaction of hydroethidine with endogenous
superoxide anions.
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On the basis of our preliminary studies, all experiments were conducted at room temperature (i.e., 20-22°C in our laboratory), exposure of hemidiaphragm preparations to ambient light was minimized, and all tissue preparations for the ethidium assay were carried out on ice in a darkened room. Because hydroethidine signals can be altered by varying muscle phosphate concentrations, we report only the ethidium signal in the present study. For each ethidium concentration assessment, ~75 mg of diaphragm tissue were weighed, pulverized under liquid nitrogen, and homogenized in saline solution. Ethanol was added to the saline mixture (1:1 vol/vol), which was then incubated on ice for 15 min. Each sample was centrifuged at 10,000 g for 15 min. The resulting supernatant was transferred to an Aminco Bowman spectrofluorophotometer (American Instrument, Silver Spring, MD) and was analyzed by using an excitation wavelength of 465 nm and an emission wavelength of 585 nm. The readings obtained were normalized for sample weight. For each experiment, the normalized value of the muscle sample frozen during preparation dissection was subtracted from the value of the sample frozen at the end of the experimental protocol, as described below. This difference, which eliminated tissue autofluorescence unrelated to ethidium formation, was converted to ethidium concentration by means of an authentic ethidium standard curve.
Experimental Protocol
Three experimental protocols were conducted in this study. Each group of studies involved infusion of hydroethidine into diaphragm preparations, application of electrical stimulation to elicit repetitive muscle contraction, and inference of generation of ROS in the diaphragm from determination of muscle ethidium formation. The details of each protocol are described below.Protocol A. To determine whether ROS generation is increased in the muscles of septic animals in response to contraction, four groups of isolated diaphragm preparations were examined in protocol A: 1) noncontracting muscle preparations dissected from control, saline-injected rats, n = 6; 2) noncontracting preparations dissected from septic rats, n = 6; 3) preparations dissected from control rats and then electrically stimulated to undergo repetitive contraction for 10 min, n = 6; and 4) preparations dissected from septic rats and then made to undergo repetitive contraction, n = 6. All experiments were conducted at room temperature (20-22°C). Septic groups received 2 ml of saline subcutaneously (to prevent dehydration) and 8 mg/kg of endotoxin intraperitoneally 18 h before being killed, whereas saline-treated groups received 2 ml of saline subcutaneously and an intraperitoneal saline injection 18 h before they were decapitated.
After dissection of each diaphragm preparation, muscle length was adjusted to the length at which force in response to twitch stimulation was maximal as measured at the central portion of the muscle (Lo), and the current was set to a supramaximal level (i.e., ~20% higher than that required to elicit a maximal twitch force). Each muscle preparation was then allowed to equilibrate at room temperature for 30 min. Hydroethidine (40 µM in 5 ml Krebs-Henseleit solution) was subsequently infused slowly into the diaphragm preparation over the course of 2 min. Because of the large vascular volume of this preparation, due to the presence of a large portion of the left rib cage, an infusion volume of 5 ml was employed to ensure complete penetration of hydroethidine into all diaphragm fibers. After an additional 10-min equilibration period, muscle force-generating capacity was assessed by sequential stimulation of the diaphragm with trains of 1-, 10-, 20-, 50-, and 100-Hz stimuli (800-ms train duration) with 5-s rests between adjacent stimulus trains. After a 30-s rest, muscles were next subjected to a 10-min repetitive stimulation trial (20 Hz, 0.25 trains/s, 500-ms train duration). At the conclusion of this trial, or at the appropriate time in time-matched, nonstimulated preparations, diaphragm muscle length was measured, this muscle was removed from the organ bath, quickly frozen with51°C spray
(Histofreeze, Fisher Scientific), placed in a preweighed tube, and
immersed in liquid nitrogen. Muscle weight was determined by reweighing the sample tube. All samples were stored at 70°C and were
used for subsequent ethidium analysis (as described in
Ethidium Analysis).
Protocol B. Two groups of muscle preparations were studied in protocol B: 1) stimulated preparations from saline-injected rats (n = 6) infused with 10 mM Tiron, an intracellular superoxide scavenger (10), and 2) stimulated preparations from rats (n = 6) to which endotoxin had been administered and which were also infused with 10 mM Tiron. This protocol was identical to that described for described protocol A, except that Tiron was infused at the time of injection of hydroethidine.
Protocol C. In protocol A studies, we found that, during stimulation trials, average force production by septic diaphragms is lower than force production by nonseptic diaphragms. To make a comparison between ROS generation by muscles from control and septic animals under conditions in which average force generation by the two groups of muscles was similar, we performed an additional group of studies. In these studies, diaphragms from control, saline-treated animals were used, and stimulation frequency was reduced so that the force-time integral (FTI, or the area under the curve of force vs. time; this can be viewed as the "total" force generated during a repetitive contraction trial) achieved during repetitive contraction trials in this latter group was similar to that for the septic group of protocol A studies. To accomplish this goal, we carried out an experimental protocol similar to that used for protocol A studies, except that stimulation frequency during the repetitive contraction trial was reduced to 5 Hz.
Data Analysis
Diaphragm force was normalized for muscle cross-sectional area by using the formula (6)
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Statistical Analysis
An unpaired t-test was used to compare single variables (e.g., ethidium levels) across animal groups to determine statistical differences. A repeated-measures ANOVA was used for comparisons in which repeated measurements of a given variable were made under different conditions (e.g., force-frequency curves from different groups). Data are presented as means ± SE. A P value of <0.05 was taken to indicate statistical significance.| |
RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Muscle Characteristics
Diaphragm weights were similar for control and sepsis groups (320 ± 8 and 322 ± 15 mg, respectively). The Lo generation by diaphragm strips was also similar for these two groups of experiments (2.39 ± 0.05 and 2.27 ± 0.05 cm, respectively).Protocol A: Diaphragm Force Generation
In protocol A, the formation of ROS in septic and control diaphragms during repetitive contraction produced in response to the same frequency and stimulation paradigm was compared. In these studies, we found that contraction time (the time required for peak force development during muscle stimulation with a single supramaximal impulse) of diaphragm preparations dissected from septic animals was similar to that of muscles from saline-treated animals (76 ± 1 and 80 ± 2 ms, respectively). Similarly, there was no significant difference in one-half relaxation time (the time required for twitch force to fall by 50% from its peak value) between diaphragm muscles from septic animals (59 ± 2 ms) and muscles from controls (63 ± 2 ms). On the other hand, the force-generating capacity of diaphragms from septic animals was severely reduced compared with forces of muscles from control animals (Fig. 2; P < 0.01 for comparison of force-frequency curves for these two groups of animals). Low-frequency force generation was especially depressed for diaphragms from septic animals. For example, the tension produced by 20-Hz stimulation was 20.12 ± 1.05 N/cm2 for diaphragms from control animals and was only 11.76 ± 2.76 N/cm2 for diaphragms from septic animals (P < 0.01 for this comparison).
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Force generation over time during repetitive contraction trials is
shown in Fig. 3. During these trials, force
generation by muscles from control animals was greater than the force
generated by muscles from endotoxin-treated, septic animals
(P < 0.01 for comparison of
force-time curves for diaphragms from control and septic groups). As a
result, the FTI (i.e., an index of the mean force developed during
trials) tended to be larger for control preparations than for
diaphragms taken from septic animals. Values were 10.1 ± 0.1 and
8.0 ± 0.2 N · min/cm2,
respectively, for these two groups of experiments
(P < 0.001).
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Protocol A: ROS Formation in the Diaphragm
Ethidium levels for noncontracting diaphragm muscle from both control and septic animals were similar and low. This indicates little ROS generation by resting diaphragms from either experimental group (i.e., 2.0 ± 0.6 and 2.8 ± 0.7 ng/mg tissue, respectively). Repetitive-contraction trials induced large increases in ethidium concentrations for diaphragms from saline-treated control animals (19.0 ± 2.8 ng/mg tissue) and even larger ethidium levels (30.0 ± 2.8 ng/mg tissue) in diaphragms from endotoxin-treated septic animals (see Fig. 4 for comparison of septic vs. control experimental groups; P < 0.02 ). This 40% increase in ethidium formation in septic compared with control groups occurred, despite the fact that diaphragms from the septic group produced ~22% less force (i.e., had 22% lower FTIs) than did muscles from saline-treated control animals.
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Protocol B: Effects of Tiron on Ethidium Formation in the Diaphragm
Administration of Tiron, an intracellular superoxide scavenger, prevented ethidium formation by contracting diaphragms from both control and septic animals, as shown in Fig. 5. This finding supports the argument that contraction-related ethidium formation in both control and endotoxin-treated groups is the result of reaction of superoxide or one of its products with hydroethidine.
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Protocol C: ROS Formation in Diaphragms with Low FTIs
Protocol A studies were designed to compare ROS formation by diaphragms from control and endotoxin-treated septic animals when both groups of muscles were activated with a comparable stimulation paradigm. Diaphragms from septic animals examined in this fashion generated less force, but more ROS, over time than did diaphragms from control animals. In protocol C studies, we altered the stimulation paradigm applied to control diaphragms to achieve a force level in these control muscles that was similar to that produced by muscles from septic animals. Results from protocol C experiments are displayed in Fig. 6. For comparison, data from protocol A studies are also displayed in this figure. As per this design, FTIs for diaphragms from control and septic groups (7.7 ± 0.2 vs. 8.0 ± 0.2 N · min/cm2, respectively) were similar for protocol C studies. More importantly, ethidium levels for contracting diaphragms from endotoxin-treated septic animals were more than twice those achieved in protocol C control diaphragms (Fig. 6 B). If we normalize ethidium levels by calculating the ratio of ethidium/FTI (E/FTI), we find that E/FTI was 1.88 ± 0.16 and 3.75 ± 0.21 ng ethidium · cm2/N · min · mg tissue for control and endotoxin-treated groups in protocol A studies (P < 0.001) and was 1.43 ± 0.11 and 3.75 ± 0.21 ng ethidium · cm2/N · min · mg tissue, respectively, for control and septic studies in protocol C (P < 0.001).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study shows that diaphragms taken from septic animals generate more ROS during contraction than do control diaphragms taken from nonseptic animals when electrically stimulated with the same contraction paradigm. This greater ROS formation occurred despite the fact that septic muscles generated less force, per contraction, than did muscles taken from control animals. When the frequency of electrical stimulation applied to muscles from control animals was reduced, so as to more closely match the forces generated by septic muscle, even larger differences in ROS formation were noted between control and septic muscle studies.
Methodological Issues
The major goal of these experiments was to determine whether, for a given contractile stimulus, muscles from septic animals generate greater amounts of ROS compared with muscles from control animals. This goal was complicated by the fact that the force-generating capacity of muscles from septic animals is impaired compared with muscles from controls. If we stimulated control and septic muscles with the same electrical paradigm, force would be different; if we chose to adjust paradigms to match average force, stimulation frequencies would be different. To further complicate matters, if we adjusted stimulation frequencies to match forces initially, this would not ensure forces that were equivalent over time, which would result in trials with both different force generation and different stimulation paradigms. To try to deal with these issues, 1) we used separate protocols (A and C) in which we either matched stimulation paradigm (protocol A) or matched an index of average force generation (protocol C), and 2) we decided to quantitate "total" force generation during trials by calculating the trial FTI (i.e., the area under the curve of force over time). We thought this latter approach was appropriate, because our technique of assessing ROS formation basically "integrates" ROS formation over the entire period of repetitive contraction. It therefore seemed logical that the most appropriate index of force generation to be used would be one that also integrates force over the entire time period of the contraction trial, i.e., the FTI. We found that with both approaches of comparing septic and control muscles (protocols A and C), septic muscles had much higher rates of formation of ROS. We should add that we did not employ force-time curves to infer the relative fatigability of control and septic muscles in this study. These curves were used only to determine FTIs of control and septic preparations.In protocol B, we examined the effects of Tiron, an intracellular superoxide scavenger and iron chelator, on free radical formation. The purpose of these studies was simply to provide additional evidence that the fluorescent marker that was used to detect formation of ROS in the present study (i.e., hydroethidine conversion to ethidium) is, in fact, a genuine marker of formation of superoxide and its reaction products.
Comparison with Previous Studies
Hussain et al. (7) were the first to demonstrate that sepsis induces significant alterations in respiratory muscle function, reducing muscle force-generating capacity to the point that respiratory failure ensues. Subsequent studies performed by our group and others have corroborated these findings and demonstrated large reductions in the force generating capacity of the respiratory muscles after endotoxin-induced sepsis (14, 16, 17, 22). The endotoxin-mediated reductions in diaphragmatic force-generating capacity (Figs. 2 and 3) found in the present study are consistent with these previous reports. That is, we found that administration of endotoxin elicited large reductions in the low-frequency force-generating capacity of the diaphragm and proportionately smaller, but still sizeable, reductions in force generation in response to higher stimulation frequencies. Also, in keeping with previous reports by our laboratory (14), sepsis did not induce significant alterations in diaphragmatic twitch kinetics (i.e., contraction and relaxation times) in the present study.Previous work has also provided a clue as to the likely mechanism by which sepsis induces alterations in respiratory muscle function, an indication that oxygen-derived free radical species play a critical role in the induction of this form of muscle dysfunction. Specifically, these reports have shown that sepsis elicits an increase in free radical-induced protein modification and lipid peroxidation in the respiratory muscles (14, 17, 22) and that administration of a variety of free radical scavengers during the induction of sepsis both suppresses these biochemical alterations and almost completely prevents the development of sepsis-induced muscle dysfunction (14, 16, 22). The scavenger species that have been previously demonstrated to provide this protective effect include superoxide dismutase, catalase, dimethylsulfoxide, and N-acetylcysteine. In addition, administration of inhibitors of nitric oxide synthase (i.e., nitro-L-arginine methyl ester) have also been shown to prevent the development of sepsis-induced diaphragmatic dysfunction (3). The fact that both inhibitors of nitric oxide formation and scavengers of a number of free radical species provide protection argues that this form of muscle damage does not result from the actions of a single free radical species but, more likely, results from the interaction of several free radical species. For example, peroxynitrite, the reaction product of superoxide and nitric oxide, may be a major cause of damage (9).
The present report extends these previous observations by providing evidence that the respiratory muscles themselves are a major source of potentially damaging free radical species during the development of sepsis. These data are also consistent with previous work that showed that free radicals are generated even in normal muscles during strenuous contractions, because ROS generation by "control" muscles increased by eightfold over baseline levels in the present experiments. In view of this latter fact, the present findings could be interpreted as an indication that sepsis simply results in an upregulation of contraction-related free radical generation by muscles. Muscles taken from septic animals generated more free radicals than did muscles from control animals for a given level of muscle contractile activity. Free radicals generated in this manner may react with and alter the function of the sarcoplasmic reticulum and other redox-sensitive organelles involved in muscle contraction, thus accounting for sepsis-related alterations in muscle function (2, 4).
It should be noted that the assay used to detect production of ROS in this study (i.e., conversion of hydroethidine to ethidium) was originally thought to be a specific index of superoxide formation (5). It is now known that a number of ROS, including superoxide, peroxynitrite, hydroxyl ions, and hydrogen peroxide, can all react, to varying degrees, with hydroethidine to generate ethidium (1). Each of these species, however, is derived directly or indirectly from superoxide ions (i.e., peroxynitrite is generated from the reaction of nitric oxide with superoxide, hydrogen peroxide is generated by the catalyzed dismutation of superoxide anions, and hydroxyl radicals are formed by the metal-catalyzed reaction of superoxide and hydrogen peroxide). As a result, superoxide formation is required to generate all of the molecular species known to produce ethidium in this assay, and, in theory, no signal would result in the absence of superoxide formation. In support of this argument, the present experimental data also demonstrate that administration of Tiron, a low- molecular-weight intracellular scavenger of superoxide (8), completely ablates ethidium formation during contraction in both control and septic muscles.
We would argue, therefore, that the present results more specifically point to an upregulation of superoxide formation during contraction in septic muscles. Moreover, in the same way that superoxide is the parent molecule for all of the molecular species detected by the ethidium assay, this compound is also a precursor for all of the molecular species (superoxide, peroxynitrite, hydrogen peroxide, hydroxyl radicals) thought to contribute to free radical-induced muscle dysfunction in sepsis.
Potential Sources of ROS in Septic Muscle
There are several potential explanations for the heightened contraction-related free radical formation by septic muscle that we observed in the present study. One possibility is that the excised muscles we studied contained trapped neutrophils, and the mechanical stresses applied to these cells during muscle contraction evoked free radical elaboration. However, we think this possibility is unlikely, because we found, in a recent study (11), that administration of apocynin, a potent inhibitor of the neutrophil cell surface free radical-generating NADPH complex, had no effect on ROS formation by septic muscle during in vitro contractions. This occurred despite the fact that comparable doses of apocynin totally suppress ROS formation by neutrophils in vitro after phorbol 12-myristate 13-acetate activation.We have recently shown (11a) that ROS formation during in vitro contraction by normal diaphragm is dependent on the activity of phospholipase A2 (PLA2) and can be completely suppressed by the administration of inhibitors of the 14-kDa isoform of this enzyme. It is also known that a number of cytokines, including TNF, can elicit increases in 14-kDa PLA2 levels in several tissues (13). It is reasonable to speculate that cytokines may upregulate tissue levels of PLA2 in the diaphragm after endotoxin administration and that the findings of the present study may be a consequence of cytokine-mediated increases in diaphragm PLA2 activity.
Sepsis may also produce an elaboration of free radical production by the mitochondrial electron-transport chain. It is known that small amounts of superoxide and hydrogen peroxide are produced as a by-product of normal mitochondrial respiration and the magnitude of this ROS "leak" can be increased experimentally by substances that completely or partially block distal components of the electron-transport chain (i.e., distal to the NADH dehydrogenase of complex I) (21). It is conceivable that a similar electron-transport-chain "blockade" occurs in sepsis and that contraction-related increases in mitochondrial respiration evoke increased ROS leak, thus accounting for the findings of the present study. It has been demonstrated that sepsis elicits increases in NOS activity in a variety of tissues (20). It is possible that contraction-related increases in cytosolic calcium are the stimuli for this development. The nitric oxide produced as a result of this heightened activity may react with superoxide to form peroxynitrite. Any one or all of the mechanisms postulated above may contribute to contraction-related ROS formation in muscle taken from septic animals, and additional studies will be needed to evaluate these various possibilities.
Potential Implications
The present findings demonstrate that free radical generation is substantially greater in septic muscles than in nonseptic muscles under conditions of comparable activation. More specifically, these elevated levels of free radical formation for septic muscles were observed during repetitive stimulation in response to trains of 20-Hz stimuli. In addition, heightened free radical formation for septic muscles was observed only with contraction, with low resting levels of free radical formation in both control and septic muscle preparations. It is difficult to compare directly results obtained during in vitro trials with conditions present under in vivo circumstances. Nevertheless, it is important to recognize that the in vivo diaphragm is normally activated at relatively low stimulation frequencies (i.e., 5-10 Hz). Higher levels of activation (e.g., with phrenic motoneuron firing frequencies that approach 20 Hz) are usually observed only in the presence of lung disease. The results of the present study are, therefore, most applicable to conditions in which sepsis both augments the free radical-generating capacity of diaphragm muscle and simultaneously increases the workload of breathing to increase the activation level of this muscle. In addition, the present findings suggest that free radical elaboration and free radical-induced muscle dysfunction can probably be minimized by placing the respiratory muscles at rest. Generation of free radicals by diaphragm muscle in sepsis should be greatly diminished when mechanical ventilation is provided and neural activation of this muscle is minimized.| |
FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Supinski, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109.
Received 15 July 1998; accepted in final form 1 June 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Al-Mehdi, A. B.,
H. Shuman,
and
A. B. Fisher.
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), a mixture of glutathione and xanthine oxidase-hypoxanthine
(
), or a mixture of Trolox and xanthine oxidase-hypoxanthine (
).
Time of incubation is displayed on
x-axis. Addition of glutathione or
Trolox failed to influence formation of ethidium.
