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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 radical-mediated lipid peroxidation takes place within the diaphragm on strenuous contraction. This phenomenon has only been demonstrated using fairly artificial experimental models and has not been studied during the type of sustained respiratory loading typically seen in patients with lung disease. The purpose of the present study was to measure the levels of several biochemical markers of protein oxidation (protein carbonyl levels) and lipid peroxidation (8-isoprostane, reduced glutathione, and oxidized glutathione levels) in diaphragms of rats subjected to chronic respiratory loading. Respiratory loading was accomplished by tracheal banding; groups of animals were loaded for 4, 8, or 12 days, and a group of sham-operated unloaded animals was used as controls. After loading, animals were killed, diaphragm contractility was assessed in vitro by using a portion of the excised diaphragm, and the remaining diaphragm and the soleus muscles were used for biochemical analysis. We found diminished force generation in diaphragms from all groups of banded animals compared with muscles from controls. For example, twitch force averaged 7.8 ± 0.8 (SE) N/cm2 in unloaded animals and 4.0 ± 0.4, 3.0 ± 0.4, and 3.4 ± 0.4 N/cm2 in animals loaded for 4, 8, and 12 days, respectively (P < 0.0001). Loading also elicited increases in diaphragmatic protein carbonyl concentrations (P < 0.001), and the time course of alterations in carbonyl levels paralleled loading-induced alterations in the diaphragm force-frequency relationship. Although loading was also associated with increases in diaphragmatic 8-isoprostane levels (P < 0.003) and reductions in diaphragm reduced glutathione levels (P < 0.003), the time course of changes in these latter parameters did not correspond to alterations in force. Soleus glutathione and carbonyl levels were not altered by banding. We speculate that respiratory loading-induced alterations in diaphragmatic force generation may be related to free radical-mediated protein oxidation, but not to free radical-induced lipid peroxidation.
free radicals; skeletal muscle; diaphragm; respiratory muscles
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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RECENT WORK INDICATES that free radicals are generated in the diaphragm when the respiratory system is acutely loaded (1-3, 6, 29). Previous evidence supporting this contention includes data from experiments in which free radical production in the diaphragms of loaded rats was measured directly using electron spin resonance techniques (3) and data from studies that detected alterations in several indexes of lipid peroxidation [i.e., increases in malondialdehyde levels, reductions in reduced glutathione (GSH) concentrations, and increases in oxidized glutathione (GSSG) concentrations] in the diaphragms of loaded animals (1, 2, 6, 29). In these latter experiments, increased lipid peroxidation was taken as an indication that loaded breathing activates the glutathione redox cycle within the diaphragm.
These previous studies, however, examined only brief periods of loading (i.e., minutes to several hours), whereas the loads imposed by lung diseases on patients are typically elevated for days to weeks. Moreover, in these previous laboratory models, massive levels of respiratory loading were studied, resulting in respiratory failure far more quickly than is usually observed in clinical disease. It is not clear from these previous data that imposition of more clinically relevant respiratory loads (i.e., lower loads for longer periods of time) would result in sufficient oxidative stress to result in any detectable lipid peroxidation or protein oxidation in the respiratory muscles. It is possible that such lower, more physiological levels of loading may fail to increase the rate of respiratory muscle free radical formation or, alternatively, may induce radical formation at such a low rate that endogenous scavenger systems (e.g., mitochondrial antioxidants) could easily protect muscles, preventing appreciable accumulation of lipid peroxidation and protein oxidation products.
The purpose of the present study was to determine whether, in fact, lower-level, chronic respiratory loading does result in sufficient oxidative stress to elicit measurable alterations of the protein and lipid constituents of the diaphragm. These studies were performed in rats in which chronic respiratory loading was accomplished using tracheal banding techniques (31). Indexes of protein and lipid modification assessed included 1) diaphragm 8-isoprostane levels, a measure of lipid peroxidation; 2) muscle GSH + GSSG concentration; and 3) diaphragm protein carbonyl levels. We compared these biochemical indexes for unloaded, control animals with animals banded for 4, 8, and 12 days.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Tracheal banding. This study was approved by the Case Western Reserve University Institutional Animal Care and Use Committee. All animals were cared for in a university-sponsored animal facility and examined daily by a veterinarian. A total of 24 rats were studied, divided evenly into unloaded, sham-operated control animals and groups of animals loaded for 4, 8, and 12 days by tracheal banding. All studies utilized adult Zivic-Miller rats that weighed 200-350 g before banding (or sham surgery). Animals were fed rat chow and water ad libitum, and their movement around their cages was unrestricted.
Food was removed from the animal cages at 5 PM on the day before surgery. On the next day, rats were anesthetized by placing them in a chamber containing halothane, with additional halothane delivered during the procedure via a face mask. Animals were subsequently injected with atropine (0.04 mg im) to reduce bronchospasm and arrhythmias due to tracheal manipulation. Tracheal banding was then performed using sterile technique (31). The neck was first prepared with betadine, a small incision was made to expose the trachea, and a small clamp was used to pass a 2-0 silk suture around the trachea. The band (a 4-mm-long, 2.5-mm-ID piece of plastic tubing) was then placed around the trachea and secured in place with silk suture. Esophageal pressure was monitored during the banding procedure by use of a water-filled polyethylene catheter (0.76 mm ID) attached to a Validyne force transducer (±100 cmH2O), and the suture tension was adjusted during load placement to approximately double the esophageal pressures generated by the spontaneously breathing animals. Powdered ampicillin was placed in the wound, and the skin incision was closed with three small sutures. Anesthesia was then withdrawn, the mouth was suctioned, and the animals were placed in an oxygen tent until vigorous spontaneous breathing efforts returned (i.e., ~15 min). This oxygen tent was infused with a supply of oxygen but was not completely closed; oxygen concentrations in this tent were ~50% by gas analysis. After completion of the surgical procedure, banded animals were given water and food ad libitum. In a few preliminary studies in which we weighed loaded animals and their food intake daily, we observed a reduction in food intake over the first 2-3 days after banding. We therefore limited the food intake of control animals in this study to approximately match the intake of banded animals (i.e., for controls, food was not given on the 1st day after surgery, 4 g were provided on day 2 and 7 g on day 3, and 20 g/day were given thereafter). All animals were weighed every other day, food supplies were weighed daily, and food intake per day was calculated. No deaths occurred in animals used for this experiment. All animals used for data collection maintained acceptable grooming, slowly gained weight, and continued to ambulate within their cages.Killing of animals.
At the end of the desired period of loading, animals were anesthetized
using halothane, the abdominal cavity was entered, and the entire
diaphragm was rapidly excised. Both soleus muscles were also removed. A
portion of the costal diaphragm was placed in a dissecting dish
containing gassed Krebs-Henseleit solution (95% oxygen-5% carbon
dioxide, pH 7.40, consisting of 135 mM NaCl, 5 mM KCl, 11.1 mM
dextrose, 2.5 mM
CaCl2, 1 mM
MgSO4, 14.9 mM NaHCO3, 1 mM
NaHPO4, 50 U/l insulin, and 16 mg/l d-tubocurare) and subsequently
used for assessment of in vitro force generation. The remaining costal
diaphragm and soleus muscles were rapidly frozen in liquid nitrogen and
stored (at 
70°C) until subsequent biochemical analysis. As
part of this procedure, frozen and unfrozen tissues were weighed, and
total diaphragm, soleus, and animal weights were recorded at the time
the animals were killed. We chose costal diaphragm to study, rather
than other portions of this muscle (e.g., crural diaphragm), because
1) muscle fibers in the costal
portion of the diaphragm are nearly parallel in orientation,
facilitating dissection and performance of in vitro physiological
assessment, and 2) the physiological
role of the costal portion of the diaphragm is well described, with
this muscle known to play an important role in enabling inspiration
during respiratory loading. The soleus, a slow-twitch limb muscle, has been extensively used in previous respiratory muscle studies as a limb
muscle "control" (28), and we chose this particular limb muscle
to study in keeping with this convention.
In vitro assessment of diaphragm force generation. For this assessment, diaphragm muscle strips were cut from the excised portion of the diaphragm placed in the dissecting bath. These strips were then mounted vertically in an organ bath containing Krebs-Henseleit solution (27°C) that was bubbled with a 95% oxygen-5% carbon dioxide gas mixture. The origin of each strip was tied to the base of the bath, and the insertion was attached to a metal rod connected to a Grass FT10 force transducer. Platinum field electrodes placed around strips were used to deliver current from a constant-current amplifier (Applied Neural Control Laboratories, Case Western Reserve University) driven by a Grass S48 stimulator. Muscle strip length was adjusted to the length at which isometric twitch tension was constant. Amplifier current level was then increased so that it was ~20% greater than that required to achieve maximal twitch forces.
Muscle force-generating capacity and in vitro fatigability were then characterized. To ensure that all in vitro measurements were done at a uniform time in relationship to in vivo load trials, measurements of contractile parameters were started exactly 20 min after the animal was killed. We then stimulated strips with single electrical impulses to determine twitch contraction time (CT, i.e., time to peak force development) and half relaxation time (RT1/2, i.e., time for peak force to fall by 50%). After a 5-s rest, the force-frequency relationship was assessed by sequentially stimulating muscles at 1, 10, 20, 50, and 100 Hz. Each stimulus frequency was applied for 800 ms, and adjacent stimulus trains were separated by 5-s rest periods. A 30-s rest period was provided after the force-frequency curve. In vitro fatigability was then assessed by stimulating strips to contract 30 times/min for a total of 5 min in response to 500-ms trains of 20-Hz impulses. After completion of this protocol, muscle strips were removed from the bath, extraneous connective tissue was removed, and the wet weight of each strip was recorded.Glutathione concentrations. GSH and GSSG levels were assayed using the HPLC method of Fariss and Reed and co-workers (10, 21). Use of this method permits a more accurate assessment of GSH and GSSG concentrations than spectrophotometric assays. Briefly, when this assay is performed, pulverized muscle is added to iodoacetic acid to form S-carboxymethyl derivatives of thiol-containing proteins; 1-fluoro-2,4-dinitrobenzene is then added to form chromophores. The resulting reaction mixture was then injected onto a Spherisorb amino column and eluted with a sodium acetate gradient in a water-methanol-acetic acid solvent. GSH and GSSG peaks areas were assessed with a variable-wavelength ultraviolet detector and subsequently integrated using a Hewlett-Packard 3390-A integrator. GSH and GSSG standards were used to quantify glutathione concentrations.
8-Isoprostane levels.
Lipid peroxidation was assessed by measuring muscle 8-isoprostane
levels (i.e., 8-isoprostaglandin
F2
) with use of a competitive
phase enzyme immunoassay (12, 19). Because of the quantities of tissue
required for this assay, soleus muscles could not be assessed using
this technique. For this determination, we pulverized ~100 mg of
diaphragm tissue under liquid nitrogen. This powder was homogenized in
methanol (20 ml/g of tissue), an aliquot of this homogenate (1 ml) was
mixed with 5 ml of 0.1 M potassium phosphate buffer, pH 7.4, and the
mixture was passed through a Sep-Pak
C18 filter (prewashed with
methanol and water). After the C18
filter was washed with additional methanol-phosphate buffer and with
ultrapure water, a sample was eluted from the filter by using an ethyl
acetate-1% methanol mixture. The ethylacetate was subsequently
evaporated under a stream of nitrogen passed through a Supelco heating
system. The residue remaining after solvent evaporation was redissolved
in buffer, placed in assay wells coated with mouse monoclonal rabbit
antibody, and mixed with rabbit antiserum to 8-isoprostane and
8-isoprostane linked to acetylcholinesterase. After incubation, wells
were washed to remove unbound reagents, and Ellman's reagent
[containing acetylthiocholine and
5,5'-dithio-bis-(2-nitrobenzoic) acid] was added to the
wells. A spectrophotometric plate reader set at a wavelength of 412 nm was used to measure the 5-thio-2-nitrobenzoic acid formed by reaction of Ellman's reagent with acetylcholinesterase. Known concentrations of
8-isoprostane were used to construct a standard curve, and tissue
8-isoprostane concentrations were calculated by reference to this curve.
Protein carbonyl determination. Protein carbonyl side group content was assessed to provide an index of free radical-mediated alterations of protein structure (14, 18, 26). For this assay, frozen muscle samples were pulverized under liquid nitrogen, and powdered tissue was homogenized in 0.05 M K2HPO4 and 5 mM EDTA, pH 7.4. Homogenates were precipitated with TCA and centrifuged. The resulting pellet was then resuspended using 2 N HCl containing 0.1% 2,4 dinitrophenylhydrazine (DNP). A second portion of the pellet was resuspended in HCl without DNP. Samples were incubated at room temperature for 60 min, and proteins were reprecipitated with TCA. The precipitate was washed three times with 1:1 ethanol-ethyl acetate and then dissolved in 6 M guanidine-HCl. The absorbance of the DNP-derived sample minus the absorbance of the nonderived protein was taken as an index of protein carbonyl group content by using a molar extinction coefficient of 21,000.
Analysis of airway characteristics. We quantitated the effective load placed on animals as a result of the banding procedure in three ways. First, at the point of banding, esophageal pressures were monitored and band "snugness" was adjusted to attain an approximately twofold increase in esophageal pressure swings with respiration.
Second, we measured the long and short internal axis dimensions of the banded and unbanded portions of the trachea immediately after the animals were killed in all animals and calculated cross-sectional areas (CSAs) assuming an elliptical tracheal shape. Third, we characterized the pressure-flow relationships of the excised tracheae of all animals and analyzed these relationships by curve fitting individual data sets to laminar flow equations [i.e., pressure = K1(flow) + K2, where K1 represents "airway resistance" and K2 represents "opening pressure"] and to a mixed laminar-turbulent airflow model [i.e., with use of Poiseuille's equation: pressure = R1(flow2) + R2(flow)]. We found that the "fit" to the laminar equation was relatively good for all experiments, with the correlation coefficient for linearity averaging 0.94 ± 0.03. Fit to a mixed laminar-turbulent model was not as satisfactory, with correlation coefficients for linearity averaging only 0.65 ± 0.06 for data sets. Because there was a poor fit of data to the mixed laminar-tubular model, only laminar equation curve-fitting results are reported.Force analysis. Force was normalized for CSA simply by dividing force (in newtons) by the CSA (in cm2), with CSA calculated from
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Statistical analysis. An ANOVA was used to compare variables (e.g., 8-isoprostane concentrations) across groups of animals, with post hoc testing (Student-Newman-Keuls) to determine statistical differences between individual groups.
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 for control and banded groups of animals). Values are means ± SE. P < 0.05 was taken as indicating statistical significance.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animal weight over time.
We calculated the food ingested by animals on a daily basis and weighed
all animals every other day after operative procedures. Figure
1 displays food intake and animal weight
over time after surgery in all groups of animals. As a result of the
precautions taken, food intake was very similar in all groups, with
essentially no intake on the day immediately after surgery, gradually
increasing intakes over the next 2-3 days, and a relatively
constant level of food intake of ~20 g/day by day
4 of the postoperative period (Fig. 1,
top). Body weight fell over the
first 4 days after surgery and then remained relatively stable (Fig. 1,
bottom).
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Airway characteristics.
CSAs of the banded portions of the trachea were reduced by ~65% in
4-, 8-, and 12-day banded study groups (Table
1) compared with the size of the same
portion of the trachea in unbanded control animals. We also found that
the CSA of the trachea in sham-operated controls (0.126 ± 0.010 cm2) was similar to the size of
the unbanded portions of the trachea in animals banded for 4 days
(0.110 ± 0.004 cm2), 8 days
(0.119 ± 0.010 cm2), and 12 days (0.119 ± 0.016 cm2).
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Muscle lengths and weights.
The gross morphological characteristics of in vitro muscle strips from
the four experimental groups were similar, with strips from all groups
having roughly the same lengths and weights (Table 2).
Total diaphragm and soleus weights were also similar for the four
experimental groups.
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In vitro diaphragm force generation. Diaphragm CT and RT1/2 were similar for muscles from the four groups of animals. Specifically, CT averaged 62 ± 2, 62 ± 2, 67 ± 5, and 61 ± 2 ms in control and 4-, 8-, and 12-day banded animals, respectively; RT1/2 was 60 ± 3, 53 ± 3, 66 ± 5, and 56 ± 3.5 ms, respectively.
On the other hand, twitch force was reduced for diaphragms from all three loaded groups compared with unloaded control animals, averaging 7.8 ± 0.8, 4.0 ± 0.4, 3.0 ± 0.4, and 3.4 ± 0.4 N/cm2 in control and 4-, 8-, and 12-day banded groups, respectively (P < 0.001 for comparison of controls with the banded groups). Force, plotted as a function of stimulation frequency, is displayed in Fig. 2. The resultant diaphragm force-frequency curves for all banded groups of animals were appreciably downward shifted compared with the control animal diaphragm force-frequency relationship, with quantitatively similar force reductions observed for diaphragms from all three banded groups (P < 0.01 for comparison of the curve from the control group with the 3 banded groups).
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Muscle glutathione, 8-isoprostane, and protein carbonyl
concentrations.
Diaphragm GSH and GSH + GSSG levels of animals loaded for 8 days were
significantly lower than diaphragmatic GSH levels for unloaded control animals (Table
3;
P < 0.01), but diaphragm GSH and GSH + GSSG concentrations of 4- and 12-day banded groups were not
significantly different from those of controls. Diaphragm GSSG
concentrations for control and all three banded groups were similar.
Diaphragm GSSG/GSH ratios were significantly greater in 8-day banded
animals than in controls (P < 0.05;
Table 3). GSH, GSSG, and GSH + GSSG concentrations and GSSG/GSH ratios
for soleus muscles were similar in the four experimental groups (Table 3).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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These data indicate that sustained respiratory loading of rats accomplished using tracheal bands causes pronounced reductions in the force-generating capacity of the diaphragm, with especially marked reductions in the forces generated in response to low-frequency electrical stimulation (i.e., 1-20 Hz). Moreover, tracheal banding also appears to elicit alterations in several biochemical indexes of oxidative stress, resulting in modification of diaphragmatic lipid and protein constituents. Specifically, we found that loading resulted in a decrease in diaphragm concentrations of GSH, an increase in diaphragm GSSG/GSH ratios, an increase in diaphragm 8-isoprostane levels, and an increase in protein carbonyl levels.
The time course of alterations in GSH and 8-isoprostane levels during loading seemed to differ from the pattern of alteration of force generation. Although low-frequency force production was severely reduced by 4 days into banding, diaphragm GSH levels were most different from controls in 8-day banded animals and diaphragm 8-isoprostane levels were most different from control values in 12-day banded animals.
Alterations in force-generating capacity with respiratory loading. We found that tracheal banding elicited a reduction in diaphragm strength (i.e., low-frequency force-generating capacity was reduced) and a reduction in muscle fatigability (i.e., improvement of in vitro fatigability of 12-day banded animals compared with controls). Although reductions in low-frequency force generation were quantitatively similar in the three loaded groups, our in vitro index of fatigability appeared to gradually change the longer banding was maintained. In addition, in vitro diaphragm fatigability appeared to gradually decrease over time during loading, despite evidence of continued or increasing lipid and protein oxidation.
This observed dissociation between alterations in strength (decrease) and fatigability (increase) over time during sustained loading in the present study is consistent with a recent report by Prezant et al. (20), who observed that 24 wk of tracheal banding elicited a reduction in low-frequency diaphragm force-generating capacity and a reduction in diaphragm fatigability. In this study by Prezant et al., reductions in low-frequency force appeared to be a consequence of a loading-induced shift in the fiber composition of the diaphragm (with a shift that reduced "fast"-twitch fiber content and increased "slow"-twitch fiber content) (20). In the present study, however, the observed reduction in force 1) occurred more rapidly than would be expected on the basis of a fiber type shift, 2) was characterized as a reduction in low- and high-frequency force-generating capacity by the diaphragm, and 3) preceded changes of in vitro fatigability. Moreover, the alterations observed in the present study are similar to those we previously observed after much shorter periods of respiratory loading (i.e., 1- to 2-h inspiratory resistive loaded breathing trials produce reductions in the diaphragm force-frequency relationship that mirror those found at 4 days in the present experiment) (6, 29). With these considerations taken into account, it seems likely that the reduction in force observed in the present study represents a form of loading-induced muscle dysfunction akin to that seen after acute loaded breathing trials. The changes we observed for in vitro diaphragm fatigability after 12 days of loading cannot, however, be explained in this fashion. Instead, this latter alteration (i.e., reduced fatigability over time with sustained loading) is likely to represent some form of muscle adaptation. For example, the continued stress of loading could lead to adaptive upregulation of mitochondrial function over time, improving energy generation capacity and thereby resulting in less hydrogen ion and inorganic phosphate accumulation during a short period of increased contractile activity (e.g., during an in vitro repetitive contraction trial) and less fatigue.Loading-related alterations in biochemical indexes. Recent studies have suggested that free radical-induced oxidative modification of cellular constituents within the respiratory muscles during strenuous contraction contributes to the development of muscle fatigue (9, 23, 27). This assertion is supported by three lines of evidence: 1) the finding that free radical scavenger administration alters the rate of development of respiratory muscle fatigue during strenuous repetitive contraction trials (22, 25, 30), 2) the observation that short-term repetitive strenuous contraction of the respiratory muscles (in response to electrically stimulated contractions of in situ muscle preparations or during the application of exogenous inspiratory resistive loads to intact animals) is associated with an increase in the concentrations of several biochemical markers of oxidative stress (i.e., malondialdehyde, 8-isoprostane, and glutathione oxidation) within the respiratory muscles (1-4, 6, 29), and 3) the finding that scavenger administration ablates contraction-related increases in biochemical markers of oxidative stress in parallel with the effects of these agents to reduce fatigue rate (30).
All these previous experiments were performed, however, using relatively short-term trials of inspiratory loading or electrically stimulated contraction. The present study sought to extend these previous observations by determining whether markers of oxidative stress (i.e., 8-isoprostane, GSH-GSSG relationships, and protein carbonyl concentrations) rose over time during the type of sustained inspiratory loading typically observed in patients with lung disease and, furthermore, whether these marker alterations varied in parallel to loading-induced alterations in measures of diaphragmatic contractile function (i.e., the force-frequency relationship and in vitro measures of diaphragm fatigability). The finding that GSH concentrations were decreased in the diaphragms of chronically loaded animals in the present study is, in fact, consistent with observations in previous studies examining much shorter periods of inspiratory loading (1, 2). For example, Anzueto et al. (1) found that acute loading of anesthetized animals evoked a fall in diaphragmatic GSH and a rise in diaphragmatic GSSG levels. Subsequent work by our laboratory examining the loading responses of unanesthetized decerebrate animals also demonstrated that diaphragm GSH levels fall, GSSG increases, and the GSSG/GSH ratio rises under a variety of acute loading conditions (5, 28). The fact that diaphragmatic GSSG levels failed to increase during sustained loading in the present study differs, however, from our previous observations and those of Anzueto et al. (1) regarding the effect of acute loading on the diaphragm. One potential explanation for this discrepancy is that GSSG is carried across cell membranes by what appears to be a saturable transport process (8). As a result, it is possible that GSSG transport from cells may be time dependent, with sustained loading affording sufficient time for GSSG levels to fall to near "baseline levels," whereas more acute loading does not. We also found that diaphragmatic 8-isoprostane levels increased in response to sustained loading in the present experiment. This latter substance is a by-product of free radical-mediated peroxidation of lipid components of membranes (16) and has been demonstrated to be a marker of free radical generation in the lung after hyperoxia-induced injury and in the liver after carbon tetrachloride administration (12, 17). Increases in diaphragmatic 8-isoprostane concentrations have also recently been observed after the short-term application of inspiratory resistive loads in decerebrate rats (13). Importantly, in the present study, there appeared to be a difference in the time course of changes in 8-isoprostane and in glutathione metabolism with sustained loading, and, moreover, the time course of changes over time of these two indexes failed to mirror loading-induced alterations in the diaphragm force-frequency relationship. The fact that neither of these indexes appeared to parallel alterations in diaphragmatic force generation over time argues that lipid peroxidation is not linked to alterations in diaphragmatic force generation over time during chronic respiratory loading. On the other hand, we found that the time course of changes in protein carbonyl concentrations over time during sustained loading did parallel reductions in diaphragmatic force-generating capacity. Moreover, the increase in diaphragm carbonyl content after sustained respiratory loading in the present experiment was striking, with levels 2-2.5 times greater than controls in diaphragms of 4-, 8-, and 12-day loaded animals. These observations raise the theoretical possibility that oxidative modification of one or more cellular proteins may have been responsible for the observed loading-induced reductions in force generation. In fact, such a phenomenon would be in keeping with recent experiments examining the direct effect of free radical-generating solutions on the function of various components of the skeletal muscle contractile apparatus. For example, Brotto and Nosek (5) recently found that exposure of skinned muscle cells to hydrogen peroxide resulted in significant reductions in calcium release by the sarcoplasmic reticulum. As a result, it is possible that oxidative modification of the contractile proteins or the sarcoplasmic reticulum could so alter muscle calcium handling or myofilament activation as to result in a sustained alteration in force-generating capacity. Although no previous study has examined protein carbonyl concentrations or other markers of protein oxidation in the respiratory muscles after respiratory loading, this index has been reported to change in limb muscles after exercise. Specifically, carbonyl concentrations have been found to increase in limb skeletal muscle after single bouts of exhaustive exercise and a prolonged regimen of endurance training (24). The magnitude of the increase in carbonyl concentrations observed in the present study after respiratory loading is, however, substantially greater than has been reported previously for mixed or white skeletal muscle in the limb after exercise or training (24, 32). The fact that respiratory loading elicited such large increases in carbonyl concentrations in this study may be a reflection of the nature of the load imposed by tracheal banding; banding results in a substantial, sustained increase in the respiratory workload. This may result in a significantly greater total oxidative stress than would be achieved with limb muscle exercise regimens, which typically are of limited duration. The fact that loaded breathing had no effect on glutathione concentrations or protein carbonyl levels for soleus muscles has several implications. First, this finding suggests that loading-induced protein oxidation and lipid peroxidation within the diaphragm did not occur as the result of nonspecific systemic alterations that may have accompanied loading (i.e., changes in serum catecholamine levels, alterations in cardiac output and heart rate, and changes in arterial oxygen or carbon dioxide tensions). If these various factors had played a role in initiating diaphragm protein/lipid oxidation, one would have expected a concomitant alteration in soleus carbonyl and glutathione levels. Second, this finding indicates that systemic nutritional alterations in response to surgery and loading were also unlikely to have significantly influenced our findings, since one would have expected systematic changes in soleus glutathione and carbonyl concentrations over time if this factor was important. As a result, the observed alterations in diaphragm glutathione, carbonyl, and 8-isoprostane levels produced by respiratory loading should represent a direct consequence of the increased work done by this muscle.Potential implications. Although the present data are consistent with the possibility that inspiratory loading-induced protein oxidation is linked to loading-related reductions in the diaphragm force-frequency relationship, these findings alone do not prove a causal relationship between these two loading-related phenomena. "Proof" of such linkage will require additional experimentation identifying the specific protein being modified and determining whether interventions (e.g., free radical scavengers) that block protein modification prevent contractile dysfunction. Nevertheless, the present work provides the first information indicating that 1) protein and lipid peroxidation can be evoked in the diaphragm by a model of respiratory loading (tracheal banding) that approximates the character of the sustained stress that lung diseases typically place on the respiratory muscles of patients, 2) alterations in diaphragmatic force in response to sustained loading do not appear to be related to lipid peroxidation within this muscle, and 3) changes in an index of protein oxidation do parallel alterations in diaphragm strength over time during this form of sustained respiratory loading. We speculate that similar phenomena may also occur in patients with an increased respiratory workload due to lung disease.
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FOOTNOTES |
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Address for reprint requests: G. S. Supinski, MetroHealth Medical Center 2500 MetroHealth Dr., Cleveland, OH 44109.
Received 26 December 1995; accepted in final form 27 October 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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) and 4- (
), 8- (
), and 12-day tracheal banded animals (
).
Bottom: weight loss after surgery for
4 experimental groups.
