INTRODUCTION

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Keywords: free radicals; nitric oxide; sarcolemma; mechanical stress; respiratory muscles

Respiratory failure is a frequent occurrence in patients with severe sepsis, and has been shown to be a major contributor to the high mortality associated with this condition (1). The diaphragm is the primary muscle of respiration, and severe dysfunction of the diaphragm, consisting of decreased maximal force production as well as an increased susceptibility to fatigue, has been documented in animal models of sepsis (2). Indeed, experimental animals with septic shock die of respiratory failure which is related not to pulmonary disease per se, but rather to an inability of the diaphragm to ventilate the lungs (3). Therefore, respiratory muscle dysfunction is likely to be important in the pathogenesis of respiratory insufficiency in septic patients. Release of endotoxin (i.e., lipopolysaccharide [LPS]) from gram-negative bacteria is considered a seminal factor underlying the development of the sepsis syndrome and diaphragm contractile failure (3, 7).

Oxidative stress has been strongly implicated in the development of diaphragm dysfunction during sepsis (4, 6, 8, 9). Recent studies have suggested that nitric oxide (NO) may be one of the principal mediators of this phenomenon (5, 10). In normal skeletal muscle, NO is produced at low levels by constitutively expressed neuronal and endothelial isoforms of NO synthase (nNOS and eNOS, respectively) (14, 15). However, high level expression and enzymatic activity of the inducible isoform (iNOS) have been demonstrated in the diaphragm of rats injected with endotoxin (5, 10). In addition, inhibition of NOS activity prevents endotoxin-induced decreases in diaphragm force production (10, 13). We have recently reported that in rats exposed acutely to endotoxin, increased iNOS expression in the diaphragm is associated with widespread damage to the myofiber surface membrane (sarcolemma), which can be significantly alleviated by pharmacologic inhibition of NOS activity (12). Taken together, the above observations strongly support the proposition that diaphragm dysfunction as well as sarcolemmal injury under septic conditions are mediated, at least in part, through increased NO production. It is generally believed that much of the tissue damage observed in the setting of high ambient NO levels is related to interaction of the latter with superoxide to form peroxynitrite (16, 17).

The cellular mechanisms potentially responsible for diaphragm dysfunction during sepsis include oxidative/nitrosative modification of critical proteins involved in excitation- contraction coupling or cross-bridge cycling (14, 18, 19), lipid peroxidation of membranes (17), DNA strand breakage (20), and inhibition of mitochondrial respiration (5). In addition, although the mechanism underlying endotoxin-induced diaphragm sarcolemmal injury is unknown, in erythrocytes oxidative stress during sepsis is associated with hyperfragility of the cell membrane (21). Moreover, there is evidence that peroxy- nitrite and its precursors, NO and superoxide, play a role in mediating these changes (21). If also present in muscle cells during sepsis, membrane hyperfragility of this nature could greatly favor the development of diaphragm sarcolemmal injury by lowering the threshold for contraction-induced membrane disruption (25, 26).

The main objective of the present study was to determine whether reducing the level of diaphragm muscle activity through the use of mechanical ventilation can provide protection against diaphragm sarcolemmal injury and attendant contractile dysfunction during sepsis. We hypothesized that such protection could be mediated by at least two mechanisms. First, mechanical ventilation could protect the diaphragm by decreasing the level of mechanical stress imposed on diaphragm myofibers suffering from sepsis-induced hyperfragility of the sarcolemma. Second, mechanical ventilation could reduce exposure of the diaphragm to NO and other reactive species, derived from either inflammatory cells or myofibers per se. With respect to the latter, Nethery and coworkers (27) have recently demonstrated that increased contractile activity by the septic diaphragm significantly augments free radical formation in the muscle. Accordingly, in this study we have determined the relationship between sarcolemmal injury and markers of oxidative stress in the septic rat diaphragm under conditions of spontaneous breathing as well as during controlled mechanical ventilation. In addition, to determine whether oxidative stress leads to an increased susceptibility to mechanical stress-induced sarcolemmal injury, we have employed a tissue culture model which permits independent modulation of oxidative and mechanical stresses imposed on muscle cells.

	METHODS

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Animal Procedures

Pathogen-free adult male Sprague-Dawley rats (450 to 500 g) were randomly assigned to one of three groups: (1) a spontaneously breathing control group (CON), (2) a spontaneously breathing endotoxemic group induced by administration of E. coli LPS, and (3) a mechanically ventilated endotoxemic group (LPS + MV). The protocol was approved by the local institutional animal care and use committee.

Surgery was performed under sterile conditions. All animals were anesthetized by intraperitoneal administration of sodium pentobarbital and tracheostomized. The left carotid artery was cannulated with the catheter directed retrograde into the aortic arch to allow continuous monitoring of blood pressure, withdrawal of arterial blood samples, and injection of LPS as previously described (12). In the LPS + MV group, an endotracheal tube was connected to a pediatric ventilator (Babylog 8000; Drager, Lubeck, Germany) equipped with standard sterile tubing and filters. The ventilator was placed in the assist-control mode with a trigger threshold of 0.25 cm H₂O, and the initial settings were as follows: respiratory frequency, 90/min; tidal volume, 8 ml/kg; fractional inspired oxygen (FI_O2), 0.21; and positive end-expiratory pressure (PEEP), 1.5 cm H₂O. The ventilation was adjusted to ensure an arterial PCO₂ of 37 to 42 mm Hg at the beginning of the protocol. Absence of respiratory muscle effort was confirmed by the absence of triggering as well as the stable and reproducible shape of the tracheal pressure waveform throughout the experiment.

To produce endotoxemia in the LPS and LPS + MV groups, the aorta was infused with LPS (20 mg/kg of serotype 055:B5; Sigma Chemical Co., St. Louis, MO) over a 10-min period; the CON group underwent the same procedure with an equivalent volume of saline. After LPS or saline administration, animals were maintained in the anesthetized state until being euthanized 4 h later. The selection of this time point was based on our previous demonstration of NOS-dependent diaphragm injury and significant abnormalities of diaphragm myofiber electrophysiology at 4 h after LPS administration (12). After euthanasia, one randomly selected hemidiaphragm was used for morphologic assessment of diaphragm injury or immunohistochemical studies, whereas the opposite hemidiaphragm was obtained for measurements of muscle mechanics or biochemical studies.

Detection of Diaphragm Sarcolemmal Injury in Vivo

The severity of sarcolemmal injury to diaphragm myofibers was evaluated by exposing the muscles to a low-molecular-weight (formula weight [FW] = 631) fluorescent tracer dye (Procion Orange; Sigma) in vitro, as previously described in detail (28). Using epifluorescence, a minimum of 300 myofibers from randomly selected microscopic fields were then counted on frozen sections, and for each diaphragm at least three tissue sections were analyzed and averaged.

Diaphragm Muscle Mechanical Properties

Contractile properties were measured on excised diaphragm muscle strips placed in a perfused tissue bath chamber maintained at 25° C, as previously described in detail (29).

Indicators of Oxidative/Nitrosative Stress

iNOS Expression. Immunodetection and densitometric quantification of iNOS expression was performed as previously described in detail (12). Immunostaining for iNOS was also performed as previously described (10).

Protein carbonyl content. Protein carbonyl formation was employed as a general marker of oxidative stress (30, 31). The OxyBlot protein oxidation detection kit (Intergen Company, Purchase, NY) was used for immunodetection of carbonyl groups according to the manufacturer's instructions. The immunoreactive proteins were then visualized by enhanced chemiluminescence and quantified by densitometry.

Tissue Culture Experiments

Detection of sarcolemmal injury in cultured myotubes. Fluorescein isothiocyanate-labeled dextran (FITC-Dx, Sigma), a low-molecular-weight (FW = 10,000) tracer molecule that enters into myotubes with plasma membrane disruptions, was used to detect sarcolemmal damage in cultured myotubes as previously described by Clarke and Feeback (32). All experiments were performed at 7 d after switching L6 rat myoblasts (American Type Culture Collection, Rockville, MD) to reduced serum conditions. The amount of FITC-Dx entry into myotubes was quantitated by lysis of the cells with 0.1% sodium dodecyl sulfate (SDS) and determination of lysate fluorescence levels on a spectrophotofluorometer (Delta Scan Model 4000; Photon Technology International, London, Ontario), with detection wavelength set at 520 nm after excitation at 490 nm (32). Quantitation of FITC-Dx in the myotube lysates was based on a standard curve of FITC-Dx suspended in Dulbecco's phosphate-buffered saline (D-PBS) containing 0.1% bovine serum albumin (BSA):0.1% SDS. Total protein was ascertained on the same myotube lysate samples, and FITC-Dx release values were normalized to protein content to correct for any variability of cell number between wells.

Muscle cells were grown on special plates in which the base of the wells consisted of a type I collagen-coated, flexible substratum made of silicone (Bioflex; Flexcell Corp., Hillsborough, NC). To apply mechanical stress to L6 myotubes, the plates were placed in a Flexercell Strain Unit (Model FX-3000, Flexcell Corp.). The latter allowed the flexible substratum (and hence the cells) to be repetitively stretched to a defined length using a computer-controlled vacuum applied to the base of the wells (32). Myotubes were exposed to repetitive 10% elongation at a frequency of 1.5 Hz for 4 h in the presence of FITC-Dx. To study the interaction between oxidative and mechanical stress, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) (Biomol Research Laboratories, Inc., Plymouth Meeting, PA) was also added to the medium. This compound releases NO and superoxide anion in approximately equal amounts to generate peroxynitrite, which is considered the main arbiter of NO-mediated oxidative damage to tissues (16, 17, 33). At the end of the foregoing protocols, myotubes were washed in D-PBS and lysed with 0.1% SDS to release incorporated FITC-Dx tracer, and the amount of tracer released was quantitated by spectrophotofluorometry as described earlier. For each independent experiment, values were expressed as a percentage of the mean value obtained from a simultaneously processed control plate (six wells) that was not exposed to mechanical stress or SIN-1.

Statistical Analysis

All data are reported as means ± SE and were analyzed with a statistical analysis program (Statistix Analytical Software, St. Paul, MN). For the animal protocols, differences between groups were initially tested by analysis of variance (ANOVA), with post hoc application of the Tukey test where appropriate. For the tissue culture experiments, comparisons between groups were performed in the same manner or by using Student's t test for unpaired samples. Statistical significance was defined as p < 0.05.

	RESULTS

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Hemodynamic and Gas Exchange Parameters

Endotoxin administration led to an acute and transient decrease in mean arterial blood pressure (to approximately 50% of CON values), with the nadir occurring at approximately 15 min after LPS injection (data not shown). There were no differences in the blood pressure response between the LPS and LPS + MV groups, and in both of these cohorts arterial pressure returned to CON levels by approximately 1 h after endotoxin administration. Table 1 shows arterial blood gas values in the three groups of experimental animals at the end of the 4-h protocol. As can be seen, both groups receiving endotoxin demonstrated a mild metabolic acidosis, as indicated by reductions in HCO₃. In addition, the spontaneously breathing LPS group tended to show a lower arterial PCO₂ value than the other two groups, consistent with the presence of hyperventilation. Arterial PO₂ was within normal limits for all groups.

Prevention of Endotoxin-Induced Diaphragm Injury by Mechanical Ventilation

Excised diaphragms were exposed to a low-molecular-weight tracer dye which is unable to penetrate into the cytoplasm of normal myofibers with an intact sarcolemma (12, 28). Representative photomicrographs demonstrating the protective effects of mechanical ventilation against endotoxin-induced diaphragm sarcolemmal injury are shown in Figure 1A. Note that although there was intense tracer dye staining of the interstitial connective tissue space of the diaphragm in CON animals (top), no dye uptake was found within the cytoplasm of individual myofibers. In the spontaneously breathing LPS group (middle), numerous diaphragm myofibers demonstrated cytoplasmic staining of varying intensities, consistent with the presence of different degrees of impaired membrane integrity. In marked contrast, tracer dye entry into diaphragm myofibers after LPS administration was much reduced by the concomitant use of mechanical ventilation (bottom). Quantitative data for the percentage of diaphragm myofibers exhibiting morphologic evidence of sarcolemmal injury are shown in Figure 1B. As can be seen, in the spontaneously breathing LPS group, sarcolemmal damage was observed in almost one-quarter of diaphragm myofibers. There was a significantly lower prevalence of such damaged myofibers in the LPS + MV group, which did not differ statistically from values obtained in the CON group.

View larger version (57K): [in this window] [in a new window]   Figure 1.   Effects of mechanical ventilation on diaphragm sarcolemmal injury during sepsis. (A) Representative photomicrographs of the degree of sarcolemmal damage present in CON (upper panel ), LPS (middle panel ), and LPS + MV (lower panel ) groups. In the CON group, very little tracer dye uptake was observed within individual myofibers, although intense staining of extracellular connective tissue indicates good dye diffusion throughout the tissue. In contrast, numerous myofibers were unable to exclude the tracer dye in the LPS group, indicating a widespread loss of sarcolemmal integrity. This LPS-induced sarcolemmal damage was greatly reduced by the use of mechanical ventilation. (B) Percentage of myofibers exhibiting sarcolemmal injury in the diaphragm for the three experimental groups. All values are group means ± SE. *p < 0.05 versus CON; p < 0.05 versus LPS.

Alleviation of Endotoxin-Induced Diaphragm Weakness by Mechanical Ventilation

There were no differences in diaphragm twitch kinetics (contraction and half-relaxation times) among the three experimental groups (data not shown). Figure 2 shows group mean data for isometric contractile force parameters. Maximal twitch force (Pt) and tetanic force (Po) were both significantly depressed in the spontaneously breathing LPS group, whereas these abnormalities were partially reversed in the LPS + MV group. Furthermore, as shown in Figure 2C, LPS administration caused a major reduction in diaphragm force production over the entire range of tetanic stimulation frequencies examined. Although the improvement was not sufficient to completely restore diaphragm force-generating capacity to CON levels, mechanical ventilation significantly mitigated the LPS-induced force deficit at all stimulation frequencies except at 10 Hz (where a similar trend toward increased force was nonetheless observed). Measurements of diaphragmatic fatigue resistance are depicted in Figure 3. Absolute force levels during fatiguing stimulation (Figure 3A) paralleled changes in the force-frequency relationship, but there were no differences in diaphragm fatigue resistance once the force loss was normalized to its initial level (Figure 3B). Finally, it is important to note that in the absence of LPS administration, mechanical ventilation per se had no effect on maximal force production by the diaphragm (25.9 + 1.3 N/cm²) or any of the other contractility parameters mentioned earlier (n = 4 rats, data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 2.   Effects of mechanical ventilation on diaphragm force-generating capacity during sepsis. (A) Maximal isometric twitch force data. (B) Maximal isometric tetanic force data. (C ) Force-frequency relationship of the diaphragm in the three experimental groups. All values are group means ± SE (n = 6 per group). *p < 0.05 versus CON; p < 0.05 versus LPS. , Control; , LPS; , LPS + MV.

View larger version (14K): [in this window] [in a new window]   Figure 3.   Effects of mechanical ventilation on diaphragm fatigue resistance during sepsis. (A) Force generation over time during repetitive contractions, expressed in terms of absolute specific force values. (B) Force generation over time during repetitive contractions, expressed as a percentage of the initial force level. All values are group means ± SE (n = 6 per group). *p < 0.05 versus CON; p < 0.05 versus LPS. , Control; , LPS; , LPS + MV.

Effect of Mechanical Ventilation on Endotoxin-Induced iNOS Expression in the Diaphragm

Figure 4 shows the results of Western analysis for iNOS expression in the diaphragm of the CON, LPS, and LPS + MV groups. In nonendotoxemic CON animals, there was little or no visible iNOS expression in the diaphragm. In contrast, in diaphragms of the spontaneously breathing LPS group there was an easily detectable signal for iNOS protein at 4 h after LPS administration. However, the level of iNOS expression in the LPS + MV group was also significantly elevated compared with CON and did not differ from the spontaneously breathing LPS group. To determine whether the localization of iNOS expression might differ between the spontaneously breathing and mechanically ventilated rats exposed to LPS, immunohistochemistry was also performed. As shown in Figure 5, anti-iNOS immunoreactivity was found primarily within inflammatory cells located in blood vessels and in the perivascular regions of the muscle. Importantly, the same pattern of iNOS localization was found in both the LPS and LPS + MV groups. Therefore, these data suggest that the protective effects of mechanical ventilation were not related to a downregulation or altered localization of iNOS expression in the diaphragm.

View larger version (26K): [in this window] [in a new window]   Figure 4.   Effects of mechanical ventilation on iNOS expression in the diaphragm during sepsis. (A) Representative Western blot analysis of iNOS expression in diaphragm protein samples obtained from the three experimental groups. (B) Densitometric quantitation of iNOS protein. Note that use of mechanical ventilation did not blunt the LPS-induced upregulation of iNOS expression in the diaphragm. All values are group means ± SE. *p < 0.05 versus CON.

View larger version (110K): [in this window] [in a new window]   Figure 5.   Immunohistochemical detection of iNOS within diaphragm tissues. (A) Representative diaphragm section from the CON group: there was no detectable anti-iNOS immunoreactivity. (B) Representative diaphragm section from the LPS group: anti-iNOS immunoreactivity (red staining) was observed primarily in inflammatory cells present within blood vessels and in the perivascular regions of the muscle. (C ) Representative diaphragm section from the LPS + MV group: same pattern of anti-iNOS immunoreactivity as seen in the LPS group.

Effect of Mechanical Ventilation on Endotoxin-Induced Protein Carbonyl Formation in the Diaphragm

Detection of protein carbonyl formation in the diaphragm by Western analysis is shown in Figure 6. Nonendotoxemic CON diaphragms demonstrated several immunoreactive bands, consistent with many proteins in normal muscle being in a partially oxidized state as previously noted by others (34). As shown in Figure 6A, the electrophoretic fractionation pattern was similar among the three experimental groups. However, in quantitative terms the degree of protein carbonyl formation was significantly higher in the LPS and LPS + MV groups than in CON, and this was mostly due to increased signal intensity for bands migrating at approximately 38 and 35 kD. Moreover, there was a significant correlation between carbonylation of these protein species and iNOS expression within the diaphragm (see Figure 7), suggesting that NO overproduction was likely involved in the increased oxidative stress found in the LPS and LPS + MV groups.

View larger version (20K): [in this window] [in a new window]   Figure 6.   Effects of mechanical ventilation on protein carbonyl formation in the diaphragm during sepsis. (A) Representative immunoblot detection of carbonyl compounds present in diaphragm protein samples obtained from the three experimental groups. In particular, note the increased signal intensity for bands migrating at approximately 38 and 35 kD in the LPS as well as the LPS + MV samples. (B) Densitometric quantitation of total protein carbonyl formation. Note that LPS administration significantly increased the magnitude of diaphragm protein carbonyls; mechanical ventilation did not significantly affect this phenomenon. All values are group means ± SE. Data are expressed as a percentage of the mean value obtained from CON samples run on the same blot. *p < 0.05 versus CON.

View larger version (14K): [in this window] [in a new window]   Figure 7.   Correlation between protein carbonyl formation and iNOS expression in the diaphragm. Diaphragm iNOS expression was significantly correlated with carbonyl formation in protein species migrating at approximately 38 and 35 kD (depicted in A and B, respectively), which also showed the greatest increases after LPS administration. , Control; , LPS; , LPS + MV.

Direct Effects of Oxidative and Mechanical Stress on Muscle Cell Sarcolemmal Integrity

By unloading the respiratory muscles, mechanical ventilation reduces the diaphragm myofiber mechanical stresses that are inherently associated with spontaneous breathing efforts. Therefore, we reasoned that the protective effects of mechanical ventilation on the diaphragm during sepsis could be due to decreased mechanical stress placed on the sarcolemma. To determine whether the sarcolemma is hyperfragile in the setting of increased oxidative stress, we employed an in vitro system.

Figure 8 demonstrates that application of the NO and superoxide-generating compound SIN-1 to cultured myotubes caused a loss of myotube membrane integrity in a concentration-dependent manner, as indicated by abnormal cellular entry of FITC-Dx. Exposure to 0.25 mM SIN-1 did not cause significant sarcolemmal injury on its own. However, when the same dose of SIN-1 was combined with augmented mechanical stress (via imposition of 10% strain on myotubes), the amount of injury was significantly greater than observed with the use of 10% strain alone. Thus, a low amount of oxidative stress that was insufficient to disrupt membrane integrity under static conditions was nonetheless able to significantly increase the susceptibility of muscle cells to sarcolemmal injury induced by mechanical stress. Therefore, it appears that oxidative stress induces hyperfragility of the sarcolemma, thereby leading to a synergistic relationship between oxidative and mechanical stresses that greatly favors the development of sarcolemmal injury when the two factors are simultaneously present.

View larger version (17K): [in this window] [in a new window]   Figure 8.   Oxidative stress increases the susceptibility of muscle cells to mechanical stress-induced sarcolemmal injury. Oxidative stress was achieved by exposure to SIN-1, whereas mechanical stress was increased by means of imposition of 10% strain on cultured myotubes. Impaired myotube sarcolemmal integrity was indicated by abnormal cellular entry of FITC-Dx. Note that although exposure to 0.25 mM SIN-1 did not cause significant sarcolemmal injury on its own, it greatly increased the amount of injury incurred by 10% mechanical strain. All values are group means ± SE (n = 18 wells per condition, 3 independent experiments). Data are expressed as a percentage of the mean value obtained from a simultaneously processed control plate not exposed to SIN-1 or mechanical strain. *p < 0.05 versus control values; p < 0.05 versus 10% strain alone.

	DISCUSSION

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To our knowledge, this is the first study to specifically investigate the effects of mechanical ventilation on diaphragm injury and contractility during endotoxemia, as well as its impact on oxidative stress to the diaphragm in this setting. The principal findings of our study are as follows: (1) the use of mechanical ventilation during acute endotoxemia almost completely eliminated microscopic evidence of sarcolemmal damage within the diaphragm; (2) mechanical ventilation significantly improved force production by the septic diaphragm; (3) the aforementioned benefits were not associated with alterations in indices of oxidative/nitrosative stress within the diaphragm, which remained abnormally elevated in endotoxemic rats despite the use of mechanical ventilation; and (4) in an in vitro system that allows one to specifically modulate the oxidative and mechanical environment to which muscle cells are exposed, oxidative and mechanical stresses acted together in a synergistic fashion to greatly favor the development of sarcolemmal injury.

Comparison with Previous Studies

A large number of previous studies have examined the effects of endotoxemia and other sepsis models on diaphragm contractility in spontaneously breathing animals. Hussain and coworkers (3) initially reported that injection of endotoxin in dogs produced profound respiratory muscle dysfunction, which ultimately led to ventilatory failure and death of the animals. Subsequent studies have shown that scavengers or inhibitors of several reactive species, including NO, can significantly alleviate endotoxin-induced diaphragm contractile impairment (4, 6, 8, 13). The source of NO and other reactive species in the septic diaphragm appears to be infiltrating leukocytes as well as diaphragm myofibers themselves (9, 10, 13). Boczkowski and coworkers (10) observed that iNOS was expressed in inflammatory cells and not within diaphragm myofibers at an early time point (6 h) after intraperitoneal LPS injection, which is consistent with the findings in this study. However, these researchers also reported that diaphragmatic contractile impairment only occurred once iNOS was expressed in diaphragm myofibers (10). In contrast, we observed a major depression of diaphragm contractility during the period in which diaphragm iNOS expression was limited to infiltrating white cells. Although the precise reason for this difference between our study and the report of Boczkowski and coworkers (10) is not entirely clear, we speculate that methodologic considerations, such as the different routes of administration and dosages of LPS employed, may be the explanation. In addition, it is noteworthy that our data are in keeping with the previous in vitro observation that cardiomyocyte contractility is depressed by NO production from adjacent (in this case endothelial) cells (35).

Another study of relevance to our investigation is that of Leon and coworkers (2), which also examined diaphragm function in mechanically ventilated rats after endotoxin administration. However, because no spontaneously breathing endotoxemic animals were included in the study design, the impact of mechanical ventilation per se on septic diaphragm function was not evaluated. These investigators reported a significant reduction in diaphragm force-generating capacity, limited to high (> 30 Hz) frequencies of stimulation, despite the use of mechanical ventilation during endotoxemia (2). Furthermore, the diaphragm of endotoxemic animals was more susceptible to muscle fatigue during repetitive stimulation in their study (2). In contrast, we observed a reduction in force-generating capacity over the full range of the force-frequency relationship, and found no differences in fatigue resistance between septic (either spontaneously breathing or mechanically ventilated) and nonseptic diaphragms. The different effects of endotoxemia on the force-frequency relationship observed in the two studies may be explained on the basis of the different time points examined. In this regard, Shindoh and coworkers (36) examined rat diaphragm force production at several time points up to 6 h post-endotoxin administration, and found that the force-frequency curve was maximally depressed at 4 h after endotoxin delivery. In addition, Leon and coworkers (2) assessed diaphragm fatiguability in vivo, where hemodynamic and systemic metabolic factors may have had important effects on diaphragm endurance. Studies of diaphragm strip preparations performed in vitro are presumably less affected by such factors and may be a better reflection of myofiber properties per se. The lack of effect of endotoxemia on diaphragm fatigue resistance found in our study is consistent with previous investigations in which the same in vitro diaphragm strip methodology was employed (9, 10, 36).

Mechanism of Diaphragm Protection during Mechanical Ventilation

In a previous investigation, we observed an apparent relationship between sarcolemmal injury and iNOS expression in the muscles of septic rats, and pharmacologic inhibition of NOS activity was able to largely prevent the development of diaphragm sarcolemmal injury in this setting (12). Based on these observations, we concluded that NO overproduction likely plays a central role in the pathogenesis of diaphragm injury during sepsis. However, here we report that despite a very substantial protective effect with regard to both sarcolemmal injury and contractility, use of mechanical ventilation did not reduce iNOS expression within the diaphragm of septic rats. Furthermore, the carbonyl immunoblot assay findings strongly suggest that oxidative stress occurred within the septic diaphragm irrespective of the use of mechanical ventilation, which is also consistent with the iNOS data. Indeed, persistent upregulation of iNOS expression with attendant oxidative stress could help explain the fact that impaired contractility of the muscle in sepsis was only partially mitigated by mechanical ventilation in our study. Taken together, our data argue against any major effect of mechanical ventilation on the level of exposure of the septic diaphragm to NO and other reactive species. However, if not due to a reduction in oxidative stress, through what mechanism did mechanical ventilation prevent sarcolemmal injury in the diaphragm?

One possibility to be considered is that mechanical ventilation exerted its protective effects by altering diaphragm blood flow. Diaphragm blood flow increases several-fold during endotoxemic shock in spontaneously breathing animals (7, 37). Under these conditions, placing the diaphragm at rest through the use of mechanical ventilation significantly reduces diaphragm blood flow and thereby allows a greater proportion of cardiac output to be redirected to other vital organs (37). Although such a reduction in diaphragm blood flow could favor energetic depletion of the muscle, the decreased diaphragm muscle workload associated with mechanical ventilation should also reduce energy demands. To the extent that in vitro fatiguability was unaffected by the use of mechanical ventilation in our study, these two opposing influences of mechanical ventilation on diaphragm energetics may have neutralized one another. It is also conceivable that a reduction in diaphragm blood flow could decrease exposure of the muscle to bloodborne mediators of diaphragm injury. Arguing against this, however, is the fact that indices of oxidative/nitrosative stress were not reduced by the use of mechanical ventilation in our study. Because immunohistochemical staining revealed iNOS to be expressed almost exclusively by leukocytes residing within the diaphragm, this also suggests that mechanical ventilation failed to have a major effect on the level of recruitment of activated inflammatory cells to the muscle.

The in vitro findings of our study support another explanation, which is that the beneficial effects of mechanical ventilation on diaphragm sarcolemmal injury were related to a reduction in the mechanical load faced by a structurally weakened sarcolemma. Hence, our data indicate that in the presence of increased oxidative stress such as that associated with sepsis, muscle cells develop an abnormally heightened vulnerability to mechanical stress-induced sarcolemmal damage. Mechanical stress is normally imposed on the sarcolemma during muscle contractions (38), particularly under high workload conditions (25). Previous investigations have shown that the pressure- time index of the diaphragm (PTIdi) is increased approximately 2-fold after LPS administration at a dose sufficient to induce septic shock in experimental animals (7, 37). Therefore, under septic conditions there is an increase in respiratory workload, which when combined with oxidative stress-mediated hyperfragility of the sarcolemma would be predicted to favor the development of diaphragm injury. Conversely, mechanical ventilation would be expected to prevent this adverse interaction by taking over the respiratory workload and thus eliminating contraction-related myofiber mechanical stresses.

Although major alterations in membrane mechanical properties such as reduced cellular deformability and pathologic hyperfragility have previously been linked to oxidative stress in red blood cells (21, 22), to our knowledge this is the first investigation to report the existence of a similar phenomenon in skeletal muscle cells. It is interesting to note that levels of oxidative stress that greatly increased the susceptibility of muscle cells to mechanical stress-induced sarcolemmal injury in vitro did not alter membrane integrity on their own. This is entirely consistent with the in vivo data, in which septic diaphragms with clear evidence of increased oxidative stress also failed to demonstrate sarcolemmal lesions unless additionally subjected to the myofiber mechanical stresses associated with spontaneous breathing efforts.

An interesting finding in our study was that protein carbonyl formation in the septic diaphragm appeared to be relatively selective for protein species migrating at approximately 38 and 35 kD. In addition, there was a significant correlation between iNOS expression and oxidative modification of these proteins, thus supporting a role for NO in these changes. Differential susceptibility of skeletal muscle proteins to carbonyl formation has been described under other pathologic conditions (34). Cell membranes are composed mostly of phospholipids and proteins, with the latter being glycosylated in many instances. Carbonyl groups can be added to proteins as the result of a direct attack by reactive oxygen species or through their interaction with reactive carbonyl compounds generated by glycoxidation and lipoxidation reactions (30). Therefore, protein carbonyl formation potentially serves not only as an index of free radical-mediated alterations to proteins, but also as a more general indicator of oxidative modifications to carbohydrates and lipids (30, 31). In addition, protein carbonyl levels in the diaphragm are positively correlated with force impairment in rats subjected to a high respiratory workload (39). We speculate that preferentially carbonylated proteins observed in the septic diaphragm could represent components of either the membrane or the underlying cytoskeletal network, because defects in both of these elements have been associated with an increased vulnerability to contraction-induced sarcolemmal disruption (28, 40). Specific identification of these protein species with enhanced carbonyl formation will require further study.

Study Limitations

Finally, certain methodologic issues and limitations of the present study should be addressed. First, we did not perform continuous electromyographic recordings of the diaphragm to ensure that muscle activation was completely abolished by mechanical ventilation. However, the absence of triggering of the ventilator as well as the stable and stereotypically reproducible shape of the tracheal pressure waveform allow us to conclude that any mechanical consequences of residual diaphragm activity, if present at all, were minimal. Second, there was no attempt to match the work of breathing in control and spontaneously breathing septic animals. Previous work indicates that the PTIdi is increased by about 2-fold after LPS administration (7, 37), and that this is far below the threshold level of work that is required to produce diaphragm injury in nonseptic animals (41, 42). Thus, Jiang and coworkers (41, 42) have shown that resistive loading that increases the PTIdi by approximately 15-fold is still insufficient to induce acute morphologic injury or force loss in the diaphragm within a time frame similar to that in our study. Similarly, in another study by our group (26), the cumulative diaphragm injury which resulted from repeated bouts of resistive loading (with the PTI increased approximately 10-fold) imposed over several consecutive days amounted to far less than found in the present study. A third issue is the dose of LPS administered in this study, which may be considered supraphysiologic. Importantly, we have previously compared LPS administered in this manner with a rat model of subacute peritonitis and found that both models produce equivalent amounts of diaphragm myofiber injury (12). Lastly, as is the case with all reductionist approaches, our in vitro method for imposing mechanical stress on muscle cells mimics but does not precisely reproduce the in vivo situation. For example, the 10% strain level imposed on muscle cells in our in vitro model is greater than normally experienced by the diaphragm in vivo. In addition, an actively contracting muscle is subjected not only to stretch but also to radially directed mechanical stresses (38). On the other hand, because myofiber mechanical stress in vivo is greatly increased by the presence of active contractions, the in vitro muscle stretch imposed in our study likely produces a global level of mechanical stress that is reasonably close to the in vivo situation. This is supported by the observation that in vitro passive muscle stretch at this level and in vivo contractile activity have been shown to induce many of the same adaptive cellular responses (43).

Conclusion and Implications

In summary, we have shown that mechanical ventilation prevents sarcolemmal injury and significantly improves force-generating capacity of the diaphragm in endotoxemic rats. Based on our findings, it appears unlikely that these benefits can be attributed to a reduction in oxidative stress. Rather, our data suggest that exposure of the diaphragm to oxidative stress during sepsis increases sarcolemmal fragility and hence the vulnerability of muscle cells to mechanical stress-induced sarcolemmal disruption. Under these conditions, mechanical ventilation could prevent injury by reducing the diaphragm myofiber mechanical stresses inherently associated with spontaneous breathing efforts. Further work will be needed to more clearly define the precise nature of the interactions between oxidative and mechanical stress-induced damage, including the identity of oxidatively modified proteins that are critical for preventing contraction-induced sarcolemmal injury. In addition, the clinical utility of employing noninvasive forms of mechanical ventilation, as a method for preventing respiratory muscle injury and ventilatory failure in septic patients, warrants further investigation.