Abstract
Skeletal muscle failure is a frequent manifestation of sepsis that affects prognosis and rehabilitation by impairing respiration and ambulation. Animal studies have shown that the inducible NO synthase (NOS2) is expressed in skeletal muscles during sepsis, likely affecting muscular function, by promoting the formation of the strong oxidant peroxynitrite. In contrast, whether human skeletal muscle expresses a functional NOS2 in similar conditions is unknown. We studied NOS2 expression (mRNA and protein) and activity and its role in contractile function in samples from rectus abdominis muscle obtained during surgical procedure in 16 septic patients and in 21 controls. Peroxynitrite formation was detected by immunohistochemical detection of nitrotyrosine residues. The main results of this study are as follows: (1) A significant increase in NOS2 mRNA, protein, and activity was found in muscles from septic patients, the expression of NOS2 protein positively correlating with sepsis severity. (2) Contractile force was significantly lower in septic than in control muscles. This phenomenon was not reverted by muscle incubation ex vivo with the NOS inhibitor l-NMMA, indicating that NO was not involved in force reduction at the time of biopsy. (3) NOS2 expression in skeletal myocytes was strongly co-localized with nitrotyrosine, revealing muscular peroxynitrite generation during the septic process, before the muscle was biopsied. Exposure of control muscles to an amount of peroxynitrite similar to that generated in septic muscles during the septic process resulted in a nonreversible reduction in force generation. These results suggest that NOS2 could be involved in the decreased muscular force of septic patients via the local generation of peroxynitrite.
The incidence of sepsis is continuously increasing. Despite improved therapy and better understanding of the mechanisms underlying the pathogenesis of sepsis, mortality rate remains greater than 40% (1). Death is mostly related to the failure of vital organs including liver, kidney, and heart. Sepsis also induces a severe and persistent alteration of skeletal muscle characterized by an increase in muscle catabolism, resulting in muscle wasting, and a decrease in muscular force (2, 3). Muscular alterations could have a very negative clinical impact because they may compromise the survival and the delay of recovery of septic patients. Indeed, failure of the respiratory muscles can delay weaning from mechanical ventilation (4), thus predisposing to other intensive care unit-acquired complications, and impairment of the locomotor muscles can prolong bed rest, thus predisposing to pulmonary thromboembolic disease (5). Therefore, identification of pathophysiological mechanisms for muscular failure in sepsis could help to develop new therapeutic strategies that can reduce sepsis morbidity and mortality.
Several studies in experimental animals have shown that nitric oxide (NO) synthesized by the inducible isoform of the NO synthase (NOS2) is an important mediator of the decreased force of respiratory and locomotor muscles in sepsis (3, 6, 7). This is, at least in part, related to muscular oxidative stress, secondary to the local generation of peroxynitrite (8), a highly reactive oxidant formed by the reaction of NO with superoxide anion (9). However, to the best of our knowledge, no data are available in the current literature concerning the expression and the role of NOS2 in skeletal muscle contractile failure in human sepsis. This point deserves particular attention since the differences in NOS2 expression and/or activity between animals and humans (10-13) do not allow to extrapolate data from animals to humans.
The aims of the present study were therefore to investigate in the rectus abdominis muscle of septic and control patients (1) the cellular expression and activity of NOS2, (2) whether NOS2 expression was associated with peroxynitrite formation, and related to the severity of illness, and (3) the role of NOS2 on the decreased muscular force observed in septic patients, with a particular attention to the role of peroxynitrite. We choose the rectus abdominis muscle because a biopsy of this muscle is rather easy to obtain during common abdominal surgical operations.
Two groups of consecutive patients undergoing abdominal or thoracic surgery were included in the study. One group (n = 16, septic group) consisted of patients with sepsis based on the following criteria of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (14). The source of sepsis was either chest (n = 7) or intraabdominal infection (n = 9) and required surgical treatment (Table 1). All cases of chest infection were mediastinitis secondary to cardiac surgery with (n = 5) or without bypass (n = 2). To overcome the systemic inflammatory state related to bypass surgery that could influence NOS2 expression in skeletal muscle (15), patients with mediastinitis were included when the delay between bypass surgery and the surgery for infection exceeded 7 d. The other group (n = 21, control group) consisted of patients undergoing elective laparotomy (n = 10) for various nonseptic conditions, or elective cardiac surgery (n = 11). Patients with cancer or chronic heart failure, or patients treated with glucocorticoids or salicylic acid at the time of surgery were not included in the study because these conditions could potentially modulate NOS2 expression in muscle (16-20). To assess the clinical condition, the SAPS II score (21) was calculated for each patient immediately before surgery.
| Sex | Age (yr) | Site of Infection | Cause of Infection | SAPS II* | Bacteria | Positive Blood Culture | Outcome (Length of stay in ICU [d]) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | M | 78 | Peritonitis | Colorectal anastomotic | 47 | Escherichia coli, | Yes | Alive (24) | ||||||||
| leak | Enterococcus faecium | |||||||||||||||
| 2 | F | 68 | Peritonitis | Colon perforation | 32 | Escherichia coli | Yes | Alive (12) | ||||||||
| 3 | M | 44 | Peritonitis | Colon perforation | 28 | Escherichia coli, | Yes | Alive (7) | ||||||||
| Klebsiella pneumoniae | ||||||||||||||||
| 4 | M | 54 | Pancreatic | Acute pancreatitis | 17 | Klebsiella pneumoniae, | Yes | Death (36) | ||||||||
| abcess | Enterobacter cloacae | |||||||||||||||
| 5 | M | 40 | Peritonitis | Perforate appendicitis | 16 | Pseudomonas aeroginosa | No | Alive (30) | ||||||||
| 6 | F | 51 | Peritonitis | Colon perforation | 57 | Escherichia coli | No | Alive (53) | ||||||||
| 7 | F | 71 | Peritonitis | Colon anstomotic leak | 35 | Escherichia coli | No | Alive (4) | ||||||||
| 8 | F | 61 | Peritonitis | Colon perforation | 20 | Escherichia coli | No | Alive (35) | ||||||||
| 9 | F | 60 | Peritonitis | Colon perforation | 25 | Bacteriodes ovatus | No | Alive (7) | ||||||||
| 10 | M | 88 | Mediastinitis | Postcardiosurgical | 60 | Staphylococcus epidermidis | Yes | Alive (30) | ||||||||
| 11 | M | 43 | Mediastinitis | Postcardiosurgical | 36 | Staphylococcus aureus | Yes | Alive (23) | ||||||||
| 12 | M | 72 | Mediastinitis | Postcardiosurgical | 57 | Staphylococcus aureus | Yes | Alive (35) | ||||||||
| 13 | M | 69 | Mediastinitis | Postcardiosurgical | 26 | Staphylococcus epidermidis | No | Alive (9) | ||||||||
| 14 | M | 73 | Mediastinitis | Postcardiosurgical | 52 | Staphylococcus epidermidis | Yes | Alive (5) | ||||||||
| 15 | M | 68 | Mediastinitis | Postcardiosurgical | 58 | Staphylococcus epidermidis | No | Death (3) | ||||||||
| 16 | M | 76 | Mediastinitis | Postcardiosurgical | 46 | Enterococcus faecalis, | No | Alive (5) |
The study was performed with the approval of the local board governing research on human subjects at our institutions (Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédicale, Hôpital Saint-Louis, Paris, France). All patients gave informed written consent.
A biopsy specimen was obtained from the rectus abdominis muscle during the initial phase of the operation. To study the effect of the systemic and not the local inflammation on muscle NOS2 expression and activity, septic patients in whom the muscle was in contact with the site of infection were not included in the study. Tissue samples (∼ 500 mg) were quickly frozen in liquid nitrogen and stored at −80° C for less than 3 mo for mRNA and protein analysis. In some cases, an additional biopsy was obtained to perform immunohistochemical detection of NOS2 and nitrotyrosine residues. This biopsy was put into tissue glue (PolyFreeze; Polysciences Inc., Warrington, PA) on a cork holder, and quickly frozen in isopentane cooled with liquid nitrogen. Then it was stored at −80° C until analysis. Finally, in a subset of control and septic patients, one or two additional biopsies were obtained to evaluate muscular force ex vivo.
The various assays and the reading of immunostained biopsies were performed in a blinded fashion. Each sample from control and septic patients was coded by one of the investigators (J.B.), who was not directly involved in performing the different assays. This investigator was the only participant in the study who knew the codes until the end of the study. The codes were revealed once all the measurements were performed. Analysis of immunostained biopsies were performed by three persons independently. Concordance in this blind analysis was obtained in 96% of the cases. In the remaining cases, analysis was performed simultaneously and an common conclusion was attempted.
Detection of NOS2 mRNA by RT-PCR. Total RNA was extracted using the Trizol Reagent, following the manufacturer's instructions, and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for NO synthase type II (NOS2) was then performed in the presence of a defined amount of specific RNA mutant as an internal standard. The NOS2 internal standard (a kind gift from Dr. S. Nadaud) was subcloned into a pSP(64) poly(A) vector (Promega, Charbonnières, France). A cRNA was then synthesized in vitro as sense probe from 10 μg of the PvuI-linearized plasmid using SP6 RNA polymerase in the presence of 10 μCi of [α-32P]UTP. The concentration of the transcript was determined after measurement of the radioactivity incorporated into RNA product. For NOS2, the primers chosen were, for sense, 5′-AAGACCCAGTGCCCTGCTTT-3′ and for antisense 5′-CGCAAACATAGAGGTGGCC-3′, to allow the distinct amplification of NOS2 mRNA (388 bp) and of the internal standard (452 bp). These primers were chosen to encompass several introns to avoid amplification of contaminating genomic DNA. Total RNA was reverse transcribed with a fixed amount of the specific synthetic RNA and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Cergy Pontoise, France). The single-strand cDNA synthesis was carried out in 20 μl of reaction buffer (20 mM Tris–HCl [pH 8.3], 50 mM KCl, 4 mM MgCl2, 1 mM diethylnitrophenyl thiophosphate [dNTP], 10 mM dithiothreitol [DTT], 0.2 μM oligo-p[dT]). The reaction mixture was incubated 10 min at 25° C, and then 60 min at 37° C. The resultant cDNA was amplified using 2.5 U of Taq DNA polymerase (Boerhinger, Meylan, France), and 0.5 μM of the sense and antisense primers in 50 μl of 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 4 mM MgCl2, 1 mM dNTP, and 0.01% gelatine. Twenty-eight amplification cycles were undertaken as follows: denaturation at 94° C for 1 min, annealing at 62° C for 1 min, and extension at 72° C for 1 min. The final extension was carried out for 10 min. To quantify NOS2 mRNA levels, a trace amount of [32P]dCTP (deoxycytidine triphosphate) was included in the PCR reaction. After PCR amplification, the PCR products were separated on a 5% polyacrylamide gel, and radioactive signals were analyzed using a computer-based imaging system (Fuji Bas 1000; Fuji Medical Systems, Clichy, France).
Detection of NOS2 protein by Western blot and dot blot analysis. Western and dot blot experiments were performed as described previously (3). Two different antibodies recognizing different regions of the NOS2 protein were used. The first was a mouse monoclonal antibody directed against a 21-kD protein fragment corresponding to amino acids 961–1144 of mouse NOS2 (Transduction Laboratories, Lexington, KY). This antibody has been previously shown to react specifically with NOS2 in human macrophages (22) and human colon adenomas (23). A second antibody was a rabbit polyclonal antibody directed against amino acids 3–22 at the amino terminus of human NOS2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Both antibodies were used at a 1:1,000 dilution. Immunoreactive proteins were then visualized by chemiluminescence.
Proteins from rat hepatocytes transfected with the human NOS2 cDNA and expressing NOS2 protein (24), and from human and rat alveolar macrophages stimulated with 10 μg/ml lipopolysaccharide (LPS) + 10 ng/ml interferon-gamma (IFN-γ) (25), were used as positive controls.
Human alveolar macrophages were obtained as previously described (25). Briefly, bronchoalveolar lavages (BAL) were obtained from three patients with mild chronic bronchitis. These patients had normal chest X-ray and computed tomography scan, normal lung function data, and no respiratory infection within the last 3 mo. All of them were ex-smokers. All patients gave informed written consent. This part of the study was performed with the approval of the local board governing research on human subjects at our institutions (Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédicale, Hôpital Bichat, Paris, France). BAL fluid was centrifuged at 600 × g for 5 min at 4° C. The pellet was resuspended in complete RPMI medium in order to have a maximum of 4 × 106 cells/ml. Alveolar macrophages were allowed to adhere on culture dish by differential adherence during 2 h at 37° C in a humidified 5% CO2/95% air incubator. Nonadherent cells were removed by washing with phosphate-buffered saline (PBS), and then macrophages were incubated for 24 h in complete culture medium alone (control macrophages), or with 10 μg/ml LPS + 10 ng/ml IFN-γ (stimulated macrophages). Twenty-four hours later macrophages were scrapped and resuspended in lysis buffer to perform Western blot and to measure NOS2 activity. NOS2 activity (see Methods below) was 1.2 ± 0.8 and 4.1 ± 2.2 pmol/mg protein/min in control and stimulated macrophages, respectively (p < 0.05). It was inhibited by adding N-monoethyl l-arginine (l-NMMA) to the reaction media (1 mM, 97.2 ± 2.1% of inhibition, as compared with untreated samples).
NOS2 protein localization by immunohistochemistry. Immunohistochemistry was performed as described previously (3) using the same anti-NOS2 antibodies used for the Western blots. Antibodies dilution were 1/100 and 1/200 for the Transduction Laboratories and Santa-Cruz Laboratories antibodies, respectively. The specificity of the immunostaining was evaluated by omission of the primary antibody, as well as by using pooled nonimmune rabbit immunoglobulin G (IgG) instead of the primary antibody at equivalent protein concentration and processed as above.
NOS2 activity measurements. Calcium-calmodulin-independent NO synthase activity, which reflects NOS2 activity (26), was measured by the conversion of l-[3H]arginine in l-[3H] citrulline, according to the method described by Bredt and Snyder (27). Initial experiments showed that the reaction was linear for 60 min, and that it was concentration-dependently inhibited by addition of l-NMMA to the reaction buffer. Indeed inhibition of NOS2 activity, expressed as percentage of the untreated sample's activity, was 4.1 ± 1.5; 45.2 ± 0.9 and 93.6 ± 3.7 with 100 nM, 10 μM, and 1 mM l-NMMA, respectively (n = 4 for each concentration of l-NMMA).
Peroxynitrite detection. Generation of peroxynitrite in the muscle was evaluated by immunohistochemical detection of nitrotyrosine residues (9). The first antibody (1:100 dilution) was a monoclonal antinitrotyrosine antibody (U.B.I., Lake Placid, NY). In addition to the negative controls described for NOS2 immunostaining, the specificity of the nitrotyrosine antibody was verified by treating tissue sections with 100 mM sodium hydrosulfite for 20 min to reduce sample nitrotyrosine to aminotyrosine (28). Sodium hydrosulfite was prepared immediately before use in 10 mM NaHCO3, pH 9–10.
Muscular specimen preparation. To measure contractile function, the biopsies were quickly transferred to a dissecting dish containing oxygenated Krebs solution at pH 7.4. Muscle specimens were then temporarily preloaded at 10 mN to avoid contracture until further analysis (29). Contractile function was evaluated as described previously (3).
Role of NO and peroxynitrite on contractile function. Protocol 1: Effect of l-NMMA on the force of the rectus abdominis muscle of control and septic patients. To evaluate the effect of NO on muscular dysfunction in septic patients, muscular bundles from control or septic patients were exposed to Krebs solution alone and Krebs solution containing 10 mM of the NOS inhibitor l-NMMA (30); the order of exposure was randomly determined. Force was measured at the end of a 30-min exposure.
Protocol 2: Effect of peroxynitrite on the force of the rectus abdominis muscle of control patients. To mimic the potential effect of peroxynitrite on skeletal contractile function in sepsis, bundles of control patients were exposed to Krebs solution containing 5 mM of the peroxynitrite donor 3-morpholinosydnonimine (SIN-1 [9, 31]) (n = 8) during 15 min. At the end of the incubation period, bundles were exposed to Krebs solution alone during 75 min (recovery period). Force was evaluated at the end of the incubation period and each 15 min during the recovery period. Another group of bundles (n = 5) was treated with a pulse of 1 mM peroxynitrite; force was evaluated 15 min later, and then each 15 min during a 75-min period. In previous experiments, we verified that 1 mM pH-inactivated peroxynitrite and 0.3 M NaOH (the solvant for peroxynitrite) did not modify muscular (n = 3 in each case). After exposure to peroxynitrite, bundles were prepared for immunohistochemical detection of nitrotyrosine residues, as previously described. Five fibers exposed to Krebs solution alone were used as controls.
l-[3H]Arginine was from NEN/DuPont (Boston, MA). Tetrahydrobiopterin was supplied by Research Biomedical Inc. (Natick, MA). Sodium dodecyl sulfate (SDS), glycerol, 2-mercaptoethanol, and bromophenol blue were obtained from Rio-Rad (Richmond, CA). Culture media, supplements, and fetal bovine serum were from Flow Labs (Irvine, UK). Tissue culture plasticware was supplied by Costar Corp. (Cambridge, MA). Peroxynitrite was prepared as described previously (32) and stored in 0.3 M NaOH. The concentration of peroxynitrite was determined spectrophotometrically at 302 nm. Other reagents were from Sigma Chemical Co. (La Verpillière, France).
Values are given as mean ± SEM. The differences between means were analyzed by the Mann–Whitney U test (33). The relationship between NOS2 protein expression and sepsis severity was assessed by linear regression analysis. Paired comparisons of force modification after the exposure of muscle bundles to the different pharmacological agents were made with the Wilcoxon test. Significance for all statistics was accepted at p < 0.05.
Characteristics of septic patients are given in Table 1. The mean age between control and septic patients did not differ (58 versus 64 yr, respectively). Sepsis-related organs injury was reflected by a significantly higher simplified acute physiology score (SAPS II) in septic as compared with control patients (38 ± 4 versus 11 ± 1, p < 0.001).
NOS2 mRNA. NOS2 mRNA was detected by semiquantitative RT-PCR. The primer pair for amplification of our internal standard showed the presence of a 452 bp in all of the mRNA samples tested (Figure 1A). the PCR reaction products using human NOS2-specific primers showed a clear band at the predicted size of 388 bp in muscles from septic patients, whereas a very slight band was observed in samples from controls (Figure 1A). The NOS2 mRNA molecules/μg of total RNA ratio increased 7-fold (p < 0.05) in the septic group as compared with the control group (Figure 1B).
Fig. 1. (A) RT-PCR analysis of NOS2 mRNA levels in the rectus abdominis muscle of two different representative control and septic patients (C and S, respectively). Fifty nanograms of total RNA with 10,000 molecules of internal standard were assayed. The RT-PCR products were loaded onto 5% polyacrylamide gel. MWM indicates molecular weight marker. (B) Mean (± SEM) values of NOS2 mRNA molecules normalized by microgram of total mRNA in control and septic groups.
[More] [Minimize]NOS2 protein. Whole muscular homogenates from most of the septic patients (12 of 16) but none of the control patients expressed NOS2 protein as detected by Western blot, with a molecular weight identical to NOS2 protein expressed by rat hepatocytes transfected with the human NOS2 cDNA, and by in vitro LPS + IFN-γ-stimulated human and rat alveolar macrophages (Figure 2A). No band was detected in cells transfected with the empty vector (data not shown) and in nonstimulated alveolar macrophages (Figure 2A). Quantification of NOS2 protein expression by Dot blot confirmed Western blot results and showed a mean value (arbitrary units, AU, maximum 100) of 38.7 ± 10.1 AU in septic patients versus 3.7 ± 1.2 AU in controls (p < 0.01, Figure 2B).
Fig. 2. (A) Representative Western blot analysis of NOS2 protein in the rectus abdominis muscle. Proteins from muscular homogenates of two different representative control and septic patients (C and S, respectively), from rat hepatocytes transfected with the human NOS2 cDNA and expressing the protein (hrNOS2 [29], from rat alveolar macrophages stimulated with 10 μg/ml LPS + 10 ng/ml IFN-γ (rAM), and from human alveolar macrophages nonstimulated and stimulated similarly to rAM (hAM− and hAM+, respectively) were separated by 7.5% SDS–PAGE and transferred to a PVDF membrane. A main band with an estimated molecular weight of 130 kD corresponding to NOS2 is present in samples from the muscles of both septic patients, from rat hepatocytes, and from human and rat LPS + IFN-γ-stimulated alveolar macrophages. By contrast, no band is present in the muscles from both control patients. Similar results were obtained with the two anti-NOS2 antibodies utilized (data not shown). Therefore, to avoid duplication, only results obtained with the antibody from Transduction Laboratories are shown (B) Muscular NOS2 expression quantified by dot blot in control and septic patients (arbitrary units, maximum 100). Each bar represents the mean ± SEM values of NOS2 protein expression of each group.
[More] [Minimize]Localization of NOS2 protein. Histologic analysis of the muscles of septic and control patients showed no inflammatory infiltrate and an absence of tissue edema. An increased NOS2 staining was reproducibly observed in skeletal muscle myocytes of all septic patients expressing NOS2 protein in Western blot, but in none of the control patients (Figure 3). NOS2 staining appeared in a diffuse cytoplasmic pattern and occupied a variable portion of the myocyte surface. No NOS2 staining was observed when the anti-NOS2 antiserum was omitted (Figure 3). No tissue section showed positive immunostaining with nonimmune serum (data not shown).
Fig. 3. Immunohistochemical detection of NOS2 and nitrotyrosine in the rectus abdominis muscle of a representative septic and control patient. NOS2 and nitrotyrosine staining were colocalized in skeletal myocytes from the septic patient. These immunostainings were not observed when the anti-NOS2 or the antinitrotyrosine antibody was omitted. A comparable section from a control patient showed no NOS2 staining and a very slight staining for nitrotyrosine. Similar results were obtained with the two anti-NOS2 antibodies utilized (data not shown). Therefore, to avoid duplication, only results obtained with the antibody from Transduction Laboratories are shown. Original magnification: ×200.
[More] [Minimize]NOS2 activity. Induction of muscular NOS2 activity, measured by the conversion of l-[3H]arginine to l[3H]citrulline in the absence of calcium and calmodulin in the reaction buffer, paralleled induction of NOS2 protein. Indeed, l-[3H]citrulline formation was significantly higher in septic patients as compared with the low levels observed in the control patients (32.41 ± 6.62 versus 1.87 ± 1.27 pmol/mg protein/min, respectively, p < 0.01, Figure 4).
Fig. 4. Muscular NOS2 activity in the different groups of patients. NOS2 activity was expressed as pmol of l-[3H]citrulline produced by milligram of protein and by minute of incubation. Each bar represents the mean ± SEM of each group.
[More] [Minimize]Correlation between NOS2 protein expression and sepsis severity. NOS2 protein expression was positively related to patient's severity scores (r = 0.84; p < 0.001; Figure 5) but not with age, sex, catecholamines treatment, the type of bacteria, or the source of infection (data not shown).
Fig. 5. Relationship between the disease severity score SAPS II and the intensity of muscular NOS2 expression quantified by dot blot.
[More] [Minimize]Immunohistological analysis for nitrotyrosine residues showed results that paralleled those for NOS2. Indeed, extensive nitrotyrosine staining was colocalized with NOS2 immunostaining (Figure 3), no staining being observed when the antinitrotyrosine antibody was omitted (Figure 3). Furthermore, the specificity of the antinitrotyrosine antibody was confirmed by the marked reduction in the intensity of the immunostaining after reducing nitrotyrosine to aminotyrosine by treating tissue sections with sodium hydrosulfite (data not shown).
No difference was observed in muscle bundles dimensions between control and septic patients. Values of bundles weight ranged from 61 to 159 mg and length from 14 to 28 mm.
Effect of l-NMMA on force generation in muscles from control and septic patients. The force generated by the rectus abdominis muscle was reduced in septic patients. Indeed, maximal tetanic tension (tension observed at 30 Hz) was significantly lower in bundles from septic than from control patients (p < 0.05; Figure 6). Similar results were observed at 10 Hz stimulation (data not shown). Exposure of bundles from septic and control patients to the NOS inhibitor l-NMMA did not modify force (Figure 6).
Fig. 6. Force of the rectus abdominis muscle (expressed in grams per gram of bundle weight) obtained at 30-Hz stimulation in bundles from control and septic patients exposed to Krebs solution alone (solid bars) or Krebs solution containing 10 mM l-NMMA (open bars). Each bar represent the mean ± SEM of each group. NS, no significant difference.
[More] [Minimize]Effects of peroxynitrite on force generation in muscles from control patients. Force was similar in the different groups of bundles before application of the pharmacological agents, being 20.2 ± 4.5 and 56.8 ± 7.6 g/g for 10 and 30 Hz, respectively. Both the peroxynitrite donor SIN-1 and peroxynitrite itself elicited a significant decrease in force. Thus, tension obtained at 30 Hz stimulation was decreased by 20.3 ± 6.4% (p < 0.05) and 45.2 ± 79% (p < 0.01) of basal value after exposure of the bundles to SIN-1 or peroxynitrite, respectively (Figure 7A). Force values did not change during the 75 min of the recovery period as compared with values obtained at the end of the incubation period. Similar results were observed at 10-Hz stimulation (data not shown).
Fig. 7. (A) Force of the rectus abdominis muscle (expressed as a percentage of basal value) obtained at 30-Hz stimulation in bundles from control patients after a 15-min exposure to either Krebs solution alone or Krebs solution containing 5 mM SIN-1; or either 15 min after exposure to a single 1 mM peroxynitrite pulse. Each bar represent the mean ± SEM of each group. (B) A positive nitrotyrosine immunostaining was observed in a bundle after exposure to 1 mM peroxynitrite whereas no such immunostaining was observed after exposure to Krebs solution alone.
[More] [Minimize]The reduction of force after exposure to peroxynitrite was accompanied by a positive nitrotyrosine immunostaining, whereas no such immunostaining was observed in bundles exposed to Krebs solution alone (Figure 7B).
This study shows that NOS2 mRNA, protein, and activity were significantly higher in the rectus abdominis muscle of septic as compared to control patients. To our knowledge this is the first evidence of NOS2 expression and its functional consequences in the setting of human sepsis.
The slight signal from NOS2 mRNA and protein detected in samples from control patients was reproducibly observed. This expression is unlikely to be related to the underlying clinical condition as diseases known to induce NOS2 expression such as chronic heart failure or cancer (17, 18) were excluded. Moreover, as recently reported, a slight constitutive NOS2 expression not related to the presence of inflammatory processes may occur in human skeletal muscle (34).
A potential residual activation of NOS2 after bypass surgery might have amplified NOS2 signals in some septic patients. Although we choose to include patients only after a delay greater than 7 d between scheduled surgery and septic complication, a participation of the bypass-induced inflammation cannot be excluded (15). However, in the absence of complications, inflammation decreases in few days after bypass surgery. In addition to the systemic inflammation, a local compartmentalized inflammation related to the nidus of infection can occur. However, we did not include patients in which the muscle was in contact with the site of infection. Furthermore, histological analysis of the muscles included in the study revealed no inflammatory changes, ruling out a local inflammation of the muscle as a cause of NOS2 up-regulation. Consequently, increased NOS2 expression in skeletal muscle was very likely part of the systemic manifestations of sepsis. This was further confirmed by the positive close correlation between NOS2 protein expression and SAPS II score. Indeed, SAPS II score integrates different indexes indicative of multiple organ dysfunction, and has been associated with the systemic production of inflammatory mediators such as bactericidal/ permeability-increasing protein and soluble E-selection (35).
Despite the large number of studies on NOS2 expression and function in experimental animals, available data in human sepsis are very scarce. To our knowledge only two studies in the current literature directly investigated tissue NOS2 expression in human sepsis (36, 37). Both of them were performed in samples from the myocardium obtained postmortem (n = 5 and 3 septic patients, respectively) and reported NOS2 mRNA and/or protein expression in cardiac myocytes. However, they did not provide data on NOS2 activity. Furthermore, because these studies were performed on autopsy samples, they might not reflect the in vivo characteristics of the protein because of postmortem protein degradation. The results of the present study therefore provide definite evidence that significant amounts of NOS2 mRNA, protein, and enzymatic activity were present in a tissue from alive septic patients.
The force of the rectus abdominis was reduced in septic patients. This is, to the best of our knowledge, the first direct demonstration of a reduction in the force of a skeletal muscle from septic patients. This finding is in line with data previously reported in experimental animals (2, 6).
In spite of the marked increase in expression and activity of NOS2, the decrease in submaximal and maximal force of the muscles of septic patients was not reversible by the incubation with the NO synthase inhibitor l-NMMA, indicating that NO was not directly involved in force reduction at the time of sepsis the muscle was biopsied (an average of 12 h after the onset of sepsis). However, we cannot rule out an acute effect of NOS2-synthetized NO on the contractile process in the early stages of sepsis, before the muscle was biopsied (38). It must be noted that muscular failure during sepsis is a multifactorial phenomenon, which involves several mechanisms acting on muscular excitation–contraction coupling either directly or indirectly via modifications of muscular metabolism and hemodynamic parameters. Therefore, products other than NO, such as prostaglandins, proinflammatory cytokines, or reactive oxygen species could also be involved in the decreased force of the muscles from septic patients (see [39] for review). In this way, it is interesting to note that continuous NOS2-dependent NO production from the early stages of sepsis may also result in generation of the strong oxidant peroxynitrite, which can induce long-lasting alterations of the contractile machinery, no more reversible by further inhibition of NO synthesis. Indeed, submicromolar concentrations of NO can compete with endogenous superoxide dismutase for superoxide anion, by a rapid reaction with a near diffusion controlled rate constant, that generates the potent oxidizing agent peroxynitrite (9), which can directly attack many biological targets (40) including contractile proteins (41, 42). In addition, peroxynitrite readily nitrates tyrosine residues in proteins, which could result in long-lasting modifications of protein structure and function (43, 44). In vivo, the half-life of nitrotyrosine bonds appears to be > 3 d (45), indicating a constant generation of peroxynitrite will lead to accumulation of tyrosine-nitrated proteins. Supporting a role of peroxynitrite generated from the beginning of the septic process in contractile failure, we have previously shown in the rat diaphragm that treatment of the animals with l-NMMA from the beginning of an endotoxemic process prevented both peroxynitrite generation and submaximal and maximal force decrease (8).
To evaluate if peroxynitrite was generated in the muscles of septic patients, we performed immunohistochemical detection of nitrotyrosine residues (9). The results of these experiments showed that the expression of NOS2 was associated with the local formation of nitrotyrosine, thus revealing local peroxynitrite formation. It must be noted, however, that in vitro studies have identified additional mechanisms of nitrotyrosine formation different from that of peroxynitrite. The most important of such mechanisms is a peroxidase-dependent nitrite oxidation (46, 47). This mechanism could be of importance at loci of inflammatory-immune processes when myeloperoxidase and/or eosinophil peroxidase are secreted from activated granulocytes. Since no infiltration by granulocytes or other inflammatory cells was observed in the muscle of septic patients, nitrotyrosine can be accepted as a specific stable marker for the formation of peroxynitrite in the present study (9). In this way, it is noteworthy that NOS2 and nitrotyrosine were strictly colocalized in septic myocytes, i.e., myocytes expressing NOS2 also showed nitrotyrosine immunostaining and, conversely, absence of NOS2 expression was associated with an absence of nitrotyrosine staining, thus strongly suggesting that NOS2 was directly involved in peroxynitrite generation. As peroxynitrite is the product of the reaction between NO and superoxide anion (9), colocalization implies that NOS2-synthetized NO was directly involved in superoxide generation, as demonstrated by Poderoso and coworkers (48). Alternatively, colocalization between muscle NOS2 and nitrotyrosine immunostaining could be ascribed to simultaneous release of NO and superoxide anion by NOS2, as recently reported by Xia and Zweier in murine macrophages (49).
Because oxidating and nitrotyrosinating properties of peroxynitrite can alter proteins involved in the contractile process, such as actin (41), and the sarcoplasmic reticulum Ca-ATPase (42), we then evaluated if this molecule was involved in the reduction of muscular force observed in septic patients, as previously demonstrated in rat muscles (50). This was performed by incubating muscles of control patients with the peroxynitrite donor SIN-1 (9, 31), using a protocol that allows muscles to be exposed ex vivo to an amount of peroxynitrite similar to that generated in vivo during sepsis. Indeed, although no direct measure of peroxynitrite generation rates in human muscle during sepsis has yet been made, an estimation of this parameter can be made based on the chemistry of NO and peroxynitrite. In fact, because the reaction of NO with superoxide anion to yield peroxynitrite is a near diffusion-controlled reaction (9), a simultaneous increase in superoxide and NO steady-state concentrations will increase the rate of peroxynitrite formation, NO and superoxide reacting quantitatively with each other. Therefore, assuming that NO production by NOS2 in muscle homogenates was 0.4 10−8 M s−1 (calculated from Figure 1), a similar rate of peroxynitrite generation, as previously shown in the rat diaphragm (8), can be expected. As demonstrated by Brunelli and coworkers (51), for fast-decaying molecules, such as peroxynitrite (half-life < 1 s at pH 7.0 and 37° C), the chemical effects must be analyzed in terms of the total amount of molecules added to the system, i.e., concentration multiplied by time. In this way, we utilized a protocol of SIN-1 administration (5 mM SIN-1 during 15 min) that allowed an exposure of muscle proteins to 750 μM of peroxynitrite (31). This concentration of peroxynitrite is in the range of the one generated in the muscles of septic patients during 12 h (0.4 × 10−8 M s−1 × 60 s × 720 min), this time corresponding to the average estimated period of sepsis before the rectus abdominis was sampled. This protocol of SIN-1 incubation resulted in a significant and nonreversible decrease in muscular force. Furthermore, a similar decrease in force was observed with a single pulse of 1 mM peroxynitrite, thus confirming the results obtained with SIN-1. Because peroxynitrite vehicle (NaOH 0.3 mM) and decomposed peroxynitrite did not modify muscle force, and because application of 1 mM peroxynitrite resulted in nitration of tyrosine residues in muscle fibers, in a pattern similar to that in the muscles of septic patients, we can estimate that exposure of the muscle ex vivo to an amount of peroxynitrite close to what the septic muscles were exposed to in vivo resulted in a significant decrease in force. Therefore, NOS2-derived peroxynitrite could be considered a final mediator of a deleterious effect of increased NOS2 activity on force generation in septic muscles. It must be noted, however, that an intervention devoted to block in vivo NOS2-derived peroxynitrite formation was impossible to perform in the present study, and thus, the definitive proof of causality between NOS2 induction, peroxynitrite formation, and muscular failure in septic patients remains to be provided. Other mechanisms such as impairments in cellular respiration, glucose metabolism, and/or intracellular Ca2+ kinetics (see [39] for review) could also participate in a deleterious effect of the increased NOS2 expression and activity in muscle function.
We conclude that sepsis induces the expression of a functional NOS2 in human skeletal muscle, along with the local formation of peroxynitrite. These findings could help to better understand the effects of sepsis on the skeletal musculature in humans.
The authors are very grateful to Dr. Maria Cecilia Carreras (Laboratory of Oxygen Metabolism, University of Buenos Aires, Argentina) for helpful discussion of the manuscript and to Pr. Bruno Crestani and Dr. Claudine Peiffer (INSERM U408, Paris, France) for their encouraging comments. The expert technical assistance of Mrs. Laure Guillaudeu and Corinne Rolland is greatly acknowledged.
Supported by the Assistance Publique-Hôpitaux de Paris (Grant CRC-97-141) and by an award (S.L.) and a grant (D.P.) from Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche.
| 1. | Wheeler AP, Bernard GRTreating patients with severe sepsis. N Engl J Med3401999207214 |
| 2. | Ruff RL, Secrist DInhibitors of prostaglandin synthesis or cathepsin B prevent muscle wasting due to sepsis in the rat. J Clin Invest73198414831486 |
| 3. | Boczkowski J, Lanone S, Ungureanu-Longrois D, Danialou G, Fournier T, Aubier MInduction of diaphragmatic nitric oxide synthase after endotoxin administration in rats. J Clin Invest98199615501559 |
| 4. | Murciano D, Boczkowski J, Lecocguic Y, Milic-Emili J, Pariente R, Aubier MTracheal occlusion pressure: a simple index to monitor respiratory muscle fatigue during acute respiratory failure in patients with chronic obstructive pulmonary disease. Ann Intern Med1081988800805 |
| 5. | Anderson FA Jr, Wheeler HB. Venous thromboembolism: risk factors and prophylaxis. Clin Chest Med 1995:16:235–251. |
| 6. | El Dwairi Q, Comtois A, Guo Y, Hussain SN. Endotoxin-induced skeletal muscle contractile dysfunction: contribution of nitric oxide synthases. Am J Physiol 1998;274:C770–C779. |
| 7. | Gath I, Cloos EI, Gödtel-Armbrust U, Schmitt S, Nakane M, Wessler I, Föstermann UInducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for contractile function. FASEB J10199616141620 |
| 8. | Boczkowski J, Lisdero C, Lanone S, Samb A, Carreras MC, Boveris A, Aubier M, Poderoso JJEndogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia. FASEB J13199916371646 |
| 9. | Beckman JS, Koppenol WHNitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol2711996C1424C1437 |
| 10. | Albina JEOn the expression of nitric oxide synthase by human macrophages: Why no NO? J Leukocyte Biol581995643649 |
| 11. | Weinberg JB, Misukonis MA, Shami PJ, Mason SN, Sauls DL, Dittman W, Wood ER, Smith GK, MacDonald B, Bachus KE, et al.. Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages. Blood86199511841195 |
| 12. | Thiemermann TNitric oxide and septic shock. Gen Pharmacol291997159166 |
| 13. | Nussler AK, Di Silvio M, Liu ZZ, Geller DA, Freeswick P, Dorko K, Bartoli F, Billiar TRFurther characterization and comparison of inducible nitric oxide synthase in mouse, rat, and human hepatocytes. Hepatology21199515521560 |
| 14. | Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJDefinitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest101199216441655 |
| 15. | Wan S, LeClerc JL, Vincent JLInflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies. Chest1121997676692 |
| 16. | Adams V, Yu J, Möbius-Winkler S, Linke A, Weigl C, Hilbrich L, Schuler G, Hambrecht RIncreased inducible nitric oxide synthase in skeletal muscle biopsies from patients with chronic heart failure. Biochem Mol Med611997152160 |
| 17. | Riede UN, Föstermann U, Drexler HInducible nitric oxide synthase in skeletal muscle of patients with chronic heart failure. J Am Coll Cardiol321998964969 |
| 18. | Buck M, Chojkier MMuscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. EMBO J15199617531765 |
| 19. | Geller DA, Nüssler AK, Di Silvio M, Lowenstein CJ, Chern HO, Davies P, Pitt BR, Simmons RL, Billiar TRCytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci USA901993522526 |
| 20. | Farivar RS, Brecher PSalicylate is a transcriptional inhibitor of the inducible nitric oxide synthase in cultured cardiac fibroblasts. J Biol Chem27119963158531592 |
| 21. | Le Gall JR, Lemeshow S, Saulnier FA new simplified acute physiology score (SAPS II) based on a European/North American multicenter study. JAMA270199329572963 |
| 22. | Watkins SC, Macaulay W, Turner D, Kang R, Rubash HE, Evans CHIdentification of inducible nitric oxide synthase in human macrophages surrounding loosened hip protheses. Am J Pathol150199711991206 |
| 23. | Ambs S, Merriam WG, Bennett WP, Felley-Bosco E, Ogunfusika MO, Oser SM, Klein S, Shields PG, Billiar TR, Harris CCFrequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression. Cancer Res581998334341 |
| 24. | Geller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di Silvio M, Freeswick P, Wang SC, Nakayama DK, Simmons RL, Snyder SH, Billiar TRMolecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci USA90199334913495 |
| 25. | Crestani B, Cornillet P, Dehoux M, Rolland C, Guenounou M, Aubier MAlveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. J Clin Invest941994731748 |
| 26. | Morris SM, Billiar TRNew insights into the regulation of inducible nitric oxide synthesis. Am J Physiol2661994E829E839 |
| 27. | Bredt DS, Snyder SHIsolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA871990682685 |
| 28. | Kooy NW, Royall JA, Ye YZ, Kelly DR, Beckman JSEvidence for in vivo production in human acute lung injury. Am J Respir Crit Care Med151199512501254 |
| 29. | Coirault C, Riou B, Bard M, Suard I, Lecarpentier YContraction, relaxation, and economy of force generation in isolated human diaphragm muscle. Am J Respir Crit Care Med152199512751283 |
| 30. | Reif DW, McCreedy SAN-Nitro-l-arginine and N-monomethyl-l-arginine exhibit a different pattern of inactivation toward the three nitricoxide synthases. Arch Biochem Biophys3201995170176 |
| 31. | Darley-Usmar V, Hogg N, O'Leary V, Wilson M, Moncada SThe simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Rad Res Commun171992920 |
| 32. | Ishida H, Ichimori K, Hirota Y, Fukahori M, Nakazawa HPeroxynitrite-induced cardiac myocyte injury. Free Rad Biol Med201996343350 |
| 33. | Winer, BG. Statistical principles in experimental design. New York: McGraw-Hill; 1971. |
| 34. | Park CS, Park R, Krishna GConstitutive expression and structural diversity of inducible isoform of nitric oxide synthase in human tissues. Life Sci591996219225 |
| 35. | Froon A, Bonten M, Gaillard C, Greve JW, Dentener MA, de Leeuw PW, Drent M, Stoberringh EE, Buurman WAPrediction of clinical severity and outcome of ventilator-associated pneumonia. Am J Respir Crit Care Med158199810261031 |
| 36. | Thoenes M, Föstermann U, Tracey WR, Bleese NM, Nussler AK, Scholz H, Stein BExpression of inducible nitric oxide synthase in failing and non-failing human heart. J Med Cell Cardiol281996165169 |
| 37. | Fukuchi M, Hussain SNA, Giaid AHeterogeneous expression and activity of endothelial and inducible nitric oxide synthases in end-stage human heart failure. Circulation981998132139 |
| 38. | Kobzik L, Reid MB, Bredt DS, Stamler JSNitric oxide in skeletal muscle. Nature3721994546548 |
| 39. | Hussain SNARespiratory muscle dysfunction in sepsis. Mol Cell Biochem1791998125134 |
| 40. | Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BAApparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA87199016201624 |
| 41. | Hantler PD, Gratzer WBEffects of specific chemical modification of actin. Eur J Biochem6019756772 |
| 42. | Viner RI, Hühmer AFR, Bigelow DJ, Schöneich CThe oxidative inactivation of sarcoplasmic reticulum Ca2+-ATPase by peroxynitrite. Free Rad Res241996243259 |
| 43. | MacMillan-Crow LA, Crow JP, Kerby JD, Beckman JS, Thompson JANitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci USA9319961185311858 |
| 44. | Eiserich JP, Estevez AG, Bamberg TV, Ye YZ, Chumley PH, Beckman JS, Freeman BAMicrotubule dysfunction by posttranslational nitrotyrosination of a-tubulin: anitric oxide-dependent mechanism of cellular injury. Proc Natl Acad Sci USA96199963656370 |
| 45. | Kamisaki Y, Wada K, Ataka M, Yamada Y, Nakamoto K, Ashida K, Kishimoto YLipopolysaccharide-induced increase in plasma nitrotyrosine concentrations in rats. Biochim Biophys Acta136219972488 |
| 46. | Van der Vliet AJ, Eiserich JP, Halliwell B, Cross CEFormation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. J Biol Chem272199776177625 |
| 47. | Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, van der Vliet AFormation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature3911998393397 |
| 48. | Poderoso JJ, Carreras MC, Schöpfer F, Lisdero CL, Riobo NA, Giulivi C, Boveris AD, Boveris A, Cadenas EThe reaction of nitric oxide with ubiquinol: kinetic properties and biological significance. Free Rad Biol Med261999925935 |
| 49. | Xia Y, Zweier JLSuperoxide and peroxinitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci USA94199769546958 |
| 50. | Supinski G, Stofan D, Callahan LA, Nethery D, Nosek TM, Di Marco APeroxynitrite induces contractile dysfunction and lipid peroxidation in the diaphragm. J Appl Physiol871999783791 |
| 51. | Brunelli L, Crow JP, Beckman JSThe comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch Biochem Biophys3161995327334 |
