Endotoxemia or sepsis causes respiratory muscle dysfunction, which, when it includes the diaphragm (1–7), may contribute to acute respiratory failure. Endotoxin increases inducible nitric oxide synthase (iNOS) protein expression that correlates with reduction of force-generating capacity in the diaphragm of rats (3,4). Inhibition of iNOS induction by dexamethasone or iNOS activity by NG-monomethyl-l-arginine successfully prevents reduction of diaphragmatic contractility in endotoxemic/septic rats (3,8). Incubation of diaphragm strips isolated from septic rats with S-methylisothiourea, an iNOS inhibitor, restores the decline in force generation (4). These findings show that nitric oxide (NO) overproduced via iNOS is responsible for the pathogenesis of endotoxemia/sepsis-induced diaphragmatic dysfunction, although the precise mechanism underlying infection-induced impairment of contractile profile and endurance capacity in the diaphragm remains to be elucidated. Most NO-mediated cell/tissue injuries are caused by peroxynitrite, an NO-derived free radical. This compound with powerful oxidative activity at least causes lipid peroxidation of the muscle membrane resulting in alteration of action potential propagation and excitation-contraction coupling, and consequently impairs diaphragm contractility (5–7). ONO1714, a potent selective iNOS inhibitor, has been recently developed (9). Inhibition of iNOS activation by this drug may also be able to attenuate sepsis-induced diaphragmatic dysfunction. To test this hypothesis, therefore, we assessed diaphragmatic contractility and endurance capacity in vitro using muscle strips excised from the costal diaphragm of septic hamsters receiving or not receiving ONO1714. Furthermore, to elucidate the mechanism underlying attenuation of diaphragmatic dysfunction with the iNOS inhibitor, we determined diaphragm malondialdehyde (MDA) levels and iNOS activity (reflecting local NO production), and plasma nitrite/nitrate (NOx) concentrations. Measurement of MDA production is important as an index of free radical-mediated membranous lipid peroxidation. In many previous studies, NO overproduction through iNOS activation has been systemically assessed by plasma NOx levels because rapid metabolism of NO offers difficulty of direct measurement for local NO production.
The current study was approved by the animal care review board of Kobe University School of Medicine. The care and handling of the animals were in accord with National Institutes of Health guidelines. Fifty male Golden-Syrian hamsters weighing between 120 and 135 g were randomly divided into five groups (Groups Sham, Sham-ONO1714high, Sepsis, Sepsis-ONO1714low, and Sepsis-ONO1714high;n = 10 each group). Hamsters in Groups Sham and Sham-ONO1714high underwent sham laparotomy, and animals in Groups Sepsis, Sepsis-ONO1714low, and Sepsis-ONO1714high underwent laparotomy followed by cecal ligation and puncture (CLP). Groups Sham and Sepsis received intraperitoneal injection of saline, whereas Groups Sham-ONO1714high, Sepsis-ONO1714low, and Sepsis-ONO1714high received intraperitoneal injection of ONO1714 (Ono, Osaka, Japan) in doses of 0.3, 0.1, and 0.3 mg/kg, respectively. ONO1714 dissolved in saline, or saline, was intraperitoneally given 10 min before surgery. The animals were killed 24 h after operation. The second author conducted all operations using general anesthesia with sevoflurane.
The CLP technique has served as a septic model (10). In the current study, the cecum was devascularized and ligated tightly at its base with a 3–0 silk thread without obstructing the bowel. The cecum was then punctured once with a sterile 18-gauge needle on the antimesenteric border. Gentle pressure was applied to the cecum until a small amount of feces exuded. This procedure ensured the puncture hole would not close. In previous experiments, dietary intake was obviously reduced in septic hamsters after laparotomy. Thus, feeding was restricted in all groups after surgery to avoid differences in nutrition supplement among the groups because of difference in oral intake: sunflower seeds 2 g were placed in the cage for each hamster.
At 24 h postsurgery, the hamsters were killed by cervical dislocation under general anesthesia with sevoflurane. Within 1 min of death, blood samples were obtained by aspiration from the left ventricle to determine the plasma concentrations of endotoxin and NOx. Thereafter, the left hemidiaphragm was removed and placed in a dissecting dish containing oxygenated (95% O2 and 5% CO2) Krebs-Henseleit solution (pH, 7.40, NaCl 135 mM, KCl 5 mM, glucose 11.1 mM, CaCl2 2.5 mM, MgSO4 1 mM, NaHCO3 14.85 mM, NaHPO4 1 mM, and insulin 50 U/L). Pancuronium 2 μM was added to the solution to eliminate indirect muscle activation mediated by nerves. A part of the right hemidiaphragm was also removed, frozen in liquid nitrogen, and stored at -70°C for subsequent MDA analysis. Another part of the right hemidiaphragm was fixed by 10% formaldehyde solution and embedded in paraffin wax for immunohistochemical experiment. A muscle strip was dissected from the left hemidiaphragm and mounted in an organ bath containing Krebs-Henseleit solution at 22°C. The origin of each muscle was secured by a steel hook embedded in the bath, and the tendinous insertion of each strip was secured to another hook tied to a silk thread attached to a force transducer. Strips were stimulated with supramaximal currents (1.2 to 1.3 times the current required to elicit a maximal tension) delivered via platinum field electrodes. Current (0.2 ms duration in pulses) was supplied by an electrical stimulator (DPS-1100D; Dia Medical System Co., Tokyo, Japan). The muscle tension was amplified by an AC strain amplifier.
Strips were allowed to equilibrate for 15 min in the organ bath; muscle length was then adjusted to the length at which twitch tension development was maximal. Muscle length was measured with a micrometer. Muscle contractile characteristics were assessed from measurements of twitch kinetics, the diaphragm force-frequency relationship, and of diaphragm fatigability during a series of repetitive rhythmic contractions. Twitch kinetics were assessed by measuring maximum rate of muscle tension development (dp/dtmax) and the time required for peak tension to decrease by 50% (half relaxation time) during single muscle twitches. The diaphragm force-frequency relationship was assessed by sequentially stimulating muscles at 1, 10, 20, 50, and 100 Hz. Each stimulus train was applied for 800 ms, and adjacent trains were applied at 5-s intervals. After completion of the force-frequency, a 30-s rest period was provided. Muscle fatigability trial using rhythmic contraction for 5 min was then started. Rhythmic contraction was induced by applying trains of 20-Hz stimuli (train duration, 500 ms; duty cycle, 0.50 ms) at a 60 train/min rate. On completion of this protocol, the muscle strip was removed from the bath and weighed.
The blood taken within 1 min after death was immediately centrifuged at 3000 rpm for 10 min at 4°C. Plasma was then stored at -70°C until assayed. Plasma endotoxin concentrations were measured by using an endotoxin-specific test (Endospecy®; Seikagaku, Tokyo, Japan) (11). The lower limit of sensitivity of the method is 5 pg/mL. Plasma NOx concentrations were also determined with an automatic analyzer (NOX 1000m®; Tokyo Kasei, Tokyo, Japan) using the Griess reaction (12).
MDA was assayed on diaphragmatic samples using thiobarbituric assay (13,14). In brief, muscle samples were homogenized with cold 1.15% KCl to make a 10% homogenate. A 0.1-mL aliquot of this homogenate was then added to 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (pH adjusted to 3.5), and 1.5 mL of 0.8% aqueous thiobarbituric acid. The mixture was made up to 4 mL with distilled water and then heated for 60 min in a water bath at 90°C. After cooling, this solution was mixed with 5 mL of butanol and 1 mL of distilled water and centrifuged at 2500 rpm for 20 min. The supernatant was read at 532 nm on a spectrophotometer. Absorbance values were compared with standard curves constructed using MDA produced in response to known concentrations of tetramethoxypropane (2.5, 5.0, 7.5, and 10 n). Final MDA concentrations are reported as nanomoles of MDA per gram of wet weight of tissue.
Immunostaining for nitrotyrosine has been used as a putative evidence of peroxynitrite because of its rapid decomposition. Thin sections (5 μm) of each formalin-fixed, paraffin-embedded tissue were cut onto commercially available slides (Superfrost/plus; Fisher, Pittsburgh, PA), deparaffinized, and cleansed through a series of xylene and alcohol washes. After deparaffinization, endogenous peroxidase was quenched with 0.5% hydrogen peroxide in phosphate-buffered saline for 30 min. Antigen retrieval was performed by heating the sections using a microwave oven in citrate buffer. Nonspecific adsorption was minimized by incubating the section in 5% normal goat serum (Sigma, St. Louis, MO) in phosphate-buffered saline for 20 min. The sections were then incubated overnight at 4°C with 1:200 dilution of primary polyclonal antinitrotyrosine antibody (#06–284; Upstate Biotech, Lake Placid, NY). The slides were probed with biotinylated goat antirabbit secondary antibody (Vector, Burlingame, CA) followed by treatment with the streptavidin-biotin-horseradish peroxidase complex (Amersham, Buckinghamshire, UK). Peroxidase-stained sections were developed with 0.5 mg/mL 3,3′-diaminobenzidine (Sigma) and counterstained with hematoxylin stain. This antinitrotyrosine antibody only recognizes nitrated proteins without cross-reaction with other tyrosine proteins or phosphotyrosine (T. Kasamatsu, Cosmo Bio, Tokyo, Japan, personal communication). Immunohistochemical staining was qualitatively assessed by two independent observers who were unaware of group assignment. We also calculated the ratio of immunostain-positive cells to total cells for semiquantitative analysis by inspecting five high power fields of each immunostaining sample with an optical microscope.
Another set of hamsters (110–140 g, n = 25) was prepared and divided into the same 5 groups for determination of diaphragmatic iNOS activity. The animals were killed by cervical dislocation under general anesthesia with sevoflurane 24 h after operation. The diaphragm was quickly excised, cleaned of connected tissue, and frozen at -80°C in liquid nitrogen until assay of iNOS activity determined using commercial NOS quantitative assay kit (Bioxytech; OXIS International, Portland, OR). Briefly, frozen diaphragm was homogenized in 20 volumes of homogenization buffer (pH 7.4, 25 mM HEPES buffer, 1 mM EDTA, 1 mM EGTA). The crude homogenates were centrifuged at 4°C for 5 min at 15000 rpm and the supernatants were collected. Diaphragmatic samples 10 mL were added to reaction buffer (50 μL) of the following composition: pH 7.4, 25 μM Tris/HCl buffer, 60 mM valine, 1 mM NADPH, 1 μM flavin adenine dinucleotide, 1 mM flavin mononucleotide, 3 μM tetrahydrobiopterin, 1 μL of 120 μM stock l-[3H]-arginine (Amersham-Pharmacia), and 2 μM EGTA (except for assay of a positive control). The samples were incubated for 30 min at 25°C and the reaction was terminated by the addition of ice-cold (2°C) stop buffer (pH 5.5, 50 mM HEPES, 5 mM EDTA). To obtain free l-[3H]citrulline for the determination of enzyme activity, equilibrated resin was added to elimninate excess l-[3H]-arginine. The supernatant was assayed for l-[3H]citrulline using liquid scintillation counting. Enzyme activity was expressed as counts per minute per mg total protein. Protein concentration was measured by the Bradford technique (Protein Assay Kit; Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. NOS activity in the positive control was measured in the presence of 0.6 mM CaCl2 and rat brain homogenate instead of diaphragmatic samples. NOS activity in the presence of 1 mM of NG-nitro-l-arginine methyl ester served as a negative control. The iNOS activity was calculated as the difference between samples assayed in the presence of EGTA and that measured in the presence of NG-nitro-l-arginine methyl ester.
Muscle strip cross-sectional area was calculated by dividing muscle mass by the product of fiber length and muscle density (1.06 g/cm3) (15). Force generation was normalized as force per unit cross-sectional area (kg/cm2). The data among the groups were analyzed by analysis of variance with Scheffépost hoc testing. The within-group (over time) data were statistically analyzed using repeated-measures analysis of variance followed by Scheffépost hoc test. P < 0.05 was deemed statistically significant.
Autopsy examination revealed that all hamsters in the three septic groups had panperitonitis. However, we found no mortality in any of the groups. There were no differences in the body weights immediately before death among the groups.
Muscle strip lengths and weights excised were similar for five groups (data not shown). As shown in Table 1, dp/dtmax of diaphragmatic contraction was decreased in Group Sepsis compared with that in Group Sham. ONO1714 dose-dependently mitigated reduction of dp/dtmax. In the diaphragm from septic hamsters, half relaxation time was prolonged (Table 1). ONO1714 in a dose of 0.3 mg/kg shortened this prolongation although a smaller dose of ONO1714 failed to do so. Twitch tensions (by stimulation of 1 Hz frequency) were significantly lower in strips from the septic hamsters than in those from the Sham group (Table 1). ONO1714 attenuated impairment of the twitch tensions in a dose-dependent manner (Table 1).
Diaphragm Force-Frequency Relationship and Fatigability
Sepsis decreased the tensions generated in response to all frequencies of stimulation. ONO1714 significantly blunted the sepsis-induced reduction of force-frequency relationship in a dose-dependent fashion (Fig. 2 and Table 1). Figure 1 shows representative recordings of tensions over time during the fatigue trial (until 65 s) in Groups Sham, Sepsis, and Sepsis+ONO1714high. The time until tension decreased to 50% of the initial value (T50%) was used as an index of diaphragmatic endurance. In the diaphragm from septic hamsters, T50% was shortened more in the diaphragms isolated from Group Sepsis than in those from Group Sham (Table 2). ONO1714 dose-dependently returned T50% toward the level observed in the diaphragms from sham hamsters.
Diaphragm MDA and iNOS Activity and Plasma Endotoxin and NOx
The MDA levels were larger in diaphragmatic samples taken from Group Sepsis than in those taken from Group Sham (Table 2). ONO1714 significantly attenuated sepsis-induced increases of MDA. CLP procedure increased plasma endotoxin levels (Table 2). Plasma NOx levels were significantly increased in septic hamsters, suggesting sepsis-induced systemic NO production (Table 2). This increase was dose-dependently attenuated with ONO1714. Diaphragmatic iNOS activity was increased in Group Sepsis compared with Group Sham (Table 2). This increase was attenuated by ONO1714 in a dose-dependent fashion.
Immunohistochemical analysis of nitrotyrosine revealed that no staining was found in the diaphragm isolated from Group Sham (Fig. 3A). In Group Sepsis, positive immunostain for nitrotyrosine, found in inflammatory cells in endomysial and perivascular spaces, was the most intense (Fig. 3B). A large dose of ONO1714 reduced the intensity to minimal level (Fig. 3C) although the immunostaining remained moderate/intense in the small-dose group. Ratio of immunostain-positive cells was increased in septic hamsters (Table 2). ONO1714 reduced the ratio in a dose-dependent fashion (Table 2).
We confirmed that sepsis caused diaphragmatic dysfunction (impairment of contractility and endurance capacity) as manifested by a reduction of the twitch kinetics and tetanic tensions of the muscle, a downward shift in force-frequency relationship, and a reduction of T50% during fatigue trial. ONO1714 successfully produced a dose-dependent attenuation of sepsis-induced diaphragmatic dysfunction.
Many mediators responsible for sepsis-induced diaphragmatic muscle failure have been proposed (1–7). NO overproduced by iNOS is involved in the pathogenesis of diaphragmatic muscle failure (3,4). Several studies demonstrate that endotoxin/sepsis induced iNOS expression in inflammatory cells and myofibers in the diaphragm (4). This expression is more in the diaphragm than in the soleus (16). Peroxynitrite, formed by NO and superoxide that is concurrently overproduced in sepsis (17), plays a central role in NO-mediated cell/tissue damages including myofiber injury because NO per se is not a strong oxidant. The mechanisms for peroxynitrite-induced cell damage include lipid peroxidation (as a result of oxidative modification), protein modification (as a result of nitration of tyrosine residues), DNA strand breakage, and inhibition of mitochondrial respiration (18–21). Membranous lipid peroxidation by peroxynitrite probably alters action potential propagation and excitation-contraction coupling in the muscle. This speculation is supported by the study in which CLP-induced peritonitis caused diaphragmatic sarcolemmal damage mediated by NO with altered resting membrane potential in myofibers (8). The membrane injury affects intracellular metabolism sufficiently to impair contractile protein function, thereby reducing diaphragmatic muscle force generation and relaxation. Contrary to these reports concerning unfavorable effects of iNOS, a recent experiment using iNOS knockout mice has documented that iNOS may play a protective role in attenuating the inhibitory influence of endotoxin on diaphragm contractility (22).
In the current study, sepsis increased plasma NOx levels and iNOS activity and immunostaining intensity of nitrotyrosine (peroxynitrite footprint) in the diaphragm. Furthermore, sepsis also increased MDA levels in the diaphragm. Attenuation of diaphragmatic dysfunction with ONO1714 was accompanied by reduction of these variables. These findings suggest that ONO1714 attenuated sepsis-induced diaphragmatic muscle failure, in part, by inhibiting diaphragmatic lipid peroxidation caused by NO-derived free radicals (peroxynitrite). Reduction of immunostaining for nitrotyrosine in the diaphragm supports this mechanism. These results coincide with the other studies using treatment of iNOS inhibition (3,4,8) :NG-monomethyl-l-arginine, in particular, successfully attenuates diaphragm sarcolemmal injury (8).
A large amount of superoxide and NO are readily and excessively generated in mitochondria (23). Therefore, large concentrations of peroxynitrite are expected to be produced in or near the organella. This may be able to affect mitochondrial respiration and intracellular constituents including contractile proteins of the muscle cells. Sarcoplasmic reticulum in the muscle cells is also one of plausible intracellular targets of peroxynitrite. Twitch kinetics are thought to be a function of the rate of release and reuptake of calcium from sarcoplasmic reticulum in the muscle cells (24). Our findings of twitch kinetics suggest that sepsis produces damage of this intracellular organelle. In particular, sepsis-induced prolongation of half relaxation time indicates impairment of reuptake of intracellular calcium ion by sarcoplasmic reticulum. However, positive immunostaining for nitrotyrosine was not observed in the myocytes in the septic animals. To elucidate mechanisms of successful treatment with ONO1714 other than inhibition of membranous lipid peroxidation, further studies are required to assess modification of myofibril protein or organelle inside the myocytes.
ONO1714 is a new competitive inhibitor of iNOS with high safety margin (maximum tolerated dose/50% inhibitory dose for NOx accumulation = 5000) (9). The drug exhibits 10-fold selectivity for iNOS (inhibitor constant of 1.88 nM) over endothelial constitutive NOS (inhibitor constant -endothelial constitutive NOS = 18.8 nM) (9). ONO1714 also has approximately 34-fold higher selectivity for iNOS than NG-monomethyl-l-arginine, and 233 times as high inhibitory potency for human iNOS as NG-monomethyl-l-arginine (inhibitory concentration of 50%: ONO1714 = 12 nM;NG-monomethyl-l-arginine = 2.8 μM) (9). Furthermore, ONO1714 is thought to possess higher selectivity for iNOS than aminoguanidine, a relatively selective iNOS inhibitor (4∼9-fold selectivity for iNOS over endothelial constitutive NOS). These are advantageous pharmacological characteristics of ONO1714.
Sepsis may indirectly affect diaphragmatic contractility and fatigability by depletion of energy as a result of decreased oral intake. In the current study, the body weights of the hamsters were similar among all the groups because of strict dietary control. Thus, it is unlikely that the iNOS inhibitor attenuated the diaphragmatic dysfunction by improving oral ingestion in the hamsters with sepsis. Even pretreatment with ONO1714 (0.3 mg/kg) was not able to completely prevent sepsis-induced diaphragmatic dysfunction, although this regimen successfully reduced iNOS activity and NO production close to control levels. NO overproduced from iNOS is not the sole cytotoxic mediator responsible for the diaphragmatic muscle failure (1,3). A number of mechanisms have been proposed to account for this observation, including an imbalance between energy use and supply as well as direct cytotoxic effects of various mediators (e.g., oxygen free radicals, cytokines) released as a part of the systemic inflammatory response associated with sepsis (25). ONO1714 is unable to inhibit all types of inflammatory mediators. Thus, there may be a limit to complete prophylaxis against diaphragmatic dysfunction using the iNOS inhibitor. A better prophylactic remedy may be provided by a novel strategy consisting of a concomitant use of ONO1714 and drugs that suppress other inflammatory mediators. Untoward effects of NO inhibition in clinical practice may include increased incidence of infection because NO is thought to play an important role in the host defense system. However, the current study is unable to provide an obvious solution to this problem because the effect of iNOS inhibition with ONO1714 on the bactericidal system in this setting was not investigated. However, advantages of NO inhibition therapy in severe infectious conditions include maintenance or restoration of hemodynamic stability. Determination of benefit/risk ratio in ONO1714 treatment for sepsis-induced diaphragmatic dysfunction deserves further study.
In conclusion, we have shown that ONO1714 dose-dependently attenuated sepsis-induced impairment of diaphragm function (contractility and fatigability). The attenuation may be attributable, in part, to reduction of diaphragmatic lipid peroxidation by NO-derived free radicals (e.g., peroxynitrite). These data indicate that ONO1714 may have promise in preventing diaphragmatic dysfunction in critically ill patients with severe infection or those with risk factors for sepsis. A therapeutic effect of ONO1714 on sepsis-induced diaphragmatic dysfunction once developed remains to be elucidated before clinical application.
We would like to express our gratitude to Ono Pharmaceutical Co. for a generous supply of ONO-1714.
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