Role of heme oxygenases in sepsis-induced diaphragmatic contractile dysfunction and oxidative stress

Esther Barreiro1,2, Alain S. Comtois2, Shawn Mohammed2, Larry C. Lands3, and Sabah N. A. Hussain2

1 Department of Respiratory Medicine, Hospital del Mar-Municipal Institute of Medical Research, Pompeu Fabra University, 08003 Barcelona, Spain; 2 Critical Care and Respiratory Divisions, Royal Victoria Hospital and Meakins-Christie Laboratories; and 3 Respiratory Medicine Department, Montreal Children's Hospital, McGill University, Montreal, Quebec H3A 1A1, Canada


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Heme oxygenases (HOs), essential enzymes for heme metabolism, play an important role in the defense against oxidative stress. In this study, we evaluated the expression and functional significance of HO-1 and HO-2 in the ventilatory muscles of normal rats and rats injected with bacterial lipopolysaccharide (LPS). Both HO-1 and HO-2 proteins were detected inside ventilatory and limb muscle fibers of normal rats. Diaphragmatic HO-1 and HO-2 expressions rose significantly within 1 and 12 h of LPS injection, respectively. Inhibition of the activity of inducible nitric oxide synthase (iNOS) in rats and absence of this isoform in iNOS-/- mice did alter sepsis-induced regulation of muscle HOs. Systemic inhibition of HO activity with chromium mesoporphyrin IX enhanced muscle protein oxidation and hydroxynonenal formation in both normal and septic rats. Moreover, in vitro diaphragmatic force generation declined substantially in response to HO inhibition both in normal and septic rats. We conclude that both HO-1 and HO-2 proteins play an important role in the regulation of muscle contractility and in the defense against sepsis-induced oxidative stress.

nitric oxide; nitric oxide synthase; diaphragm


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HEME OXYGENASES (HOs) are the rate-limiting enzymes of the initial reaction in the degradation of heme, which yields equimolar quantities of biliverdin IXa, carbon monoxide (CO), and free iron (8). Biliverdin is subsequently converted to bilirubin, and free iron is rapidly incorporated into ferritin. So far, three HO isoforms have been identified: HO-2 and HO-3 are constitutively expressed in various cells, whereas HO-1 is transcriptionally activated in response to a variety of stimuli including bacterial lipopolysaccharide (LPS), heat stress, hypoxia, and exposure to nitric oxide (NO) (1, 7, 9). There is increasing evidence that HOs play important roles in the cellular defense against oxidative stress and the deleterious effects of proinflammatory cytokines and LPS. Indeed, pharmacological inhibition of HO activity renders rats more sensitive to LPS-induced mortality (27). Similarly, mice that are deficient in HO-1 are very susceptible to the effects of reactive oxygen species (ROS) and develop a high mortality in response to LPS administration (28). In endothelial cells, gene transfer of HO-1 protects against oxidant-induced lung injury (36). The antioxidant properties of HOs are believed to be mediated by products of HO activity including bilirubin, CO, and ferritin (26, 30).

Little is known about the existence and functional significance of HOs in skeletal muscles. Baum et al. (4) described the presence of HO-2 protein inside normal mammalian skeletal muscles and reported that this protein colocalizes with neuronal nitric oxide synthase in close proximity to the nonjunctional sarcolemma. HO-2 has also been identified at the neuromuscular junctions of normal skeletal muscles (20). More recent studies have indicated that HO-1 is expressed at relatively low levels in normal in vivo skeletal muscles and in vitro cultured myoblasts and that HO-1 expression is elevated in response to increased muscle activation or exposure to exogenous hemin and NO (13, 35). The functional significance of HOs in skeletal muscle was not addressed in these studies. Very recently, Taille et al. (32) described for the first time the involvement of HOs in LPS-induced oxidative stress and contractile dysfunction of the ventilatory muscles. These authors observed that injection of LPS in rats evokes HO-1 induction in the ventilatory muscles of rats with no change in HO-2 expression. It was also reported that inhibition of HO activity augments LPS-induced muscle oxidative stress and worsens LPS-induced muscle contractile dysfunction (32). Despite this recent progress, many aspects of the biological roles of HOs in the ventilatory or limb muscles remain unclear. For instance, it is unclear whether HOs play any role in regulating redox status or contractile function of normal skeletal muscles. The observations that both enzymes, particularly HO-2, are expressed at various sites within normal muscle fibers and inside blood vessels supplying muscles (16), suggests a functional role for these enzymes in normal muscle fibers. Moreover, our preliminary experiments in rats suggested that LPS evokes significant upregulation of muscle HOs, with HO-1 expression being elevated early in sepsis, whereas HO-2 is elevated within 12 h of LPS injection. These results suggest that there are differences in the response of HO-1 and HO-2 to LPS injection. Finally, there is increasing evidence that exogenous NO donors exert a significant stimulatory effect on HO expression in a variety of cells, including cultured myocytes (17). Whether or not endogenous muscle NO production, particularly in septic animals where inducible nitric oxide synthase (iNOS) is highly expressed, participates in the regulation of muscle HO expression remains unclear.

The main objectives of this study are: 1) to assess the expression, localization, and functional significance of HO-1 and HO-2 in the regulation of redox status and contractile function of normal ventilatory muscles; 2) to evaluate the influence of sepsis (induced by LPS injection) on the expression of HO-1 and HO-2 in the ventilatory muscles and to assess the contribution of these enzymes to sepsis-induced muscle oxidative stress and contractile dysfunction; and finally 3) to evaluate the involvement of the iNOS isoform in the regulation of ventilatory muscle HO-1 and HO-2 expression both under normal conditions and in response to severe sepsis.


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Reagents

Gels and loading buffers for immunoblotting were obtained from Novex (San Diego, CA). Escherichia coli LPS (serotype 055:B5), bovine serum albumin (BSA), aprotinin, leupeptin, trypsin inhibitor, pepstatin A, Tris-maleate, dithiothreitol (DTT), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). 1400W was obtained from Cayman Chemical (Ann Arbor, MI). Monoclonal antibodies for HO-1 and HO-2 were obtained from Transduction Laboratories (Lexington, KY). Pure HO-1 and HO-2 proteins were purchased from StressGen Biotechnologies (Victoria, BC, Canada). Secondary antibodies for immunohistochemistry were purchased from Jackson ImmunoResearch (West Grove, PA). Reagents for enhanced chemiluminescence detection were obtained from Chemicon (Temecula, CA). Chromium mesoporphyrin IX (CrMSPIX) was purchased from Porphyrin Products (Logan, UT). Oxyblot protein oxidation kit was purchased from Intergen.

Preparation of CrMSPIX

CrMSPIX was dissolved in 500 µl of 10% (wt/vol) ethanolamine. After addition of 7 ml of distilled water and adjustment of the pH to 7.4 with HCl, the volume was adjusted to 10 ml to obtain a final solution of 650 µM concentration. For the inhibition of HO activity, we injected animals with a single dose of CrMSPIX (5 µmol/kg body wt) in one single injection.

Animal Preparations

Rat experiments. The Animal Research Committee of McGill University approved all procedures. Pathogen-free male Sprague-Dawley rats (250-275 g) were used in all protocols. The animals were housed in the animal facility of the hospital, were fed food and water ad libitum, and were studied 1 wk after arrival. At the end of all experimental protocols, the animals were anesthetized with pentobarbital sodium (30 mg/kg), and the diaphragm (and intercostals, gastrocnemius, and soleus in a few animals) was quickly excised and frozen in liquid nitrogen. For immunostaining, the tissues were first flash-frozen in cold isopentane (20 s) and then immersed in liquid nitrogen, and stored at -80°C.

HO-1 and HO-2 expressions in normal and septic muscles. We studied eight groups (n = 3 in each group) of male rats. Group 1 animals served as control and were injected with 0.3 ml of normal saline and were killed 1 h later. All the remaining groups received intraperitoneal injection of E. coli LPS (serotype 055:B5, 20 mg/kg, Sigma) and were killed 1, 3, 6, 12, 24, 48, and 72 h later.

iNOS inhibition and HO-1 and HO-2 expressions. We evaluated the involvement of the iNOS isoform in the regulation of diaphragmatic HO-1 and HO-2 expression by studying two groups (n = 5 in each group) of male rats. Group 1 received an injection of E. coli LPS as mentioned above and was killed 24 h later. Group 2 animals received an injection of 1400W (20 mg/kg, a selective iNOS inhibitor) 30 min before LPS administration and every 8 h thereafter and were killed 24 h after LPS administration.

Effects of HO inhibition. We evaluated the functional significance of HOs in regulating redox status and muscle contractility by inhibiting HO activity with CrMSPIX. Four groups of male rats (n = 6 in each group) were studied. Group 1 animals served as controls and were injected with 0.3 ml of normal saline and killed 1 h after the injection. Group 2 animals were injected with CrMSPIX (5 µmol/kg body wt ip) followed 1 h later by intraperitoneal injection of 0.3 ml of normal saline. These animals were killed 1 h after saline injection. Group 3 animals were injected with E. coli LPS (20 mg/kg) and were killed 24 h later, whereas CrMSPIX (5 µmol/kg body wt ip) was injected 1 h before LPS injection in group 4 animals. Animals in that group were killed 24 h after LPS injection.

Mice experiments. To further evaluate the role of iNOS in the regulation of muscle HO-1 and HO-2 expressions, we studied adult (8- to 12-wk-old male) mice genetically deficient (knockout) in iNOS. B6/129 hybrid iNOS knockout (iNOS-/-) mice were generated as previously described (21). We also studied wild-type B6/129 hybrid (iNOS+/+) mice (Jackson Laboratories, Bar Harbor, ME) bred to serve as experimental controls. In each genotype (iNOS+/+ and iNOS-/-), HO-1 and HO-2 protein expressions were studied in four groups of mice (n = 5 in each group). Group 1 animals were injected with saline and served as control, whereas group 2, 3 and 4 animals received injection of E. coli LPS (20 mg/kg ip) and were killed 6, 12, and 24 h later. The diaphragm was excised and frozen in liquid nitrogen as described above.

Immunoblotting

Frozen muscle samples were homogenized in 6 vol/wt ice-cooled homogenization buffer (10 mM Tris-maleate, 3 mM EGTA, 275 mM sucrose, 0.1 mM DTT, 2 µg/ml leupeptin, 100 µg/ml PMSF, 2 µg/ml aprotinin, and 1 mg/100 ml pepstatin A, pH 7.2). Samples were then centrifuged at 1,000 g for 10 min. The pellet was discarded, and the supernatant was designated as crude homogenate. Total muscle protein level in each sample was determined with the Bradford technique (Bio-Rad). Crude homogenate samples (80 µg/sample) were mixed with SDS sample buffer, boiled for 5 min at 95° C, and were then loaded onto 8 or 10% Tris-glycine sodium dodecylsulfate polyacrylamide gels (SDS-PAGE) and separated by electrophoresis (150 V, 30 mA for 1.5 h). Pure HO-1 and HO-2 proteins were used as positive controls. Proteins were transferred electrophoretically (25 V, 375 mA for 2 h) to methanol-presoaked polyvinylidene difluoride (PVDF) membranes and then blocked with 1% BSA for 1 h at room temperature. The PVDF membranes were subsequently incubated overnight at 4°C with primary monoclonal anti-HO-1 and anti-HO-2 antibodies. After three 10-min washes with wash buffer on a rotating shaker, the PVDF membranes were further incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody. Specific proteins were detected with an enhanced chemiluminescence (ECL) kit (Chemicon) The blots were scanned with an imaging densitometer, and optical densities (OD) of positive HO-1 and HO-2 protein bands were quantified with SigmaGel software (Jandel Scientific, Chicago, IL).

Protein Oxidation

We measured protein oxidation in muscle samples by evaluating the levels of protein carbonyl using immunoblotting. Protein carbonyls are formed by a variety of oxidative mechanisms and are sensitive indexes of oxidative injury (22). The conventional assay for protein carbonyls is a colorimetric procedure that measures the binding of dinitrophenylhydrazine (DNP); however, Western blotting using anti-DNP antibody provides a much more sensitive method than colorimetric procedures. We used an Oxyblot kit, which contains all solutions required for the derivatization of the samples as well as the antibodies required for the immunodetection. Briefly, 15 µg of protein were used per derivatization reaction. Proteins were then denatured by addition of 12% SDS. The samples were subsequently derivatized by adding 10 µl of 1× DNP solution and incubated for 15 min. Finally, 7.5 µl of neutralization solution and 2-mercaptoethanol were added to the sample mixture. Both derivatized and un-derivatized (negative control) muscle samples were then separated on SDS-PAGE and transferred onto PVDF membranes, as described in Immunoblotting. Membranes were probed with polyclonal anti-DNP moiety antibody. Positive proteins were detected with HRP-conjugated anti-rabbit secondary antibody and an ECL kit as described. Total levels of carbonyls in each muscle sample were calculated by adding OD of individual positive protein band.

Measurements of 4-Hydroxy-2-Nonenal

Peroxidation of membrane lipids results in free radical-mediated fragmentation of polyunsaturated fatty acids and the formation of various aldehydes, alkenals, and hydroxyalkenals. 4-Hydroxy-2-nonenal (HNE) is an alpha ,beta -unsaturated aldehyde and represents the most cytotoxic product of lipid peroxidation. We used a well-characterized polyclonal antibody to detect HNE protein adducts in the ventilatory and limb muscles of normal and septic rats (31). Crude muscle homogenates were separated on SDS-PAGE and transferred to PVDF membranes as described in Immunoblotting. Membranes were probed with polyclonal anti-HNE antibody (Calbiochem, San Diego, CA), and specific proteins were detected with HRP-conjugated antibody and an ECL kit. Adding OD of individual positive protein band calculated the total level of HNE in each muscle sample.

Immunohistochemistry

Frozen tissues sections (5-10 µm thickness) were adsorbed to microscope slides and dried. The sections were fixed with acetone at -20°C, rehydrated with PBS (pH 7.4), and then blocked with solutions of avidin and biotin (15 min each at room temperature) and then 3% BSA for 30 min. The sections were incubated for 1 h at room temperature with primary monoclonal anti-HO-1 and anti-HO-2 antibodies. To evaluate the negative control staining, we replaced the primary antibody with nonspecific mouse IgG. After three rinses with PBS, sections were incubated with biotin-conjugated anti-mouse or anti-rabbit secondary antibodies at room temperature for 1 h followed by exposure to Cy3-labeled streptavidin for 1 h. Sections were then washed, mounted with coverslips, and examined under a fluorescence microscope and photographed with a 35-mm camera (Olympus).

Glutathione Measurements

Frozen muscle samples were homogenized in 20 µl of 5% 5-sulfosalicylic acid (SSA)/mg tissue. The homogenate was then centrifuged at 10,000 g for 5 min at 4°C. The supernatant was diluted 1:5.5 in double distilled H2O, aliquoted, and immediately stored at -70°C, for total glutathione (GSH) measurement. GSH concentration was determined by the glutathione reductase (GR) recycling method of Tietze (34) adapted for the Cobas Mira S spectrophotometer (Roche Diagnostics, Laval, QC, Canada). Briefly, we placed 250 µl NADPH (0.3 mM), 30 µl 5,5'-dithiobis(2-nitrobenzoic acid) (6.0 mM), and 95 µl sample, standard, or 0.9% SSA into cuvettes. After a 4-min incubation period at 37°C, 15 µl of GR (2 U/100 µl) were added, and the reaction was monitored every 24 s for 12 min. Under these conditions, the method is linear for GSH concentrations between 0.1 and 6 µM.

Diaphragmatic Muscle Strip Preparation

Diaphragms of control and septic (24 h post-LPS) rats untreated and treated with CrMSPIX were surgically excised with ribs and central tendon attached and were placed in an equilibrated (95% 02-5% CO2, pH 7.38) Krebs solution chilled at 4°C that had the following composition (in mM): 118.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1 KH2PO4, 25 NaHCO3, and 11.0 glucose. From the central tendon to the rib, a muscle strip (3-4 mm wide) was dissected from the lateral costal portion of the diaphragm. The rib was left attached to the strip and was used to secure the diaphragm strip in the custom-built Plexiglas muscle chamber. The strip was mounted in a muscle chamber, and the muscle chamber was mounted vertically into a double jacket gut bath (Kent Scientific Instruments). A 4.0 silk thread was used to secure the central tendon to the isometric force transducer (Kent Scientific Instruments). Muscle strips were electrically stimulated at constant currents via platinum electrodes mounted in the muscle chamber, which were connected to a square wave pulse stimulator (Grass Instruments model S48). After an equilibration period of 30 min (temperature of 22-25°C), the organ bath temperature was increased to 35°C, and the maximum current necessary to elicit maximum force during 120-Hz stimulation frequency (600 ms duration) was then identified. Muscle length was then gradually adjusted with a micrometer to the optimal value, at which maximum isometric muscle force was generated in response to supramaximum stimulation (current of 300-350 mA, 120 Hz frequency). Force-frequency relationships of diaphragmatic strips were then constructed by varying the stimulation frequency (between 10 and 120 Hz) while maintaining supramaximal current and stimulation duration (600 ms) constant. Tetanic contractions were digitized at a frequency of 1 KHz with a personal computer and stored on the hard disk for later analysis. At the end of the experiment, the strips were blotted dry and weighed. Muscle length (cm) and weight (g) were measured and used to calculate the cross-sectional area. Isometric forces were normalized for muscle cross-sectional area estimated by using the value of 1.056 g/cm3 for muscle density. The peak force (in N/cm2) was measured for each contraction within the force-frequency curve.

Statistical Analysis

Values are presented as means ± SE. Differences in muscle force and OD of HO-1 and HO-2 proteins, total HNE, and total carbonyls among various conditions were compared with one-way ANOVA followed by Tukey's test for multiple comparisons. P values of <5% were considered significant. Statistical analyses were performed with SigmaStat software (Jandel Scientific).


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Expression and Localization of HO-1 and HO-2 in Normal Rat Skeletal Muscles

Pure HO-1 and HO-2 proteins were detected in crude diaphragmatic homogenates as clear bands with apparent molecular masses of 32 and 36 kDa, respectively (Fig. 1A). Similarly, HO-1 and HO-2 proteins were also detected in soleus, intercostal, and gastrocnemius muscle samples obtained from normal rats (results are not shown). Figure 2 illustrates the localization of HO-1 and HO-2 proteins in the diaphragm and leg muscles of normal rats. The anti-HO-2 antibody detected positive HO-2 protein staining at the subsarcolemmal region of normal gastrocnemius muscle sections (Fig. 2A). Similar positive HO-2 staining was found in close proximity to the nonjunctional sarcolemma of rat diaphragms (Fig. 2B). HO-2 protein was also present in endothelial cells of normal muscle sections. Positive HO-1 protein staining was detected with a monoclonal anti-HO-1 antibody in close proximity to the sarcolemma of gastrocnemius muscle sections (Fig. 2, C and D) and in the endothelial cells of a blood vessel traversing this muscle (Fig. 2E). Replacement of primary antibodies with nonspecific mouse IgG completely eliminated positive HO protein staining (Fig. 2F).


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Fig. 1.   A: representative immunoblots indicating the time course of heme oxygenase (HO)-1 and HO-2 protein expression in normal rat diaphragms (C) and in the diaphragms of rats killed after 1, 3, 6, 12, 24, 48, and 72 h of lipopolysaccharide (LPS) injection. +ve, Positive controls (pure HO-1 and HO-2 proteins). B: mean values (± SE) of HO-1 and HO-2 optical densities (OD) in the diaphragms of normal and LPS-injected rats. Note the differences in the time course of HO-1 and HO-2 induction in response to LPS injection.



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Fig. 2.   Localization of HO-2 protein in the gastrocnemius and the diaphragm of normal rats is shown in A and B, respectively. Note that HO-2 protein is localized in close proximity to the sarcolemma. C and D: positive HO-1 protein staining in the gastrocnemius of normal rats; E: the presence of HO-1 protein in blood vessels supplying leg muscles of normal rats; F: negative control staining in which the primary anti-HO-1 or HO-2 antibodies were replaced with nonspecific rabbit IgG.

Muscle HO Expression in Septic Rats and Mice

Figure 1 illustrates the effect of LPS injection on diaphragmatic HO-1 and HO-2 protein expression in rats. HO-1 protein levels rose rapidly by more than threefold within 1 and 3 h of LPS injection with a gradual decline thereafter. After 72 h of LPS injection, HO-1 expression was slightly higher than control values (Fig. 1, A and B). Unlike HO-1 expression, the intensity of HO-2 protein rose substantially after 12 and 24 h of LPS injection, with a return to values similar to control values after 48 h of LPS injection (Fig. 1). The changes in HO-1 and HO-2 protein expression in response to LPS injection in iNOS+/+ mice are shown in Fig. 3. Diaphragmatic HO-1 expression rose substantially within 6 h of LPS injection and remained elevated even after 24 h of LPS injection (Fig. 3, A and B). We were unable to detect HO-2 protein in immunoblots of diaphragms obtained from control and septic iNOS-/- mice.


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Fig. 3.   A: representative immunoblots of HO-1 protein in the diaphragm of wild-type [inducible nitric oxide synthase (iNOS+/+)] and iNOS knockout (iNOS-/-) mice both under control conditions (C) and 6, 12, and 24 h after LPS injection. B: mean (± SE) values of HO-1 protein ODs (expressed as percentage of control values) of diaphragmatic samples obtained from iNOS+/+ and iNOS-/- mice. **P < 0.01 compared with control values. Note that the induction of HO-1 protein in response to LPS injection was observed in both genotypes.

Role of iNOS in HO Expression

We evaluated the role of iNOS in LPS-induced HO-1 and HO-2 expressions in muscles by examining iNOS-/- mice and by injecting rats with a selective iNOS inhibitor (1400W). Figure 3 shows that diaphragmatic HO-1 expression rose substantially after 6, 12, and 24 h of LPS injection in iNOS-/- mice. The degree of HO-1 induction after LPS injection in iNOS-/- mice was similar to that observed in iNOS+/+ mice (Fig. 3). Figure 4 shows that treatment with 1400W (selective iNOS inhibitor) did not alter the induction of diaphragmatic HO-1 and HO-2 expressions observed 24 h after LPS injection.


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Fig. 4.   A: representative immunoblots of HO-1 and HO-2 proteins in the diaphragms of normal rats (C) and rats examined after 24 h of LPS injection. Also shown are diaphragm samples obtained from LPS-injected rats pretreated with a selective iNOS inhibitor (1400W). B: mean values of ODs of HO-1 and HO-2 proteins in the diaphragms of normal rats and rats injected 24 h earlier with LPS (with or without pretreatment with 1400W). **, *P < 0.01 and 0.05 compared with control rat diaphragms.

HOs and Muscle Oxidative Stress

Measurement of carbonyl groups in diaphragmatic lysates using immunoblotting indicates the presence of relatively few oxidized proteins in control diaphragms (apparent molecular masses of 65, 46, and 34 kDA; Fig. 5A). Injection of LPS resulted in a significant increase in the intensity of these preexisting oxidized proteins, as well as the appearance of new oxidized proteins with molecular masses of 236, 150, 137, and 44 kDa (Fig. 5A). Administration of CrMSPIX elicited substantial reduction in the OD of the majority of oxidized proteins both under control conditions and after LPS injection (Fig. 5, A and C). The anti-HNE antibody detected four main protein bands in control diaphragms with molecular masses of 74, 49, 44, and 40 kDa (Fig. 5B). Injection of LPS resulted in a small increase in intensity of the 74-kDa protein band. Administration of CrMSPIX in control and septic animals elicited a significant rise in total HNE intensity primarily due to the appearance of a strong positive protein band at 24 kDa (Fig. 5, B and C). Figure 6 illustrates the changes in total diaphragmatic glutathione levels both in control and septic rats. Injection of LPS resulted in a significant decline in GSH to values that are about one-third of those found in control diaphragms (Fig. 6). Injection of CrMSPIX did not alter the level of GSH in the diaphragm of control and septic rats.


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Fig. 5.   A: representative immunoblots of protein carbonyl groups in the diaphragms of control and LPS-injected rats [with or without pretreatment with HO inhibitor chromium mesoporphyrin IX (CrMSPIX)]. Note that administration of CrMSPIX augmented carbonyl group intensity both in control and septic diaphragms. B: representative immunoblots of 4-hydroxy-2-nonenal (HNE) (index of lipid peroxidation) in control and LPS-injected rats (with or without pretreatment with CrMSPIX). Note the appearance of a 22-kDa protein band in response to HO inhibition. C: mean values of total ODs of carbonyl groups and HNE in control and septic diaphragms (with and without pretreatment with CrMSPIX). *P < 0.05 compared with animals without CrMSPIX injection.



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Fig. 6.   Total diaphragmatic glutathione levels in the diaphragms of control and LPS-injected rat with or without CrMSPIX injection. Note the decline n total glutathione level in septic diaphragms. Treatment with CrMSPIX had no effect on LPS-induced decline in total glutathione.

Diaphragmatic Contractility

Figure 7 shows that LPS injection had elicited by 24 h a substantial decline in diaphragmatic force generation with mean force averaging 38, 45, 52, 45, 48, 51, and 52% of those generated in response to 1, 10, 20, 30, 50, 100, and 120 Hz of stimulation in control diaphragms. Administration of CrMSPIX elicited a significant decline in diaphragmatic force both under control conditions and in response to LPS injection (Fig. 7).


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Fig. 7.   Force-frequency relationships of in vitro isolated strips of diaphragms excised from control (open circle ) and after 24 h of LPS injection (). Also shown is the effect of CrMSPIX treatment in control () and LPS-injected () animals. Note the substantial decline in diaphragmatic force in response to CrMSPIX treatment.

HOs and iNOS Expression

We assessed the influence of HO activity on muscle iNOS expression by measuring diaphragmatic iNOS protein expression after 6 and 24 h of LPS injection (with or without CrMSPIX administration). LPS injection elicited in the diaphragm a transient expression of iNOS protein that disappeared after 24 h of LPS injection (Fig. 8). Administration of CrMSPIX had no effect on the time course of diaphragmatic iNOS expression, suggesting that muscle HO activity does not modulate iNOS expression.


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Fig. 8.   Representative immunoblots of iNOS protein in the diaphragms of control animals and animals killed after 6 and 24 h of LPS injection. Note that administration of HO inhibitor (CrMSPIX) did not alter the transient LPS-induced iNOS expression.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main findings of this study are that: 1) both HO-1 and HO-2 enzymes are constitutively expressed inside skeletal muscle fibers of rats and mice, 2) the expression of both enzymes increases significantly in response to LPS injection but on different time courses, 3) systemic inhibition of HO activity augmented muscle protein oxidation and lipid peroxidation and significantly reduced the ability of muscle fibers to generate force both in normal rats and in rats injected with LPS, and 4) the iNOS isoform is not involved in the regulation of muscle HO expression.

Expression of HO in Normal and Septic Muscles

Our study indicates that both HO-1 and HO-2 proteins are expressed, albeit at low levels, in normal limb and ventilatory muscles. This finding is in agreement with previous studies documenting the existence of these proteins in in vivo mammalian muscles and cultured myocytes (4, 20, 32, 35). In accordance with Baum et al. (4), we report here the expression of HO-1 and HO-2 in blood vessels traversing skeletal muscles.

Little is known about the factors that regulate HO expression in skeletal muscle fibers. In nonmuscle cells, many conditions such as heat shock, ischemia, hypoxia, endotoxin, proinflammatory cytokines, and hemin induce HO expression, particularly that of HO-1 (for review, see Ref. 8). In skeletal muscles, Essig et al. (13) reported that exhaustive running or artificial stimulation induces muscle HO-1 mRNA expression. Hemin is another inducer of HO-1 expression in cultured myocytes (35). We report here that diaphragmatic HO-1 and HO-2 expression in rats and HO-1 expression in mice was significantly elevated in response to LPS injection. Taille et al. (32) reported that LPS injection in rats elicits a significant induction of diaphragmatic HO-1 expression with no change in HO-2 expression. The contradictory results regarding the influence of LPS on HO-2 between our study and that of Taille et al. (32) are likely to be due to the differences in dosage of LPS used in the two studies (4 mg/kg in the Taille et al. study compared with 20 mg/kg in our study). We should emphasize that Taille et al. did not investigate the mechanisms responsible for induction of HO-1 and HO-2 expression in LPS-injected animals.

We speculate that the following mechanisms are responsible for upregulation of muscle HO expression in septic animals. First, we propose that the induction of HOs inside muscle fibers late in sepsis might be due in part to increased ROS production (23, 29), which are known to enhance HO expression (8). However, it is unlikely that ROS were involved in early (after 1 h of LPS injection) upregulation of muscle HO-1 expression since (Fig. 1). Second, HO-1 induction in septic muscles might have been mediated by stress-activated (c-fos and c-jun) and proinflammatory (nuclear factor-kappa B) transcription factors. Bindings sites for these transcription factors have been clearly identified in the promoter of HO-1 (8). In addition, tumor necrosis factor (TNF)-alpha and interleukin-1alpha activate HO-1 expression in endothelial cells, an action that is mediated via protein kinase C, Ca2+, and phospholipase A2 (33). On the basis of these observations, it is likely that LPS and proinflammatory cytokines, particularly TNF-alpha , are directly responsible for elevated muscle HO-1 expression in septic animals. Third, exposure of mesangial, endothelial, and smooth muscle cells, and cultured myocytes to exogenous NO donors has recently been shown to elicit a significant induction of HO-1 mRNA and activity (9, 11, 17, 35). Under normal conditions, NO is produced inside skeletal muscle fibers by the neuronal and endothelial NOS isoforms (19). However, muscle NO production rises significantly in septic animals as a result of iNOS induction (5, 18). We have excluded an involvement of iNOS in regulating muscle HO expression in the current study on the basis that inhibition of iNOS activity in rats and absence of iNOS expression in iNOS-/- mice did not alter the effects of LPS on HO-1 and HO-2 expressions. Fourth, it has been shown that peroxynitrite (formed from the near-diffusion reaction between NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· radicals) regulates HO-1 mRNA expression and activity in endothelial cells (15). In septic rats, tyrosine nitration (footprint of peroxynitrite formation) increases significantly in the ventilatory and limb muscles in response to LPS injection (12). On the basis of these findings, it is plausible that induction of diaphragmatic HO expression in septic animals might have been caused by increased peroxynitrite formation. We believe that this was not likely because inhibition of iNOS activity by 1400W, a procedure known to reduce peroxynitrite formation, had no effect on LPS-induced muscle HO expression.

Role of HO in Oxidative Stress

One of the major findings in our study is that administration of the HO inhibitor CrMSPIX increased diaphragmatic protein oxidation and lipid peroxidation (as measured with HNE antibody) both under normal conditions and in response to LPS injection. Taille et al. (32) described a similar rise in protein oxidation and malondialdehyde contents in response to HO inhibition in septic muscles but not in control diaphragms. We speculate that differences between our findings with respect to normal rat diaphragms and that of Taille et al. are due to the use of two different inhibitors of HO activity (CrMSPIX in our study vs. zinc protoporphyrin IX in the Taille study). Appleton et al. (3) reported that CrMSPIX is a more potent and more selective inhibitor of HO activity than zinc protoporphyrin IX. Despite this difference, both our study and that of Taille et al. suggest that muscle HO activity plays an important and protective role in attenuating muscle oxidative stress, particularly in septic animals. This suggestion is in accordance with previous findings in nonmuscle cells (15, 27). Moreover, mice deficient in HO-1 have been shown to be vulnerable to mortality and hepatic cirrhosis induced by LPS (28). The protective effects of HOs have been attributed to the antioxidant properties of the products of HO activity (bilirubin, CO, and ferritin) (14, 25, 26).

Effects of HO Inhibition on Muscle Contractility

A major finding in our study is that inhibition of HO activity evoked a significant impairment of diaphragmatic contractility (Fig. 7). Although the exact mechanisms through which HO activity influences muscle contractility remain speculative, we propose that HOs, through the antioxidant properties of their products, promote excitation-contraction coupling by inhibiting the negative effects of ROS on sarcoplasmic (SR) Ca2+ release channels and SR Ca2+ uptake (2, 6). In addition, reduction of maximum muscle force by HO inhibition suggests that HOs may also exert their effects at the level of contractile proteins. These effects may include attenuation of the deleterious effects of ROS on myofibrillar Ca2+ sensitivity and myofibrillar creatine kinase activity (24). Finally, it is possible HOs influence muscle function by promoting vascular dilation though an NO-independent activation of guanylate cyclase and a rise in guanosine 3',5'-cyclic monophosphate (10).

In summary, our results indicate the presence of HO-1 and HO-2 proteins inside ventilatory muscle fibers and that the expression of both enzymes is upregulated, albeit on different time courses, in response to LPS injection in rats. Our results also suggest that HO activity plays very important and protective roles in attenuating oxidative stress and promoting submaximum and maximum force generation both under normal conditions and in response to severe sepsis.


    ACKNOWLEDGEMENTS

The authors are grateful to L. Franchi for technical assistance and to C. Mutter and R. Carin for assistance in editing the manuscript.


    FOOTNOTES

This study is funded by a grant from the Canadian Institute of Health Research. S. Hussain is a scholar of the Fonds de la Recherche en Santé du Québec (FRSQ). L. C. Lands is a clinician-scientist of the FRSQ.

Address for reprint requests and other correspondence: S. Hussain, Rm. L3.05, 687 Pine Ave. W., Montreal, Quebec H3A 1A1, Canada (E-mail: sabah.hussain{at}muhc.mcgill.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00495.2001

Received 1 January 2001; accepted in final form 20 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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