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Am J Physiol Heart Circ Physiol 275: H1084-H1091, 1998;
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Vol. 275, Issue 3, H1084-H1091, September 1998

Increased levels of myocardial Ikappa B-alpha protein promote tolerance to endotoxin

Brian D. Shames, Daniel R. Meldrum, Craig H. Selzman, Edward J. Pulido, Brian S. Cain, Anirban Banerjee, Alden H. Harken, and Xianzhong Meng

Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Endotoxin [lipopolysaccharide (LPS)] causes tumor necrosis factor-alpha (TNF-alpha )-mediated myocardial contractile depression. Tolerance to the cardiac toxicity of LPS can be induced by a prior exposure to LPS or by pretreatment with glucocorticoids. The mechanisms by which the myocardium acquires tolerance to LPS remain unknown. LPS causes phosphorylation and degradation of inhibitory kappa B-alpha (Ikappa B-alpha ), releasing nuclear factor-kappa B (NF-kappa B) to activate TNF-alpha gene transcription. We hypothesized that LPS induces supranormal synthesis of myocardial Ikappa B-alpha protein and thus renders the myocardium tolerant to subsequent LPS. Rats were challenged with LPS after pretreatment with LPS, dexamethasone, or saline. In saline-pretreated rats, LPS caused a rapid decrease in myocardial Ikappa B-alpha protein levels, activation of NF-kappa B, and increased TNF-alpha production. These events were followed by myocardial contractile depression. After the initial decrease in myocardial Ikappa B-alpha , Ikappa B-alpha protein levels rebounded to a level greater than control levels by 24 h. Dexamethasone pretreatment similarly increased myocardial Ikappa B-alpha protein levels. In rats pretreated with either LPS or dexamethasone, myocardial Ikappa B-alpha protein levels remained similar to control levels after LPS challenge. The preserved level of myocardial Ikappa B-alpha protein was associated with diminished NF-kappa B activation, attenuated myocardial TNF-alpha production, and improved cardiac contractility. We conclude that LPS and dexamethasone upregulate myocardial Ikappa B-alpha protein expression and that an increased level of myocardial Ikappa B-alpha protein may promote cardiac tolerance to LPS by inhibition of NF-kappa B intranuclear translocation and myocardial TNF-alpha production.

nuclear factor-kappa B; tumor necrosis factor-alpha ; cardiac contractility; glucocorticoids; rat

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ENDOTOXIN [lipopolysaccharide (LPS)] causes transient myocardial contractile depression (1, 13, 28, 30), which is mediated, at least in part, by tumor necrosis factor-alpha (TNF-alpha ) (11, 18). We and others have observed (6, 23, 25, 26) that LPS induces cardiac functional tolerance to a subsequent ischemic insult. Our recent work (28) demonstrated that a prior exposure to LPS also induces tolerance to subsequent endotoxemic myocardial depression. However, the mechanisms of induced myocardial tolerance to LPS remain unknown.

LPS stimulates monocytes and macrophages by binding to CD14 receptors (43). Signals distal to the CD14 receptors activate nuclear factor-kappa B (NF-kappa B) (29). NF-kappa B is a family of proteins that share a common Rel domain that regulates nuclear translocation and gene transcription of multiple proinflammatory cytokines (36), including TNF-alpha (8, 35). Myocardial NF-kappa B is therefore a logical target for designing strategies to promote cardiac tolerance to LPS.

Cytosolic association with inhibitory kappa B (Ikappa B) prevents NF-kappa B intranuclear translocation and DNA binding. The Ikappa B family of proteins is characterized by an ankyrin repeat domain that allows for binding to the nuclear localization sequence of NF-kappa B (4). The Ikappa B-alpha gene contains an NF-kappa B binding site in its promoter, which permits regulation of Ikappa B-alpha expression by NF-kappa B (14, 22). Multiple stimuli, including LPS, activate NF-kappa B by initiating phosphorylation of Ikappa B. Phosphorylation of Ikappa B-alpha on two specific serine residues identifies it for ubiquitination and subsequent degradation by the 26S proteasome, permitting intranuclear translocation of NF-kappa B with resultant cytokine gene transcription (3, 36). LPS stimulation, both in vivo and in vitro, causes a rapid degradation with subsequent resynthesis of Ikappa B-alpha in various cell types. This resynthesis is an inducible autoregulatory pathway that functions to turn off NF-kappa B-activated gene transcription (37). The temporal profile of myocardial Ikappa B-alpha after LPS exposure has not been delineated. Examination of the temporal profile of Ikappa B-alpha in the myocardium may provide insight into the mechanisms by which the myocardium adapts to stress.

We have recently observed that glucocorticoids inhibit LPS-induced myocardial TNF-alpha production (26) and prevent endotoxemic myocardial depression (27). Glucocorticoids inhibit NF-kappa B-mediated gene transcription in cultured monocytic cells and lymphocytes by inducing Ikappa B-alpha (2, 33). However, it is unknown whether glucocorticoids upregulate myocardial Ikappa B-alpha in vivo. It has been postulated (5, 17, 38, 44) that LPS tolerance is mediated through inhibition of NF-kappa B-dependent gene transcription. We hypothesized that myocardial Ikappa B-alpha protein expression is enhanced in LPS-tolerant hearts and that upregulation of myocardial Ikappa B-alpha attenuates the NF-kappa B-mediated myocardial response to subsequent LPS.

The purposes of this study were 1) to delineate the temporal profile of myocardial Ikappa B-alpha protein expression after LPS challenge in both tolerant and naive rats, 2) to determine whether tolerance-inducing stimuli enhance myocardial Ikappa B-alpha protein expression, 3) to examine the influence of LPS pretreatment on LPS-induced myocardial NF-kappa B DNA binding, intranuclear translocation, and TNF-alpha production, and 4) to relate myocardial Ikappa B-alpha protein expression, NF-kappa B activity, and TNF-alpha content to cardiac contractile function after LPS challenge.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Male Sprague-Dawley rats, body weight 300-325 g, were quarantined and maintained on a standard pellet diet for 2 wk before initiation of the experiments. All animal experiments were approved by the Animal Care and Research Committee of the University of Colorado Health Sciences Center. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892].

Chemicals and reagents. Dexamethasone was purchased from Elkins-Sinn (Cherry Hill, NJ). The TNF-alpha assay kit was obtained from Genzyme (Cambridge, MA). All antibodies for immunoblotting and immunohistochemistry were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Ikappa B-alpha antibody is a rabbit polyclonal IgG against the carboxy terminus of human Ikappa B-alpha and cross-reacts with rat Ikappa B-alpha . The NF-kappa B antibodies are both goat polyclonal IgG raised against the carboxy terminus of the human p65 or p50 subunit of NF-kappa B, and both cross-react with the rat NF-kappa B subunits. The enhanced chemiluminescence (ECL) kit was obtained from Amersham (Arlington Heights, IL). LPS (from Salmonella typhimurium) and all other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Experimental protocols. Rats were pretreated with LPS (dissolved in bacteriostatic normal saline, 0.5 mg/kg ip, 24 h), dexamethasone (8 mg/kg iv, 30 min) or saline (0.4 ml ip, 24 h, or 0.4 ml iv, 1 h). We have previously demonstrated that this dose of LPS induces cardiac tolerance to subsequent LPS challenge (28). After pretreatment, rats were challenged with LPS (0.5 mg/kg ip). Hearts were rapidly excised after anesthetization with pentobarbital sodium (Nembutal, 60 mg/kg ip) and anticoagulation with heparin sodium (500 units ip) and were subjected to immunoblotting for Ikappa B-alpha (1-24 h after LPS challenge, 3 hearts in each group), electrophoretic mobility shift assay (EMSA) for NF-kappa B (1-4 h after LPS challenge, 3 hearts in each group), immunofluorescent localization of NF-kappa B (1-4 h after LPS challenge, 2 hearts in each group), TNF-alpha assay (1-4 h after LPS challenge, 6 hearts in each group), or isolated heart perfusion (6 h after LPS challenge, 6 hearts in each group).

Immunoblotting. After excision, each heart was flushed by retrograde perfusion through the aortic root with 10 ml of cold (4°C) PBS, and major vessels and atria were removed. Ventricular tissue was frozen with liquid N2 and stored at -70°C. Myocardium was homogenized with a Tissumizer (Tekmar, Cincinnati, OH) in 5 vol of homogenization buffer containing (in mM) 25 Tris · HCl, 2 EGTA, 1 benzamidine, and 1 phenylmethylsulfonyl fluoride (PMSF), pH 7.4. After centrifugation at 3,000 g at 4°C for 20 min, the supernatant was collected. Protein concentration was determined using the Lowry assay (21). Samples (20 µg of crude protein) were mixed with an equal volume of sample buffer (100 mM Tris · HCl, 2% SDS, 0.02% bromophenol blue, and 10% glycerol) and boiled. Electrophoresis was performed on 4-20% linear gradient SDS polyacrylamide gels. Proteins were then electrophoretically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked for 1 h at room temperature with antibody buffer (PBS containing 0.1% Tween 20 and 5% nonfat dried milk) and then incubated with primary antibody (rabbit polyclonal anti-Ikappa B-alpha , 1:500 dilution with antibody buffer) for 1 h at room temperature. Membranes were washed three times in PBS containing 0.1% Tween 20 and then incubated with peroxidase-labeled goat anti-rabbit IgG (1:10,000 dilution with antibody buffer) for 1 h at room temperature. Membranes were then washed three times, and antigen-antibody complexes were revealed by ECL.

Quantification of the immunoblot was performed by computer-assisted densitometry (NIH Application 1.599b4). Density values are expressed as a percentage of the saline control level of each experiment. All densities reported are means ± SE of three separate experiments.

EMSA. After excision, each heart was flushed and major vessels and atria were removed. Nuclear extracts were then prepared using a modified technique of Schreiber et al. (34). Hearts were homogenized in 5 vol of homogenate buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.5 M sucrose, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM benzamidine, and 1 mM PMSF. Homogenates were then centrifuged at 750 g for 10 min at 4°C to isolate crude nuclei (20). The crude nuclear pellet was then resuspended in 100 ml of ice-cold nuclear extraction buffer [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benzamidine, and 1 mM PMSF], and the tube was placed on ice for 30 min with brief, gentle vortexing every 5 min. The nuclear extract was then centrifuged at 12,000 g for 5 min at 4°C. The supernatant was collected and the protein quantified using the Lowry assay.

NF-kappa B consensus oligonucleotide (5'-AGTTGAG<UNL>GGG</UNL><UNL>ACTTTCC</UNL>CAGGC-3', binding site underlined) was 5' end labeled with [gamma -32P]ATP using T4 polynucleotide kinase. Unincorporated nucleotide was removed using a NucTrap Probe purification column (Stratagene, La Jolla, CA). Ten micrograms of nuclear protein were incubated with labeled oligonucleotide (100,000-200,000 counts/min) in binding buffer [10 mM Tris · HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 µg poly(dI-dC)-poly(dI-dC), 1% Nonidet P-40, and 4% glycerol] for 25 min at room temperature in a final volume of 25 ml. Subsequently, the free oligonucleotide and oligonucleotide-bound proteins were separated by electrophoresis on a native 4% polyacrylamide gel. The gel was then dried and exposed to an X-ray film with intensifying screens overnight at -70°C.

For supershift studies, antibodies (1 µg) to either p50 or p65 were added before the addition of labeled oligonucleotide. Binding of the antibody to NF-kappa B was indicated by a supershift in the EMSA. To further demonstrate specificity, excess unlabeled oligonucleotide was used as a specific competitor.

Immunohistochemistry. After excision, hearts were flushed as mentioned in Immunoblotting, and the ventricular tissue was embedded in tissue freezing medium. Tissue was then rapidly frozen in dry ice-cooled 2-methylbutane and stored at -70°C. Transverse 5-mm cryosections were prepared with a cryostat (IEC Minotome plus, Needham Heights, MA) and collected on poly-L-lysine-coated slides. All sections were fixed for 10 min in a 70% acetone-30% methanol mixture at -20°C. Sections were then blocked with 10% normal goat serum and incubated with primary antibody (rabbit polyclonal anti-NF-kappa B p65, 1:40 dilution with PBS containing 1% bovine serum albumin) for 1 h. After three washes with PBS, sections were incubated for 45 min with Cy3-labeled goat anti-rabbit IgG (1:250 dilution with PBS containing 1% bovine serum albumin). After being washed with PBS, sections were counterstained with fluorescein-labeled wheat germ agglutinin (5 µg/ml, for cell surface staining) and bisbenzimide (2.5 µg/ml, for nuclear staining). Sections were then mounted with aqueous anti-quenching medium. To assess the specificity of the immunostaining, adjacent sections were incubated with nonimmune rabbit IgG (5 µg/ml in PBS containing 1% bovine serum albumin) in replacement of the primary antibody and then processed identically. Microscopic observation and photography were performed with a Leica DMRXA confocal microscope (Germany).

TNF-alpha assay. TNF-alpha was measured in myocardial homogenates using an ELISA system containing a hamster anti-mouse TNF-alpha antibody (which cross-reacts with rat TNF-alpha ). Recombinant murine TNF-alpha was used as a standard. Absorbances of standards and samples were determined spectrophotometrically at 450 nm using a microplate reader (Bio-Rad, Hercules, CA). Results were recorded as optical densities and plotted against the linear portion of the standard curve. Protein levels in myocardial homogenates were measured using the Lowry assay. Results are presented in picograms of TNF-alpha per milligram of myocardial protein.

Isolated heart perfusion and assessment of cardiac function. Cardiac function was determined by an isovolumetric Langendorff technique as described previously (28) and expressed as left ventricular developed pressure (LVDP). Hearts were excised into cold (4°C) modified Krebs-Henseleit solution containing (in mM) 5.5 glucose, 119 NaCl, 1.2 CaCl2, 4.7 KCl, 25 NaHCO3, 1.18 KH2PO4, and 1.17 MgSO4. The aorta was cannulated, and the heart was perfused within 30 s after isolation. Hearts were perfused in the isovolumetric mode (70 mmHg) with the modified Krebs-Henseleit solution, which was saturated with 92.5% O2-7.5% CO2 to achieve a PO2 of 440-460 mmHg, a PCO2 of 39-41 mmHg, and a pH of 7.39-7.41 (determined by ABL-4 blood gas analyzer, Copenhagen, Denmark). A latex balloon was inserted through the left atrium into the LV, and the balloon was filled with water to achieve an LV end-diastolic pressure (LVEDP) of 5-10 mmHg. Pacing wires were fixed to the right atrium, and the heart was paced at 6.0 Hz (350 beats/min). Hearts were perfused for 15 min, and LVDP, LVEDP, and heart rate were continuously recorded with a computerized pressure amplifier/digitizer (MacLab 8, ADInstruments, Milford, MA).

Statistical analysis. Data are presented as means ± SE. ANOVA was performed to analyze differences among experimental groups. Statistical significance was accepted within 95% confidence limits.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Myocardial Ikappa B. To delineate the temporal profile of myocardial Ikappa B-alpha after LPS challenge, saline-pretreated rats were challenged with LPS and myocardial Ikappa B-alpha protein level was determined using immunoblotting. LPS challenge caused a decrease in myocardial Ikappa B-alpha within 1 h. Myocardial Ikappa B-alpha remained lower than control level at 2 h and returned to control level by 4 h. However, myocardial Ikappa B-alpha rebounded above control level at 24 h after LPS treatment (Figs. 1A and 2A). To examine the influence of LPS pretreatment on myocardial Ikappa B-alpha after subsequent LPS challenge, rats pretreated with LPS 24 h earlier were challenged with LPS and myocardial Ikappa B-alpha was determined at 1, 2, and 4 h after challenge. As presented in Figs. 1B and 2B, myocardial Ikappa B-alpha remained similar to control levels at all of these time points. To examine whether glucocorticoids also induce myocardial Ikappa B-alpha and preserve myocardial Ikappa B-alpha , rats were treated with dexamethasone and myocardial Ikappa B-alpha was determined in a group of rats at 1 h after dexamethasone treatment and in other groups of rats 1 and 2 h after subsequent LPS challenge. These results are presented in Figs. 1C and 2C. Dexamethasone induced an increase in myocardial Ikappa B-alpha . When dexamethasone-pretreated rats were challenged with LPS, Ikappa B-alpha remained similar to control levels.


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  		Fig. 1.   Immunoblotting of myocardial inhibitory kappa B-alpha (Ikappa B-alpha ) protein. Rats were challenged with lipopolysaccharide (LPS; 0.5 mg/kg ip) after pretreatment with normal saline (NS; A), LPS (0.5 mg/kg ip, 24 h; B), or dexamethasone (Dex; 8 mg/kg iv, 30 min; C). Hearts were excised at sequential time points after LPS challenge, and myocardial Ikappa B-alpha protein was probed by immunoblotting. Representative immunoblots of 3 separate experiments are shown. A: myocardial Ikappa B-alpha decreased at 1 and 2 h after LPS challenge in NS-pretreated rats. Myocardial Ikappa B-alpha normalized at 4 h and was increased above control level (NS pretreated/NS challenge) at 24 h. B: in rats pretreated 24 h earlier with LPS, myocardial Ikappa B-alpha levels at 1-4 h after LPS challenge were similar to those of LPS-pretreated/NS-challenged control. C: another group of rats was pretreated with Dex and then challenged with LPS. Dex pretreatment alone induced myocardial Ikappa B-alpha , and myocardial Ikappa B-alpha remained similar to Dex-pretreated/NS-challenged control level at 1-2 h after LPS challenge in Dex-pretreated animals. All experiments were performed at the same time, allowing comparisons among individual blots.


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  		Fig. 2.   Densitometric quantification of myocardial Ikappa B-alpha level. Quantification of immunoblots was performed by computer-assisted densitometry (NIH Application 1.599b4). Density values are expressed as percentages of NS-pretreated/NS-challenged control for each experiment. All densities reported are means ± SE of 3 separate experiments. Statistical analysis was done by ANOVA with Fisher's protected least significant differences test. A: densitometry analysis reveals that LPS causes a decrease in Ikappa B-alpha protein levels in NS-pretreated animals and then an upregulation above control levels by 24 h. B: when LPS-pretreated animals are subsequently challenged with LPS, Ikappa B-alpha levels remain similar to control levels. C: Dex pretreatment similarly increases Ikappa B-alpha levels above control levels. When Dex-pretreated animals are challenged with LPS, Ikappa B-alpha levels remain similar to control levels. * P < 0.05 vs. NS-pretreated/NS-challenged control.

EMSA for NF-kappa B. EMSA was performed on myocardial nuclear extracts after LPS challenge. NF-kappa B was activated at 1-2 h after LPS challenge in saline-pretreated rats. NF-kappa B DNA binding activity declined 4 h after LPS challenge, although not back to control levels. In LPS-pretreated rats, NF-kappa B activation after subsequent LPS challenge was attenuated (Fig. 3A). Supershift experiments revealed that both p50 and p65 subunits of NF-kappa B were involved in DNA binding (Fig. 3B). Binding specificity was confirmed by including a 100-fold excess of unlabeled consensus oligonucleotide in the DNA binding reaction. The addition of unlabeled oligonucleotide greatly diminished the intensity of the shifted band (Fig. 3B).


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  		Fig. 3.   Nuclear factor-kappa B (NF-kappa B) electrophoretic mobility shift assay. Rats were challenged with LPS (0.5 mg/kg ip) after NS or LPS pretreatment (0.5 mg/kg ip, 24 h). Hearts were excised at sequential time points after LPS challenge. Nuclear proteins were extracted, and NF-kappa B binding activity was determined. A: representative gel shows that, in NS-pretreated animals, myocardial NF-kappa B was activated primarily at 1 and 2 h after LPS challenge. NF-kappa B DNA binding activity declined at 4 h. LPS pretreatment attenuated activation of NF-kappa B by subsequent LPS challenge. B: results of supershift experiments. Myocardial nuclear extract from an NS-pretreated/LPS-challenged (2 h) animal exhibited NF-kappa B DNA binding (lane 1). Addition of antibodies to p50 (lane 2) or p65 (lane 3) caused a supershift of DNA protein complex. To further examine specificity of assay, cold oligonucleotide was added in excess. This caused a decrease in density of shifted band (lane 4).

Immunofluorescent analysis of myocardial NF-kappa B. To determine which cell types were involved in myocardial NF-kappa B activation, we examined the cellular and subcellular distribution of NF-kappa B in the myocardium by immunofluorescent localization. NF-kappa B immunoreactivity was not detected on sections incubated with nonimmune rabbit IgG (data not shown). Immunostaining with rabbit polyclonal anti-NF-kappa B p65 detected NF-kappa B immunoreactivity in both interstitial cells and myocytes. The results of NF-kappa B localization are presented in Fig. 4. Control hearts from untreated rats had minimal nuclear NF-kappa B. In saline-pretreated animals, LPS challenge resulted in the appearance of intranuclear NF-kappa B at 1 (data not shown) and 2 h after LPS challenge (Fig. 4). NF-kappa B remained in the nuclei at 4 h (data not shown). NF-kappa B translocation primarily involved cardiac interstitial cells, although intranuclear NF-kappa B was also observed in myocytes. This suggests that cardiac interstitial cells are the main source of myocardial TNF-alpha . In LPS-pretreated rats, subsequent LPS challenge resulted in a markedly reduced intranuclear translocation of NF-kappa B at 2 h (Fig. 4). This observation is consistent with the gel shift data demonstrating activation of NF-kappa B in naive rats challenged with LPS and attenuation in LPS-pretreated rats.


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  		Fig. 4.   Immunohistochemical localization of myocardial NF-kappa B. Rats were pretreated with NS or LPS (0.5 mg/kg, 24 h) before LPS challenge (0.5 mg/kg ip). Hearts were excised at various time points after LPS challenge, and cellular and subcellular distribution of NF-kappa B (arrows) was examined by immunofluorescent staining with a rabbit polyclonal anti-NF-kappa B p65 antibody (Cy3, red). Cell surface was stained with FITC-labeled wheat germ agglutinin (WGA, green), and nuclei were labeled with bisbenzimide (AMCA, blue). Control hearts from untreated rats had minimal nuclear NF-kappa B. In NS-pretreated rats, LPS challenge induced appearance of NF-kappa B in the nuclei (arrowheads) at 2 h (LPS 2 h). NF-kappa B translocation primarily involved cardiac interstitial cells, although NF-kappa B was present in cardiac myocytes (myo). In LPS-pretreated rats, intranuclear NF-kappa B was reduced at 2 h after subsequent LPS challenge (LPS + LPS 2 h).

Myocardial TNF-alpha . To examine the influence of induced Ikappa B-alpha on myocardial TNF-alpha production, TNF-alpha was measured in myocardial homogenate after LPS challenge in animals pretreated with saline, LPS, or dexamethasone. In saline-challenged rats, myocardial TNF-alpha was 5.3 ± 0.5 pg/mg. LPS challenge increased myocardial TNF-alpha at 1 and 2 h in saline-pretreated animals (26.1 ± 3.8 and 33.1 ± 6.5 pg/mg, respectively; both P < 0.05 vs. saline-challenged rats). TNF-alpha levels peaked at 2 h and declined to control level by 4 h (Fig. 5A). Pretreatment with either LPS or dexamethasone inhibited the peak myocardial TNF-alpha production (Fig. 5B). Myocardial TNF-alpha 2 h after LPS challenge was 13.9 ± 3.8 pg/mg in LPS-pretreated rats (P < 0.05 vs. saline-pretreated rats) and 12.8 ± 3.5 pg/mg in dexamethasone-pretreated rats (P < 0.05 vs. saline-pretreated rats).


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  		Fig. 5.   Influence of pretreatment with LPS or Dex on myocardial tumor necrosis factor-alpha (TNF-alpha ) production. Rats were pretreated with NS, LPS (0.5 mg/kg ip, 24 h), or Dex (8 mg/kg iv, 30 min) before challenge with LPS (0.5 mg/kg ip). A: TNF-alpha was measured in myocardial homogenates in NS-pretreated animals at 1-4 h after LPS challenge to examine time course. B: TNF-alpha was measured in myocardial homogenates of LPS- or Dex-pretreated rats at 2 h after LPS challenge to determine influence of these pretreatments. In NS-pretreated rats, LPS challenge resulted in a significant increase in myocardial TNF-alpha that peaked at 2 h; myocardial TNF-alpha levels declined back to control levels by 4 h (A). Pretreatment with either LPS or Dex attenuated maximal increase in myocardial TNF-alpha (B). Data are means ± SE; n = 6 in each group. * P < 0.05 vs. NS-pretreated/NS-challenged control. ddager  P < 0.05 vs. NS-pretreated/LPS-challenged rats (LPS 2 h).

Cardiac contractile function. To examine the influence of induced Ikappa B-alpha on contractile function, rats were challenged with LPS after pretreatment with LPS, dexamethasone, or saline, and contractile function was assessed. We have previously demonstrated that maximal contractile depression occurs at 6 h after LPS challenge (28). Therefore, hearts were excised, and contractility was assessed 6 h after LPS challenge. As shown in Fig. 6, LVDP was 99.1 ± 2.3 mmHg in saline-challenged rats. LPS challenge resulted in significant depression of myocardial contractility in rats pretreated with saline (LVDP = 57.2 ± 3.4 mmHg, P < 0.001 vs. saline-challenged animals). In contrast, pretreatment with either LPS or dexamethasone preserved cardiac contractility after LPS challenge. LVDP was 101.7 ± 3.4 mmHg in LPS-pretreated rats after LPS challenge (P < 0.001 vs. saline-pretreated rats) and 93.8 ± 2.4 mmHg in dexamethasone-pretreated rats (P < 0.001 vs. saline-pretreated rats).


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  		Fig. 6.   Influence of pretreatment with LPS or Dex on myocardial contractile dysfunction. Rats were pretreated with NS, LPS (0.5 mg/kg ip, 24 h), or Dex (8 mg/kg iv, 30 min) and then challenged with LPS (0.5 mg/kg ip). Cardiac contractile function was assessed using Langendorff technique 6 h after LPS challenge. LPS challenge caused a significant decrease in cardiac contractility in NS-pretreated animals. LPS or Dex pretreatment prevented LPS-induced myocardial contractile dysfunction. Data are means ± SE; n = 6 in each group. * P < 0.001 vs. NS-pretreated/NS-challenged control. ddager  P < 0.001 vs. NS-pretreated/LPS-challenged rat. LVDP, left ventricular developed pressure.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The myocardium overexpresses TNF-alpha in response to LPS (15), and dysregulated TNF-alpha production contributes to contractile dysfunction (11, 18, 24). NF-kappa B activity and TNF-alpha production are controlled by the molecular interaction between NF-kappa B and Ikappa B-alpha (4). In the present study, myocardial Ikappa B-alpha protein levels decreased 1-2 h after LPS treatment, returned to control level by 4 h, and increased above control level by 24 h. Coincidentally with the decrease in myocardial Ikappa B-alpha protein, NF-kappa B was activated and myocardial TNF-alpha levels increased. Cardiac contractile depression temporally followed these events. The results suggest that LPS causes a rapid degradation of myocardial Ikappa B-alpha , followed by Ikappa B-alpha resynthesis. It appears that myocardial Ikappa B-alpha degradation and/or the level of this protein is critical to subsequent cardiac NF-kappa B activation and TNF-alpha synthesis.

The results of this study show that LPS treatment increases the steady-state levels of myocardial Ikappa B-alpha protein. This increase in myocardial Ikappa B-alpha coincided with the development of cardiac tolerance to subsequent endotoxemic contractile depression. Similarly, pretreatment with glucocorticoids, which induce cardiac functional tolerance to LPS, also increased myocardial Ikappa B-alpha . Thus cardiac resistance to endotoxemic contractile depression is associated with an elevated myocardial Ikappa B-alpha protein level. It remains unknown, however, whether the increase in myocardial Ikappa B-alpha protein by either glucocorticoids or LPS is due to increased Ikappa B-alpha gene transcription, RNA stability, translation rate, or protein stability.

Interestingly, myocardial Ikappa B-alpha in LPS-pretreated animals remained similar to control level after subsequent LPS challenge. This preserved level of myocardial Ikappa B-alpha may be due to a higher baseline level of Ikappa B-alpha in LPS-pretreated animals or a combination of a higher baseline level with a decreased degradation rate. The time course utilized in this study is too limited to address this issue. The role of Ikappa B-alpha synthesis and stability in endotoxin tolerance has been examined by other investigators. In THP-1 cells, LPS tolerance is associated with a rapid regeneration of Ikappa B-alpha (19). Both induction and stabilization of Ikappa B-alpha have been observed in LPS-tolerant OVCAR-3 cells (16). Stabilization of Ikappa B was related to inhibition of an inducible Ikappa B-alpha kinase in the LPS-tolerant cells (16). It is likely that Ikappa B-alpha stabilization is involved in maintaining myocardial Ikappa B-alpha levels in the LPS-tolerant myocardium, although the mechanism responsible is unknown. An Ikappa B kinase may possibly be downregulated in LPS-tolerant hearts. Alternatively, myocardial Ikappa B-alpha may be stabilized by stress proteins, functioning as molecular chaperones, via direct protein-protein interaction (40). Indeed, we have observed an upregulation of heat shock protein 70 in LPS-tolerant rat hearts (28). Other investigators have observed that overexpression of heat shock protein 70 decreases LPS-stimulated NF-kappa B intranuclear translocation, suggesting an effect mediated through Ikappa B (10).

The results of the present study indicate that myocardial LPS tolerance is associated with inhibition of NF-kappa B and myocardial TNF-alpha production. Other investigators have observed a similar attenuation in TNF-alpha production by LPS-tolerant cultured cells (12, 31, 38, 44) and a decrease in circulating TNF-alpha in LPS-tolerant animals (7, 32). Inhibition of NF-kappa B has been proposed as a potential mechanism of LPS tolerance (5, 17, 38, 44). The results generated by this study are in agreement with these previous findings and demonstrate that cardiac tolerance to LPS involves regulation of myocardial NF-kappa B activity and TNF-alpha production.

An increased myocardial Ikappa B-alpha protein level is also associated with glucocorticoid-mediated LPS tolerance. Glucocorticoids induce Ikappa B-alpha in vitro and inhibit NF-kappa B-mediated proinflammatory cytokine production (2, 33). In this in vivo study, dexamethasone pretreatment increased myocardial Ikappa B-alpha protein levels, inhibited myocardial TNF-alpha production, and preserved myocardial contractility. Thus inhibition of NF-kappa B-mediated myocardial TNF-alpha production by induction of myocardial Ikappa B-alpha protein is a mechanism for cardiac tolerance to LPS. Regulation of Ikappa B-alpha expression and/or stability has potential clinical application in controlling the inflammatory response. Further characterization of the mechanisms of myocardial Ikappa B-alpha upregulation and/or stabilization may yield clinically accessible therapeutic strategies.

    ACKNOWLEDGEMENTS

The authors are grateful to Dr. Hazel A. Barton for assistance with EMSA, Lihua Ao for technical assistance in isolated heart perfusion and immunofluorescent staining, and Dr. Kyung Joo for preparation of figures.

    FOOTNOTES

This work was supported in part by National Institute of General Medical Sciences Grant GM-08315.

Address for reprint requests: B. D. Shames, Dept. of Surgery, Box C-320, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262.

Received 19 November 1997; accepted in final form 27 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(3):H1084-H1091
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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