Major burn trauma and the aggressive fluid resuscitation that is required to replace fluid loss into the burn wound produce myocardial, pulmonary and immune dysfunction (1–4); yet the mechanisms of downstream organ dysfunction after burn trauma remain unclear. It is likely that inflammatory mediators such as TNF-α play a significant role in postburn cardiac and pulmonary dysfunction (4–6). Recent studies have suggested that cardiomyocyte secretion of inflammatory cytokines per se contribute to the pathogenesis of cardiac contractile dysfunction after ischemia-reperfusion, hemorrhagic shock, endotoxemia and congestive heart failure (7–12). Recent evidence supporting the role of TNF-α in cardiac mechanical dysfunction has been provided by studies by Bryant and colleagues who showed that genetically engineered mice, which overexpress TNF-α specifically in cardiac myocytes and have no measurable rise in systemic TNF-α levels, develop cardiac contractile dysfunction as indicated by ventricular dilatation, decreased ejection fraction and cardiomyopathy. These data confirm that TNF-α can directly mediate cardiac dysfunction and failure (13). Other studies have confirmed the cardiodepressive effects of TNF-α by the direct application of this inflammatory cytokine to isolated ventricular muscle preparations or isolated cardiomyocytes. These studies confirm that TNF-α decreased left ventricular developed pressure and the rate of pressure rise (isolated hearts) and altered the extent of cardiomyocyte shortening (9,11,14). Still, other studies have confirmed that TNF-α added directly to cardiomyocytes produced cardiomyocyte apoptosis (15,16), providing evidence of one mechanism by which TNF-α may mediate cardiac contractile depression.
This present study was designed to determine the time course of both cardiac NF-κB nuclear translocation and cardiac myocyte secretion of TNF-α after major burn trauma. NF-κB is a transcription activator protein recognized to regulate induction of several inflammatory cytokines including TNF-α. In the latent state, NF-κB is a cytoplasmic protein composed of two subunits (p50/p65) and is complexed with the inhibitory proteins collectively termed I-κB. After cellular stimulation, I-κB is phosphorylated, ubiquitinated and degraded, allowing nuclear migration of NF-κB where this transcription factor subsequently binds to appropriate promoter regions (17,18). We hypothesized that major burn trauma and aggressive fluid resuscitation to maintain survival for 24 h after burn injury promote NF-κB activation and cardiac myocyte secretion of TNF-α. If transcription and translation of TNF-α are NF-κB dependent in the cardiomyocyte cell population, then inhibition of NF-κB nuclear translocation with the inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) would be expected to prevent burn-induced secretion of TNF-α by cardiac myocytes and to improve ventricular mechanical performance.
MATERIALS AND METHODS
Adult Sprague Dawley rats (Harlan Laboratories, Inc., Houston, TX) weighing 325–360 grams were used throughout the study. Animals were allowed 5–6 days to acclimate to their surroundings. Commercial rat chow and tap water were available at will throughout the experimental protocol. All work described herein was approved by The University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory Committee, and was performed according to the guidelines outlined in the “Guide for the Care and Use of Laboratory Animals” as published by the American Physiological Society.
Rats were deeply anesthetized with methoxyflurane, and secured in a constructed template device as previously described (1,2). The surface area of the skin exposed through the template device was immersed in 100°C water for 12 s on each side; using this technique, full-thickness dermal burns comprising 40% of the total body surface area were obtained. This burn technique produces complete destruction of the underlying neural tissue and a transient (less than 45 s) increase in internal body temperature of 1°C. Sham burn rats were subjected to identical preparation, except they were immersed in room temperature water. After immersion, the rats were immediately dried and each animal was placed in an individual cage. All burned rats were given lactated Ringer's solution (4 mL/kg/% burn, with one half of the calculated volume given IP immediately postburn and the remaining calculated volume given 8 h postburn, IP). In the sham burn group, 2 cc of lactated Ringer's solution was given IP. In a subset of rats from each experimental group (N = 5 rats/group), a polyethylene catheter (PE-50 tubing) was inserted into the left carotid artery with the tip advanced to the level of the aortic arch. Twenty-four hours after burn injury (or sham burn), hemodynamic parameters were measured (systemic blood pressure, Gould-Statham pressure transducer, Model P23ID, Gould Inc., Oxnard, CA, connected to a recorder, Model 7D Polygraph, Grass Instruments Inc., Quincy, MA; and heart rate, tachycardiograph, Model 7P4F, Grass Instruments Inc., Quincy, MA). A small sample of arterial blood was withdrawn from the arterial catheter for measuring hematocrit, arterial pH, blood gases, and circulating TNF-α (ELISA). Body temperature was measured with a rectal temperature probe (YS1-Tele Thermometer, Model 44TA, Yellow Springs, OH), and respiratory rate was monitored by counting respiratory movement.
Rats were randomly divided to receive either cutaneous burn injury over 40% of the total body surface area or sham burn injury. Subgroups of rats were sacrificed at several times after burn trauma (1, 2, 4, 8, 12, 18 and 24 h); and subgroups of sham burned rats were sacrificed at identical times to provide appropriate controls. After sacrificing rats at the designated time, hearts were extirpated and used to assess NF-κB activation, cardiomyocyte secretion of TNF-α, or ventricular function. Hearts designated to assess NF-κB activation were cleared of fat and epicardial vessels, freeze clamped in liquid nitrogen with pre-cooled tongs and stored at −80°C until nuclear extraction and electrophoretic mobility shift assay (N = 3–4 hearts/group/time period). Hearts used to prepare cardiomyocytes for assessment of myocyte TNF-α secretion were extirpated and perfused with collagenase containing buffer (N = 4 hearts/group/time period). The time course of myocardial contractile dysfunction was assessed in additional hearts extirpated from shams and burns at the times described above (N = 6–7 rats/group/time period).
To determine that cardiac myocyte secretion of TNF-α after burn trauma is NF-κB dependent, additional sham and burned animals were treated with N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), a potent inhibitor of NF-κB activation (19). Rats were given ALLN (50 mg/kg) dissolved in 200 μL ethanol IP at the time of burn injury or sham burn. All other aspects of the burn procedure, fluid resuscitation and animal care were identical to that described above. ALLN treated burns and shams were sacrificed either 4 or 24 h postburn and hearts were either freeze clamped to measure NF-κB activation (N = 3 rats/group), perfused with collagenase to measure cardiac myocyte secretion of TNF-α (N = 4 rats/group), or perfused to assess ventricular performance (N = 6 rats/group).
Cardiac NF-κB activation
Nuclear protein extraction
A modified procedure based on the method of Schreiber and colleagues (20) was used. All steps were performed on ice. Hearts were thawed in the presence of 400 μL hypotonic Tris-buffer (10 mM Tris-HCl; pH 7.8; 5 mM MgCl2; 10 mM KCl; 0.3 mM EGTA; 0.5 mM DTT; 0.3 M sucrose; 1 mM PMSF) and protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO) containing the following protease inhibitors: 20 mM 4-(2-aminoethyl)-benzenesulfaonylflouride (AEBSF), 14 μm trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane(E-64), 1300 μM bestatin, 10 μM leupeptin, 3 μM aprotinin, and 10 mM sodium EDTA). The tissue was then homogenized using a Tissuemizer (Tekmar Co., Cincinnati, OH) under standardized conditions (2 × 10 sec, 10 s pause in-between). Cells were allowed to swell for 15 min to facilitate lysis. NP-40 was added to a final concentration of 0.5%, the mixture vortexed at full speed for 20 s and centrifuged for 2 min at 8,000 × g. Nuclear proteins were extracted from the pellet with 200 μL high-salt Tris-buffer (20 mM Tris-HCl; pH 7.8; 5 mM MgCl2; 320 mM KCl; 0.2 mM EGTA; 0.5 mM DTT; 1 mM PMSF and the mixture of protease inhibitors described above) for 15 min, followed by centrifugation at 13,500 × g for 15 min. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Nuclear extracts were stored at −80°C (17).
EMSA (Electrophoretic mobility shift assay)
Double-stranded oligonucleotides corresponding to the consensus NF-κB binding site of the κ light chain enhancer (5´-AGTTGAGGGGACTTTCCCAGGC-3´) were purchased from Promega Biotech (Madison, WI). In a total volume of 10 μL, 3.5 pmol of oligonucleotide, 10 U of T4 polynucleotide kinase in 1 × forward buffer (Gibco BRL, Gaithersburg, MD), and 30 μCi of [γ-32P] ATP (Dupont NEN, Boston, MA) were incubated at 37°C for 60 min. The reaction was stopped by adding 1 μL of 0.5 mM EDTA. Volume was brought up to 50 μL with the addition of STE buffer (10 mM Tris-HCl, ph 7.5, 10 mM NaCl, 1 mM EDTA). Labeled probe was separated from unbound ATP using ProbeQuant G-50 Micro Columns from Pharmacia Biotech (Piscataway, NJ). The activity of labeled probe was determined, and the probe was stored at −20°C.
Ten μg of nuclear proteins were incubated with 500,000 cpm of probe in the presence of 2 μg salmon sperm DNA in 1 × gel shift buffer (20 mM Hepes; 7.6 pH, 50 mM KCl; 1 mM DTT; 1 mM EDTA; 5% glycerol) and water (total volume 20 μL) for 30 min at room temperature. The reaction mixtures were then separated on a non-denaturing 6% polyacrylamide gel in 0.5 × TBE (25 mM Tris-HCl; 25 mM boric acid; 0.5 mM EDTA) which had been pre-electrophoresed for 30 min at 100 V. The gel was transferred to Whatman paper (Whatman 3 M), dried in a gel dryer (Bio-Rad, Model 583) under vacuum at 80°C for 20 min and exposed to X-ray film (BioMax, Eastman Kodak, Rochester, NY) over night at −80°C. Competition analyses were performed by including a 40-molar excess of unlabeled double stranded DNA oligonucleotide in the binding reaction. Nonspecific competitor DNA contained an AP-1 binding element. Supershift was performed by incubating the 24 h burn sample with 4 μg of the appropriate antibodies (mouse monoclonal p65, goat polyclonal p50, and rabbit polyclonal Rel B and c Rel; Santa Cruz, Santa Cruz, CA) for 1 h at room temperature prior to electrophoresis.
RTPCR to measure TNF-α mRNA
Fifty milligrams of rat cardiac tissue was pulverized in liquid nitrogen using a mortar and pestle. Homogenate was added to 1mL of Trizol reagent (Life Technologies, Gaithersburg, MD) and RNA extraction continued according to manufacturer's instructions as follows. Tissue/Trizol mix was incubated at room temp for 5 min followed by the addition of 0.2 mL of chloroform. Tubes were then vigorously shaken and incubated for 3 min at room temperature. Samples were then centrifuged at 12,000 × g for 15 min at 4°C and the aqueous phase transferred to a fresh tube. To precipitate the RNA, 0.5mL of isopropyl alcohol was added, incubated for 10 min, and centrifuged at 12,000 × g for 10 min at 4°C. The pellet was washed with 1mL of ethanol by vortexing and centrifuging at 7,500 × g for 5 min at 4°C. Pellets were air-dried for 10 min and resuspended in RNase-free water and incubated at 60°C for 10 min. Samples were stored at −80°C.
Synthesis of cDNA was performed using SuperScript Preamplification System (Life Technologies, Gaithersburg, MD) according to manufacturer's instructions. 5μg of RNA was mixed with 250ng of random hexamers, and volume was increased to 12μL with DEPC-treated water (diethylpyrocarbonate, Sigma Chemical Co). Mixture was incubated for 10 min at 70°C, then placed on ice for 1min. To the RNA mix, 1X PCR buffer, 2.5mM MgCl2, 0.5mM dNTP mix, and 0.01M DTT were added and incubated at 25°C for 5 min. 200 units of SuperScript II RT was added to each tube, placed in thermal cycler (MJ Research) and incubated at 25°C for 10 min, 42°C for 50 min, 70°C for 15 min and then held at 4°C until ready to proceed. Two units of RNase H was added to each tube and incubated for 20 min at 37°C. Control cDNA was synthesized using the kit's control RNA.
A 470bp fragment (+401 to +871) of TNF-α mRNA was amplified using PCR by mixing 2μL of cDNA with 1X PCR buffer, 1.5mM MgCl2, 0.2mM dNTP mix, 10μM of each primer, and 2.5 units of Taq (all reagents from Gibco, Gaithersburg, MD). The following nucleotide sequences were used for PCR amplification F 5´-CTCAGATCATCTTCTCAAAAT-3´ and R 5´-GAGTCATTGCTCTGTGA-3´ (19). Reaction mixtures were cycled in an MJ Research thermal cycler (Waltham, MA) at 95°C for 3 min followed by 30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, with a final extension cycle at 72°C for 7 min. Products were then analyzed on a 1% Sea Kem agarose gel, stained with ethidium bromide and visualized under UV light. Control products were amplified using control primers provided in SuperScript kit.
To isolate cardiac myocytes, animals from each experimental group were heparinized at the designated time for study after burn trauma (1, 2, 4, 8, 12, 18, and 24 h postburn), decapitated, and the heart removed through a medial sternotomy using sterile techniques. The isolated heart was immediately placed in ice-cold calcium free Tyrodes solution (in mM, 136 NaCl, 5 KCl, 0.57 MgCl2, 0.33 NaH2PO4, 10 Hepes, 10 glucose). The aorta was cannulated within 60 s, and the excised heart was perfused with calcium-free Tyrodes solution using a Langendorff perfusion apparatus. Perfusion was maintained for 5 min and then switched to a collagenase solution which contained 80 mL of calcium-free Tyrodes, 40 mg collagenase A (0.05%, Boehringer Mannheim Corp., Indianapolis, IN) and 4 mg of protease (Polysaccharide XIV, Sigma Chemical Company, St. Louis, MO) with continuous oxygenation (95% O2 and 5% CO2). After this enzymatic digestion over an 8 min period, the heart was removed from the cannula and the ventricular tissue was separated from the base of the heart. This tissue was placed in a petri dish containing Tyrodes solution containing 100 μM calcium and gently minced to increase cell dispersion over 6 min. The myocyte suspension was then filtered and the cells were allowed to settle. This rinsing and settling step was repeated 3 times with 10 min between each step and with gentle swirling between each step to allow myocyte separation. The calcium concentration of the rinsing solution was gradually increased during these steps from 100 μM to 200 μM and finally to 1.8 mM. The cell viability was measured (Trypan Blue dye exclusion) and cell suspensions with greater than 85% viability were used for subsequent studies. Myocytes with a rod-like shape, clear defined edges, and sharp striations were prepared with a final cell count of 5 × 104 cells/mL (21).
Cytokine secretion by cardiomyocytes
Myocytes harvested from all experimental groups (sham, sham given ALLN, burn, and burn given ALLN) were pipetted into microtiter plates at 5 × 104 cell/mL/well (12 well cell culture cluster, Corning Inc., Corning, NY) and incubated for 18 h (CO2 incubator at 37°C). Supernatants were collected to measure myocyte-secreted TNF-α (TNF-α rat ELISA, Pierce Endogen, Rockford, IL).
To examine the cell specific effects of inhibiting NF-κB nuclear translocation on cardiomyocyte secretion of the inflammatory cytokine TNF-α, myocytes were harvested from additional rats 24 h after either burn trauma (N = 5) or sham burn injury (N = 5) as described above. Myocytes (5 × 104 cells/microtiter well) were incubated (37°C) for 60 min in Tyrodes' solution containing ALLN (40 mg/5 × 104 cells/microtiter well) for an additional 60 min, and cell viability was measured; myocytes (5 × 104 cell/microtiter well) incubated in Tyrode's in the absence of ALLN provided appropriate controls. Cells were then challenged with LPS (0, 10, or 25 μg/well) for 18 h at 37°C (CO2 incubator). After 18 h, supernatants were collected to measure myocyte secretion of TNF-α. In this manner the cell specific effects of ALLN on cytokine secretion by cardiac myocytes was assessed in vitro.
Isolated coronary perfused hearts
To assess ventricular performance, hearts were rapidly removed and placed in ice cold (4°C) Krebs-Henseleit bicarbonate buffered solution (in mM, 118 NaCl, 4.7 KCl, 21 NaHCO3, 1.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 11 glucose). All solutions were prepared each day with demineralized, deionized water and bubbled with 95% 02 – 5% CO2 (pH, 7.4; pO2, 550 mmHg; pCO2, 38 mmHg). A 17-gauge cannula, placed in the ascending aorta and connected via glass tubing to a buffer filled reservoir, allowed perfusion of the coronary circulation at a constant flow rate. Hearts were suspended in a temperature controlled chamber maintained at 38°C, and a constant flow pump (Isma Tec, model 7335-30, Cole-Palmer Instruments Co., Chicago, IL) was used to maintain perfusion of the coronary arteries by retrograde perfusion of the aortic stump cannula. Coronary perfusion pressure was measured and effluent was collected to confirm coronary flow rate. Contractile function was assessed by measuring intraventricular pressure with a saline-filled latex balloon attached to a polyethylene tube and threaded into the left ventricular chamber. Left ventricular developed pressure (LVDP) was measured with a Statham pressure transducer (Model P23ID, Gould Instruments Inc., Oxnard, CA) attached to the balloon cannula, and the rates of LVP rise (+dP/dt) and fall (−dP/dt) were obtained using an electronic differentiator (Model 7P20C, Grass Instruments, Inc., Quincy, MA) and recorded (Model 7DWL8P, Grass Recording Instruments, Quincy, MA). Data from the Grass recorder was input to a Dell Pentium computer, and a Grass PolyVIEW Data Acquisition System was used to convert acquired data into digital form.
All values are expressed as mean ± standard error of the mean. Analysis of variance (ANOVA) was used to assess an overall difference among the groups for each of the variables. Levene's test for equality of variance was used to suggest the multiple comparison procedure to be used if the ANOVA was significant. If equality of variance among the 4 groups was suggested, multiple comparison procedures were performed (Bonferroni). If inequality of variance was suggested, Tamhane multiple comparisons were performed. Probability values less than 0.05 were considered statistically significant (analysis was performed using SPSS for Windows, Version 7.5.1).
Hemodynamic and metabolic response to burn trauma
All rats survived the experimental protocol regardless of burn trauma or ALLN treatment. As shown in Table 1 (upper panel), mean arterial blood pressure was lower 24 h postburn in burned rats (113 ± 7 mmHg, P < 0.05) compared to values measured in sham burns (153 ± 5 mmHg, P < 0.05) despite aggressive fluid resuscitation throughout the postburn period. Heart rate was similar in burns (440 ± 20 bpm) and shams (450 ± 21 bpm) measured 24 h postburn. Arterial pH, PO2 and PCO2 were not significantly altered by burn trauma (Burns: 7.5 ± 0.02, 106 ± 8 mmHg, 28 ± 2 mmHg; Shams: 7.49 ± 0.01, 106 ± 3 mmHg, 31 ± 2 mmHg, respectively). Hematocrit was significantly lower in burns (30 ± 2), compared to that measured in shams (44 ± 1), and this fall in hematocrit was attributed to the aggressive fluid resuscitation after burn trauma and hemodilution. Systemic TNF-α levels were elevated 24 h postburn (baseline, 7.1 ± 0.4 pg/mL; 24 h postburn, 29 ± 4 pg/mL, P < 0.05). Administration of ALLN plus fluid resuscitation in burned rats did not alter MAP (130 ± 4 mmHg), HR (450 ± 18 bpm), arterial pH (7.54 ± .03), PO2 (118 ± 4 mmHg), PCO2 (27 ± 2 mmHg), or hematocrit (36 ± 1) compared to values measured in burned rats given fluid resuscitation alone. ALLN treatment in sham burns did not alter systemic TNF-α levels (9.6 ± 0.8) compared to levels measured in vehicle treated shams (7.1 ± 0.4 pg/mL). In contrast, ALLN treatment in burns reduced circulating TNF-α levels 24 h after thermal injury (vehicle treated burns, 29 ± 4 pg/mL versus ALLN treated burns, 15 ± 1 pg/mL, P < 0.05)
Cardiac NF-κB activation after burn trauma
Figure 1 is a representative electrophoretic mobility gel-shift assay and summarizes the time course of burn-induced cardiac NF-κB activation. NF-κB nuclear migration occurred within 4 h after burn trauma, and persisted over the entire time course of study period. As shown in Figure 2, densitometry confirmed a burn-related increase in NF-κB activation 4 h postburn through 24 h postburn; autoradiograms were scanned, and the density of the NF-κB bands was determined using Alpha Innotech's ChemiImager AlphaEase software analysis program (San Leandro, CA). As shown in Figure 3, burn-mediated activation of NF-κB (lane 1) was ablated by competition with cold NF-κB (lane 2) but not with cold AP-1 (lane 3). ALLN given in vivo ablated burn-mediated NF-κB activation (Fig. 3, lane 4). Lanes 5–8 show supershift with various NF-κB subunits.
Time course of cardiomyocyte TNF-α secretion after burn trauma
Burn trauma stimulated TNF-α secretion by cardiac myocytes as early as 1 h after injury (Fig. 4). TNF-α secretion by myocytes progressively increased after injury, achieving maximal levels 18 h postburn and persisting 24 h after burn trauma. Our data also confirm that cardiac myocytes prepared from burned animals secreted significantly more TNF-α at all times studied compared to TNF-α levels secreted by myocytes prepared from time matched sham burns. RT-PCR of rat heart RNA confirmed increased TNF-α mRNA 6 and 24 h postburn (Fig. 5). Administration of ALLN after burn trauma inhibited burn-related secretion of TNF-α by cardiomyocytes 24 h postburn (Fig. 6).
Time course of postburn cardiac contractile dysfunction
Ventricular function (Langendorff preparation) progressively deteriorated over the first 24 h after burn trauma. As shown in Table 2, left ventricular developed pressure (calculated as the difference between LV peak systolic and LV end diastolic pressure) and ±dP/dt max [the rate of LVP rise (+) and fall (−)] were significantly lower 8 h after thermal injury than values measured in time-matched shams. Left ventricular contractile dysfunction was maximal 18–24 h postburn. ALLN given with fluid resuscitation in burned rats significantly improved LVP, +dP/dt max, and −dP/dt max compared to values measured 24 h postburn in untreated burns. Ventricular function curves were significantly depressed after burn trauma while function curves were improved in burns treated with ALLN (Fig. 7). The difference in ventricular performance could not be attributed to differences in heart rates among the experimental groups; all hearts were paced in vitro at rates slightly above the capture rate (244 ± 9 to 258 ± 11 beats/min).
In vitro effects of ALLN on cytokine secretion
In a parallel experiment, hearts were harvested from burned rats 24 h after burn trauma (or sham burn for controls); TNF-α secretion by cardiomyocytes and cardiac contractile dysfunction were studied 24 h postburn since this time was shown to be the point of maximum TNF-α secretion and maximum ventricular deficits. Myocytes, prepared as described above, were pretreated with either ALLN (40 mg/5 × 104 cells) or media alone for 60 min, followed by LPS challenge (0, 10, or 25 μg LPS) for 18 h. Because the in vivo administration of ALLN could alter multiple aspects of the inflammatory cascade, the in vitro treatment of cardiomyocytes with ALLN examined the specific effects of NF-κB inhibition on myocyte secretion of TNF-α. As shown in Figure 8, myocytes from both sham and burned rats responded to LPS challenge with a dose-dependent increase in TNF-α secretion. However, myocytes from burned rats secreted significantly more TNF-α (P < 0.01) at each LPS dose compared to TNF-α levels secreted by myocytes from sham burns. In vitro ALLN treatment of cardiomyocytes inhibited LPS-induced TNF-α secretion, regardless of a previous burn injury. Thus, TNF-α levels were similar in ALLN treated myocytes prepared from both sham and burned animals.
In this present study, burn trauma promoted cardiac NF-κB nuclear migration as measured by EMSA and promoted significant TNF-α secretion by cardiac myocytes as measured by ELISA. In vivo administration of the proteosome inhibitor ALLN prevented burn-related NF-κB nuclear translocation in the myocardium, inhibited cardiac myocyte secretion of TNF-α, reduced circulating TNF-α levels and improved cardiac contractile function. Our data support and extend previous work by Schow, Joly, and colleagues who described that ALLN administration in a murine model of gram negative sepsis reduced circulating TNF-α levels (18,22). While the increase in systemic TNF-α levels 24 h after burn trauma was significant, these levels were significantly lower than plasma inflammatory cytokine levels described previously in several models of sepsis, hemorrhage or ischemia-reperfusion (7,9,23,24).
We propose that the progressive cardiac dysfunction confirmed after burn trauma in this present study and described previously in models of burn trauma in rats, mice, guinea pigs, rabbits and sheep (25–28) is related, in part, to cardiac myocyte secretion of inflammatory cytokines such as TNF-α, producing local or myocardial cytokine levels that are not easily and rapidly buffered by soluble receptors or neutralizing strategies. While ALLN effectively inhibited the burn-mediated secretion of TNF-α by cardiomyocytes, we considered that in vivo administration of the proteosome inhibitor may have altered several components of the systemic inflammatory cascade and may have downregulated synthesis of inflammatory cytokines by other peripheral tissues such as the lung and liver (6). Therefore, we examined the cardiospecific effects of ALLN by pretreating cardiac myocytes with the proteosome inhibitor followed by subsequent in vitro LPS challenge. ALLN treatment of cardiomyocytes significantly inhibited LPS-induced TNF-α secretion by this cell population, and these results are similar to data reported by Schow and Joly who showed that in vitro application of ALLN to other cell populations (i.e., RAW cells) significantly inhibited LPS-related cytokine secretion (18). However, we considered that cell signaling mechanisms that regulate cytokine production may differ in RAW cells (an immortalized murine macrophage cell line) compared to signaling mechanisms present either in vivo or in freshly prepared cells; in addition, organ to organ differences (heart vs. lung) as well as species-related differences (mouse versus rat) may occur. However, our time course studies confirmed that myocardial NF-κB activation and TNF-α secretion by cardiomyocytes preceded the development of cardiac contractile dysfunction. ALLN-mediated inhibition of myocardial NF-κB activation effectively interrupted the postburn inflammatory cascade, blunting cardiomyocyte secretion of TNF-α which is characteristic of burn trauma and improved ventricular function (5,27). Our finding that interrupting myocardial synthesis of TNF-α improved postburn cardiac contractile performance was consistent with our previous finding in a guinea pig model; in those pervious studies, the use of a different experimental method of inhibiting TNF-α was associated with improved cardiac function after burn trauma (27).
In our study, the secretogogues that mediate postburn NF-κB activation and cardiac myocyte secretion of TNF-α remain unclear. A burn-related loss of gut barrier function and bacterial/endotoxin translocation have been described (29–31). Therefore, burn trauma may promote NF-κB nuclear translocation via an LPS related mechanism. Alternatively, burn-related oxidant stress may promote cardiac NF-κB activation and secondarily promote secretion of inflammatory cytokines (32). Several labs have described oxidant-mediated activation of NF-κB (33), and we have confirmed previously that burn trauma produces significant oxidant stress (34–36). In addition, we have identified xanthine-oxidase dependent as well as leukocyte-dependent mechanisms of free radical production after major burn trauma (35). Our recent studies have shown that antioxidant vitamins prevented burn-mediated NF-κB activation and reduced cardiomyocyte secretion of the inflammatory cytokine TNF-α, suggesting oxidant-mediated nuclear emigration of NF-κB after burn trauma (37). These data are consistent with previous studies showing that either vitamin E or glutathione prevented activation of NF-κB and suggested that NF-κB activation is regulated by reactive oxygen species (ROS) (33,37). Another mechanism by which burn trauma may promote cardiac NF-κB activation is by decreasing IκBα levels in the cardiomyocyte cytosol. However in this present study, we did not measure either phosphorylation, ubiquitination, or degradation of IκB after burn trauma. The effects of burn injury on IκB handling warrant further study.
While the significant findings in this study included the burn-mediated nuclear translocation of NF-κB in the myocardium paralleled by increased TNF-α secretion by cardiac myocytes, it is clearly recognized that the transcription factor NF-κB controls gene expression of several inflammatory mediators including IL-1β, inducible nitric oxide synthase, IL-6, IL-8, and ICAM-1. In this present study, ALLN inhibition of NF-κB activation may have prevented burn-related upregulation of the ICAM gene and secondarily improved cardiac performance. This hypothesis is consistent with our previous finding that burn trauma promotes neutrophil/ICAM-1 interaction as well as neutrophil emigration into the myocardial tissue (34) and is also consistent with the finding by others that disruption of the ICAM gene in a model of TNF-α-mediated cardiac injury improved cardiac function (38).
In addition, burn-mediated TNF-α secreted by cardiomyocytes may alter several aspects of cardiac contraction and relaxation by either producing apoptosis of myocytes (15,16), activating resident neutrophils or other nonmyocyte populations which reside within the myocardium (34,38), initiating synthesis of other cardiodepressant factors such as sphingosine, or directly altering calcium binding by the contractile elements (8,39,40). It is likely that ALLN administration and NF-κB inhibition in the present study interrupted several aspects of this burn-mediated inflammatory cascade. However, our finding that nuclear migration of NF-κB and TNF-α secretion by cardiac myocytes preceded postburn cardiac contractile dysfunction provided a clearer understanding of the time course of this postburn inflammatory cascade that culminates in cardiac contraction and relaxation deficits. While a complete understanding of the contribution of NF-κB activation and TNF-α secretion to myocardial injury and dysfunction after burn trauma is lacking, it is likely that therapeutic strategies that modulate NF-κB-dependent inflammatory gene transcription and translation will have clinical utility.
In summary, data from this study established the time course of NF-κB nuclear migration in the myocardium as well as the time course of TNF-α secretion by cardiac myocytes; activation of this signaling pathway preceded the development of cardiac contraction and relaxation defects after major burn injury. The proteosome inhibitor ALLN prevented burn-induced myocardial NF-κB activation, decreased TNF-α secretion by cardiac myocytes, and improved cardiac performance. Our data suggest that cardiac myocyte secretion of inflammatory cytokines such as TNF-α occur via the proteosome pathway and contribute to postburn cardiac dysfunction.
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