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B overexpression in cardiomyocytes prevents NF-B
translocation and provides cardioprotection in trauma
1 Department of Surgery and 2 Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9160
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study examined the effects of either
I
B
overexpression (transgenic mice) or
N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) administration
(proteosome inhibitor in wild-type mice) on cardiomyocyte secretion of
tumor necrosis factor- (TNF-) and on cardiac performance after
burn trauma. Transgenic mice were divided into four experimental
groups. IB overexpressing mice were given a third-degree scald
burn over 40% of the total body surface area or wild-type littermates
were given either a scald or sham burn to provide appropriate controls.
Pharmacological studies included ALLN (20 mg/kg) administration in
either burned wild-type mice or wild-type shams. Burn trauma in
wild-type mice promoted nuclear factor-B (NF-B) nuclear
translocation, cardiomyocyte secretion of TNF-, and impaired cardiac
performance. IB overexpression or ALLN treatment of burn trauma
prevented NF-B activation in cardiac tissue, prevented cardiomyocyte
secretion of TNF-, and ablated burn-mediated cardiac contractile
dysfunction. These data suggest that NF-B activation and
inflammatory cytokine secretion play a significant role in postburn
myocardial abnormalities.
N-acetyl-leucinyl-leucinyl-norleucinal; collagenase digestion; cardiac contractile function; fura 2-AM
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MAJOR BURN TRAUMA is characterized by a rapid and continual loss of isotonic fluid into the burn wound, producing a significant decrease in circulating volume. The systemic derangements that occur secondary to fluid shifts into the burn wound include low cardiac output, decreased peripheral perfusion, impaired renal and pulmonary function, and hematological abnormalities (7, 8). Initially, the cardiac dysfunction that was shown to occur after burn trauma was attributed to this decrease in plasma volume and a Frank-Starling mechanism. However, in 1957, Dobson and Warner (14) discovered that after burn trauma, cardiac output fell significantly before changes in plasma volume. Cardiac contractile dysfunction after burn trauma was confirmed subsequently using isolated ventricular muscle preparations (17). Later studies by Baxter and colleagues (6) described a myocardial depressant factor in the serum of human burn subjects. More recently, Ferrara and colleagues (16) described the presence of a myocardial depressant factor in the lymph after experimental burn. Both experimental and clinical studies of burn trauma have correlated the extent of cardiac injury (indicated by a rise in cardiac troponin I levels in the serum) with the percentage of the total body surface area (TBSA) burn (1, 34). Cardiac troponin I is a highly specific and sensitive indicator of myocardial ischemia and injury, but is undectable in the serum of normovolemic, unburned subjects (2).
While there is considerable evidence that thermal injury impairs
cardiac systolic and diastolic function, recent attention has focused
on the hypothesis that burn trauma initiates a systemic inflammatory
response that contributes to myocardial injury and dysfunction. We and
others (27, 30, 41) have documented that cardiac myocytes
themselves secrete inflammatory cytokines such as tumor necrosis
factor- (TNF-), interleukin (IL)-1
, and IL-6 in response to a
variety of insults, including burn trauma, ischemia-reperfusion, and sepsis. Local myocardial inflammatory cytokine levels may be an important component of postburn cardiac contractile dysfunction because TNF- directly depresses cardiac contraction and relaxation and induces cardiac myocyte
apoptosis (26, 30, 32). Transgenic mice
overexpressing TNF- exclusively in the myocardium have myocardial
TNF- levels sufficient to cause dilated cardiomyopathy and severe
congestive heart failure (13). Anti-TNF- strategies
that specifically interrupt or inhibit TNF- synthesis have been
shown to provide significant cardioprotection after burn trauma as well
as after sepsis (19).
While substantial evidence continues to accumulate supporting the role
of cardiac-derived inflammatory cytokines in the myocardial systolic
and diastolic deficits that occur after major burn trauma, the signal
transduction mechanisms that regulate cytokine synthesis and secretion
after trauma remain unclear. Our (25, 29) preliminary studies suggest that burn trauma promotes nuclear translocation of the
transcription factor nuclear factor (NF)-B in the myocardium. In
unstimulated cells, NF-B exists as a latent cytoplasmic complex bound to an inhibitory protein, IB (5). In models of
sepsis, studies have shown that lipopolysaccharide (LPS) binding to
LPS-binding protein and subsequently to cell surface CD14/TLR receptors
initiates a signaling pattern mediated through the Ras/Raf-1 MAPK
pathway, leading to phosphorylation and degradation of IB and
subsequent release of NF-B (18). NF-B then
translocates to the nucleus, where it binds to specific NF-B
recognition sequences found in the 5' flanking regions of diverse genes
involved in inflammatory responses such as TNF-, inducible nitric
oxide synthase and adhesion molecules, thus regulating their synthesis
(4, 5). Recent studies have shown that measures of
increased NF-B binding activity in peripheral blood monocytes
predicted fatal outcome in septic patients. These investigators further
showed that inhibition of NF-B, by overexpression of its inhibitor
IB in an endotoxemic mouse model, increased survival
(10). Similarly, Shames and colleagues (38)
showed that high levels of IB diminished cardiac NF-B
activation and attenuated myocardial TNF- production after LPS
challenge. Therefore, NF-B may play a pivotal role in regulating myocardial synthesis and secretion of inflammatory proteins and may
provide a new target for therapeutic intervention.
The purpose of this present study was to examine the cardiac effects of
two strategies which have been shown to inhibit NF-B. If this
transcription factor plays a pivotal role in cardiac myocyte secretion
of inflammatory cytokines and burn-related cardiac dysfunction, then
strategies that inhibit this transcription factor would be expected to
improve postburn cardiac performance. We used both a pharmacological
and a transgenic approach to address this issue. Specifically, the
ability of the proteosome inhibitor
N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) to inhibit
cardiomyocyte secretion of TNF- and to ablate postburn cardiac
dysfunction was examined in C57BL/6J mice. In addition, transgenic mice
that overexpress a nondegradable (nonphosphorylatable) form of IB
(IB
N) specifically in the heart were given burn injury, and
the effects of IB overexpression on myocyte TNF- secretion and
ventricular performance were examined. The data suggest that
therapeutic strategies that specifically target activation of the
nuclear transcription factor NF-B limit burn-mediated cardiomyocyte
secretion of TNF- and improve cardiac contractile performance.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental Animals
All animals were used in accordance with the guidelines established by the University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory committee and in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animals were housed in a temperature-controlled environment with 12:12-h light-dark cycles, and food and water were available at will. Mice were acclimated for 7-10 days before inclusion in the studies described herein.To determine the cardiac effects of pharmacological inhibition of
NF-B, C57BL/6J mice were purchased from Jackson Laboratories (Bar
Harbor, ME). Adult male and female mice weighing 25-30 g were used
for the studies described. To pursue a molecular approach for
inhibiting cardiac NF-B, IBN mice were developed at
University of Texas Southwestern Medical Center at Dallas. Transgenic
mice overexpressed IBN under the control of the
cardiomyocyte-specific -myosin heavy chain promoter exclusively in
the heart but not in other tissues. Overexpression of IBN has
been shown previously to inhibit cardiac NF-B activation after LPS
exposure (23).
Burn Trauma Protocol
All mice were weighed immediately before anesthesia. After adequate anesthesia (methoxyflurane) was achieved, the side and back of each mouse were closely clipped and carefully shaved from the base of the tail to the base of the neck. The animals were then randomly assigned to sham burn or burn groups. In those animals designated for burn trauma, a cutaneous burn injury was produced >40% of the TBSA by applying brass probes (1 × 2 cm with a 3 mm thickness) heated to 100°C in boiling water to the side and back of the animals for 5 s. The TBSA was calculated using murine-specific data (22), and this calculation was verified by removing the animal pelt and measuring the actual burned area. The percentage of burned area was then calculated based on the animal's measured TBSA. The animals designated for sham burn group received identical regimens of anesthesia and handling, but no burn injury was given. After the burn trauma was completed, the mice were given lactated Ringer solution (4 ml · kg
1 · percent
burn1 ip). One-half of the calculated volume of lactated
Ringer was given immediately postburn, and the remaining fluid was
given intraperitoneally 8 h postburn. All animals received
analgesic (0.05 mg/kg im buprenorphine) every 8 h after burn
trauma. The animals were monitored closely for the first 8 h after
burn trauma to determine adequate recovery from the anesthesia, animal
responsiveness to external stimuli, the absence of pain, and the
ability to consume food and water.
Experimental Groups
Molecular approach to inhibiting NF-B.
Genetically engineered mice overexpressing IB in the heart were
randomly divided into sham and burn groups. Appropriate wild-type
groups were included, resulting in the following four experimental
groups: 1) wild-type shams, 2) wild-type mice
given burn injury over 40% TBSA and fluid resuscitation, 3)
IB overexpressing sham-burned mice, and 4) IB
overexpressing mice given burn and fluid resuscitation, as
described for group 2. All mice were euthanized 24 h
postburn, and the hearts were harvested to examine cardiac mechanical
function (Langendorff perfusion, N = 7-8
mice/group), to examine cardiac myocyte secretion of TNF-
(cardiomyocytes isolated from 4-5 mice per group), or to examine
NF-B activation (4 mice per group per time period).
Pharmacological inhibition of NF-B.
C57BL/6J mice were divided into four experimental groups. Group
1 included sham-burned mice receiving the proteosome inhibitor ALLN (20 mg/kg, Sigma; St. Louis, MO) suspended in 0.3 ml of saline and
given intraperitoneally. Group 2 included sham- burned mice receiving vehicle only (0.3 ml saline ip). Group 3 included
mice given burn injury over 40% TBSA, fluid resuscitation (lactated Ringer, 4 ml · kg1 · percent
burn1 ip), and ALLN (20 mg/kg ip as described for
group 1). Group 4 included mice receiving burn
injury and fluid resuscitation as described for group 3, but
treated with vehicle only (0.3 ml ip saline).
Measures of Cardiac Contraction and Relaxation (Langendorff Model)
To examine cardiac contraction and relaxation, mice from each experimental group were anticoagulated with 200 units of heparin sodium (Elkins-Sinn; Cherry Hill, NJ) and cervically dislocated. The heart was rapidly removed and placed in a petri dish containing ice-cold (4°C) Krebs-Henseleit bicarbonate buffered solution composed of (in mM) 118 NaCl, 4.7 KCl, 21 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11 glucose. All solutions were prepared each day with demineralized deionized water and bubbled with 95% O2-5% CO2 (pH 7.4; PO2, 500-540 mmHg; PCO2, 38 mmHg). A cannula placed in the ascending aorta was connected via glass tubing to a buffer-filled reservoir for perfusion of the coronary circulation at a constant flow rate. Hearts were suspended in a temperature-controlled chamber (38°C), and a constant flow pump (Holter model 911, Critikon; Tampa, FL) was used to maintain perfusion of the coronary arteries by retrograde perfusion of the aortic stump cannula as previously described (29). Krebs buffer was passed through a bubble trap and a heating coil before delivery to the aorta. A pressure transducer connected to the pressure tubing between the heart and the heating coil was used to measure coronary perfusion pressure. Coronary effluent was collected in a graduated cylinder and measured to confirm coronary flow rate. Contractile function was assessed by measuring left ventricular (LV) pressure (LVP) and the positive and negative first derivative of pressure over time (±dP/dt) responses to increases in either perfusate calcium or coronary flow. LVP was measured with a Statham pressure transducer (model P23 ID, Gould Instruments; Oxnard, CA) attached to an intraventricular cannula, and ±dP/dt was obtained using an electronic differentiator (model 7P20C; Grass Instruments; Quincy, MA) and recorded (model 7DWL8P; Grass Recording Instruments). Data from the Grass recorder was input to a Dell Pentium computer, and a Grass Poly VIEW Data Acquisition System was used to convert acquired data into digital form.At the time of death, blood was collected from each mouse (0.5-0.6
ml) and placed on ice until centrifugation; plasma samples were then
snap-frozen and stored at 
80°C for measurement of circulating TNF- levels with the use of ELISA kits (Endogen; Woburn, MA) and
using the protocol provided by the manufacturer.
Cardiac NF-B Activation
Nuclear protein extraction.
A modified procedure based on the method of Grabellus et al.
(20) was used. All steps were performed on ice. Hearts
were thawed in the presence of buffer A (10 mM HEPES, pH
7.9; 10 mM KCl, 1.5 mM MgCl2, 0.1% Nonidet P-40, and
protease inhibitors plus 0.5 mM DTT); the proteases included a protease
inhibitor cocktail (Sigma) containing the following: 20 mM
4-(2-aminoethyl)-benzenesulfaonylflouride, 14 µm
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane,
1,300 µM bestatin, 10 µM leupeptin, 3 µM aprotinin, and 10 mM
sodium EDTA. The hearts were allowed to thaw on ice for 20 min and were then suspended in 500 µl of buffer A per heart. The tissue
was then homogenized with the use of a dounce homogenizer by hand. The
cells were then allowed to incubate for 20 min on ice and then
centrifuged at 1,200 g for 5 min at 4°C. The supernatants (cytosolic fraction) were removed and frozen at 80°C. The remaining pellet was then resuspended in 200 µl of ice-cold buffer C
(20 mM HEPES, pH 7.9; 420 mM NaCl, 1.8 mM MgCl2, 0.2 mM
EDTA, pH 8; and 25% glycerol) and protease inhibitor as described
above plus 0.5 mM DTT. The pellets were completely resuspended by being
pipetted and were incubated for 20 min on ice with gentle resuspension of the pellets every 2 min. After incubation, the pellets were centrifuged at full speed for 5 min at 4°C. The supernatant was collected (nuclear fraction) and frozen at 80°C. Protein
concentrations were determined with the use of a protein assay
(Bio-Rad; Hercules, CA). Nuclear and cytosolic extracts were stored at
80°C.
EMSA.
Double-stranded oligonucleotides corresponding to the consensus NF-B
binding site of the -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 units of T4 polynucleotide kinase in 1× forward
buffer (GIBCO-BRL; Gaithersburg, MD), and 30 µCi of
[
-32P] ATP (DuPont NEN; Boston, MA) were incubated at
37°C for 60 min. The reaction was stopped by the addition of 1 µl
of 0.5 mM EDTA. The volume was brought up to 50 µl with the addition
of a buffer composed of 10 mM Tris · HCl (pH 7.5)
10 mM NaCl, and 1 mM EDTA. The labeled probe was separated from
unbound ATP with 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.
Western Blot Analysis
Protein extracts (20 µg), both cytoplasmic and nuclear, harvested as stated in the EMSA assay, were combined with 2 µl of 2× sample-loading buffer (0.063 M Tris · HCl, pH 6.8, 2% SDS, 5% mercaptoethanol, and 10% bromophenol blue). The samples were boiled for 5 min and resolved on a 12.5% polyacrylamide gel. The gels were then transferred to polyvinylidene difluoride membranes (Perkin-Elmer Life Sciences; Boston, MA). Membranes were blocked for 1 h at room temperature in Tris-buffered saline-Tween 20 (TBS-T; 120 mM Tris, pH 7.6, 0.9% NaCl, and 0.05% Tween 20) and 5% dried milk. Blocked membranes were then incubated overnight at 4°C with monoclonal NF-B p65 antibody or
p50 antibody (BD Transduction Laboratories; San Diego, CA) (1:100
dilution) in TBS-T and 5% milk. After being incubated, membranes were
washed for 15 min at room temperature in TBS-T, followed by five 5-min
washes in TBS-T. After the washes, the membranes were incubated for
1 h in secondary antibody (Bio-Rad) diluted to 1:2,500 in TBS-T.
Membranes were then washed as described above and exposed to a mixture
of luminol plus hydrogen peroxide under alkaline conditions
(SuperSignal West Pico, Pierce Endogen; Rockford, IL) for 5 min. The
resulting chemiluminescent reaction was detected by X-ray film.
Experiments were repeated independently three times with groups as
indicated in the results.
Primary Cardiomyocyte Preparation
All pipettes, plates, test tubes, and other equipment used for preparation and culture of cardiomyocytes were sterile. The mice were heparinized and decerebrated, and the heart was removed through a medial sternotomy with the use of sterile techniques. The isolated heart was immediately placed in ice-cold calcium-free Tyrode solution containing (in mM) 136 NaCl, 5.36 KCl, 0.56 MgCl2, 0.33 NaH2OP4, 10 HEPES, and 10 glucose. The aorta was cannulated within 60-80 s, and the excised heart was perfused with calcium-free Tyrode solution using a Langendorff perfusion apparatus. The Tyrode solution was equilibrated with 95% O2-5% CO2 during perfusion of the heart. Perfusion was maintained for 5 min and then continued for an additional 8 min with the use of a collagenase solution that contained 80 ml of calcium-free Tyrode, 20 mg collagenase A (0.05%; Boehringer-Mannheim; Indianapolis, IN), and 2 mg of protease (Polysaccharide XIV, Sigma) with continuous oxygenation. After this enzymatic digestion, the heart was removed from the cannula, and the ventricular tissue was separated from the base of the heart in a petri dish containing Tyrode solution with 100 mM calcium where gentle mincing increased cell dispersion over 5 min. The myocyte suspension was then filtered, and the cells were allowed to settle. The supernatant was removed, and the cells were resuspended in 50 ml Tyrode solution; the rinsing and settling step was repeated three 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, with calcium concentrations of 100 µM, 200 µM, and 1.8 mM. The cell viability was measured; quiescent myocytes with a rodlike shape, clear-formed edges, and clear striations were prepared with a final cell count of 5 × 104 cells/ml (26). All of the reagents used for cardiomyocyte preparations were certified as endotoxin free by the respective manufacturers.Cytokine Secretion by Cardiomyocytes
Myocytes were pipetted into microtiter plates at 5 × 104 cell/well and subsequently challenged with either diluent (Tyrode) or one of several concentrations of LPS (lot no. 6H 4053, Difco Laboratories; Detroit, MI) either 0, 10, 25, or 50 µg/ml; 12 cell preps per LPS concentration were included. Cells were incubated at 37°C in 5% CO2 for 18 h. Supernatants were collected to measure myocyte-secreted TNF- (ELISA, Endogen; Woburn, MA).
Statistical Analysis
All values are expressed as means ± SE. 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 an equality of variance among the four groups was suggested, multiple-comparison procedures were performed (Bonferroni); if inequality of variance was suggested by Levene's test, Tamhane multiple comparisons (which do not assume equal variance in each group) were performed. P < 0.05 values were considered statistically significant (analysis was performed using SPSS for Windows version 7.5.1).| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IB Overexpression or ALLN Administration Ablates
Burn-Mediated NF-B Activation
B nuclear migration
2, 4, and 24 h postburn (Fig.
1A). Cold-competition assay
with 50- and 100-fold excess NF-B oligonucleotide shifted the band, whereas cold competition assay with 40-fold excess AP-1 oligonucleotide had no effect (data not shown). As shown in Fig. 1B, ALLN
administration in burned animals inhibited burn-mediated NF-B
activation at all time periods studied. Similarly, IB overexpression
specifically in cardiomyocytes ablated burn-related NF-B activation
(Fig. 1C); there was no evidence of NF-B nuclear
translocation at any time postburn in the IB overexpressing mice.
Because multiple independent gel shifts were performed on several
animals (N = 4-5) from each experimental group at
each time postburn, the gel shifts were examined by densitometric
analysis and the data are summarized as means ± SE (shown below
each group). Western blots were also performed to confirm NF-B
activation by translocation of p50 and p65 to the nucleus. As shown in
Fig. 2, A and B,
top, representative Western blots are shown, whereas Fig. 2,
A and B, bottom, summarize the data
from multiple Western blot experiments. Translocation of p50 and p65
from the cytosol to the nucleus was confirmed 2, 4, and 24 h after
burn injury in wild-type mice; this translocation of NF-B dimer
components did not occur after burn trauma in ALLN-treated or
IB-overexpressor mice (Fig. 2).
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Cardiomyocyte Secretion of TNF- in IB Transgenic Mice
B overexpression in cardiomyocytes
altered myocyte secretion of TNF-, hearts harvested from each of the
four experimental groups were perfused with collagenase containing
buffer to prepare cardiomyocytes. As shown in Fig. 3, myocytes prepared from wild-type burns
secreted significantly more TNF- (177 ± 16 pg per 5 × 104 myocytes) compared with cytokines secreted by
cardiomyocytes prepared from wild-type sham (70 ± 4 pg per 5 × 104 myocytes, P < 0.05). These data
confirm previous reports (19, 27, 29, 41) showing, in
several species, that burn trauma promotes TNF- secretion by
cardiomyocytes per se. In contrast, cardiomyocytes prepared from
IB overexpressing mice given burn injury secreted significantly
less TNF- (15 ± 1 pg per 5 × 104 myocytes)
compared with that secreted by wild-type burns (177 ± 16 pg per
5 × 104 myocytes) (Fig. 3A).
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In vitro studies examining cardiomyocyte responsiveness to LPS
challenge (either 0, 10, 25, or 50 µg/ml) are shown in Fig. 3B. Myocytes from wild-type shams and burns responded to LPS
challenge with a dose-related increase in TNF- secretion
(P < 0.05). However, myocytes prepared from wild-type
burned mice secreted significantly more TNF- at each LPS dose
compared with responses measured in myocytes from wild-type sham-burned
mice (P < 0.05). In contrast, cardiomyocytes prepared
from IB overexpressing mice given either sham burn or burn injury
secreted minimal TNF- at all LPS doses.
Cardiomyocyte Secretion of Inflammatory Cytokines in ALLN-Treated Animals
Figure 4 summarizes TNF- levels
measured in the supernatants of myocytes prepared from 1)
vehicle-treated shams, 2) vehicle-treated burns,
3) ALLN-treated shams, and 4) ALLN-treated burns.
Cardiomyocyte secretion of TNF- was significantly higher in
vehicle-treated burns (P < 0.05) compared with that
measured in vehicle-treated shams. ALLN treatment in burn trauma
significantly reduced burn-mediated cardiomyocyte secretion of the
inflammatory cytokine (Fig. 4A). In addition, cardiomyocytes
from all four experimental groups were challenged in vitro with 0, 10, or 25 µg of LPS per 5 × 104 myocytes for 18 h.
As shown in Fig. 4B, myocytes from both vehicle-treated shams and vehicle-treated burns responded to LPS with a dose-related increase in TNF-. However, myocytes prepared from vehicle-treated burns secreted significantly more TNF- at all LPS doses compared with the responses measured in myocytes prepared from vehicle-treated shams (P < 0.05). ALLN treatment of burn trauma
significantly attenuated LPS-induced secretion of TNF- by
cardiomyocytes prepared from both burn and sham animals; myocytes from
ALLN-treated animals (both sham and burn) secreted minimal TNF- at
all LPS doses.
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Cardiac Contractile Performance in IB Transgenics and
Wild-Type Littermates
B overexpression in the heart
could alter some aspect of ventricular performance per se. To address
this issue, hearts were isolated from IB transgenic mice and
wild-type littermates in the absence of burn trauma; hearts were
perfused in vitro via Langendorff apparatus. Ventricular performances
in these two groups were compared as either the coronary flow rate or
the perfusate calcium was incrementally increased. LV function measured
in IB transgenic mice and wild-type mice was nearly identical as
coronary flow rate was incrementally increased from 1 to 4 ml/min (Fig.
5). Similarly, there were no significant differences in ventricular responsiveness to increases in perfusate calcium (from 1 to 8 mM) in IB overexpressing mice compared with
wild-type littermates (Fig. 6).
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As shown in Fig. 5, burn trauma in wild-type mice produced significant
cardiac contractile dysfunction as indicated by the reduced LVP and
±dP/dtmax responses to incremental increases in coronary flow rate and compared with responses measured in wild-type shams at each level of coronary flow. In contrast, burn trauma in the
IB overexpressing mice produced significantly less cardiac contractile dysfunction at all levels of coronary flow rate compared with that shown in wild-type burned littermates (Fig. 5). Whereas burn
trauma in wild-type littermates significantly impaired LVP and
+dP/dt responses to increases in perfusate calcium compared with that measured in wild-type shams (Fig. 6), cardiac IB
overexpression provided significant cardioprotection as indicated by
the significantly higher LVP and ±dP/dt at each
concentration of perfusate calcium compared with responses measured in
wild-type burns.
Cardiac Contractile Performance in ALLN-Treated Burns
To further explore the role of NF-B activation in postburn
cardiac contractile dysfunction and myocyte cytokine secretion, the
proteosome inhibitor ALLN was administered immediately after burn
trauma in wild-type mice (both sham and burn). Wild-type shams as well
as wild-type burns given fluid resuscitation plus vehicle (no ALLN
therapy) were included to provide appropriate time-matched controls. As
shown in Fig. 7, cardiac contraction and
relaxation deficits were evident in hearts harvested from mice given
burn injury plus vehicle and compared with function calculated for
sham-burned mice. LVP and ±dP/dtmax increased
significantly in all mice as either coronary flow rate (Fig. 7) or
perfusate calcium (Fig. 8) was increased.
However, all measures of cardiac performance were significantly lower
in vehicle-treated burns at each level of coronary flow rate or at each
level of perfusate calcium compared with values measured in the time
matched shams. In contrast, ALLN given immediately after burn trauma
significantly improved cardiac contractile responsiveness to increases
in either coronary flow rate (Fig. 7) or perfusate calcium (Fig. 8)
compared with those values measured in vehicle-treated burns. Measures of contractile performance (LVP and ±dP/dtmax)
were always higher in ALLN-treated burned mice compared with values
measured in burns given standard fluid resuscitation and vehicle.
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Circulating TNF- Levels
levels, measured 24 h postburn in wild-type mice,
were elevated (32 ± 1 pg/ml) compared with values measured in
wild-type control mice (15.9 ± 0.8 pg/ml, P < 0.05). ALLN administration (20 ± 2 pg/ml) and IB
overexpression (15 ± 1 pg/ml) attenuated the burn-related rise in
circulating TNF-.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Both the pharmacological and the transgenic approach used in this
present study inhibited NF-B activation, confirming previous reports
in this regard (23, 37). In addition, both IB
overexpression in cardiac myocytes and ALLN treatment of burned mice
decreased TNF- secretion by cardiac myocytes and provided
cardioprotection after burn trauma. Overexpression of the nondegradable
form of IB was limited to the cardiac myocytes, and we have shown
previously that this molecular approach prevented LPS-mediated
activation of NF-B in a murine model of sepsis (23).
These data are consistent with previous reports (3, 11,
12) showing that overexpressing nondegradable IB inhibits
NF-B activation. In this regard, Böhrer and colleagues
(10) showed that injection of an IB expression
plasmid in mice reduced LPS-induced NF-B activation in
polymorphonuclear blood cells and improved survival of septic mice.
It was expected that ALLN administration would reduce the burn-related
rise in circulating TNF- levels, confirming that systemic administration of this NF-B inhibitor interrupted TNF- synthesis by several cell populations, including Kupffer's cells, alveolar macrophages, and cardiomyocytes. Overexpression of IB, specifically in cardiomyocytes, was expected to have little effect on TNF- secretion by nonmyocyte cell populations, and the mechanism underlying the significant fall in systemic TNF levels seen late in IB
overexpressor burned mice is unclear.
The transgenic mouse line used in our study was characterized by
expression of a murine nonphosphorylatable nondegradable IB
coding sequence (IBN) driven by the murine -myosin heavy chain promoter. This promoter is steadily activated only in cardiac myocytes, and thus nondegradable IB is constitutively and
exclusively expressed in the heart (21). Our finding of
significant cardiac contractile dysfunction in hearts prepared 24 h after burn trauma in wild-type littermates is consistent with
previous reports (15, 19, 28, 29, 35) of cardiac
contraction and relaxation deficits after thermal trauma in rats,
rabbits, guinea pigs, sheep, and humans. Overexpression of IB in
cardiomyocytes significantly attenuated burn-induced cardiac
contractile dysfunction despite an identical percent of TBSA burn and
identical anesthetic regimens used for all burned animals. Significant
differences in cardiac performance in the burned wild-type mice
compared with the burned IB overexpressing mice could not be
attributed to either differences in in vitro handling of the hearts or
perfusion stabilization times; in addition, heart rates and coronary
flow rates were nearly identical in all burned mice.
Our pharmacological approach to inhibiting burn-mediated cardiac
NF-B activation used a new class of proteasome inhibitor shown
previously to inhibit NF-B activation in several nonmyocyte cell
types (33, 37). Our preliminary studies examined the effects of several doses of ALLN on cardiac function and cardiomyocyte viability (unpublished data). The ALLN dose selected by us was consistent with that reported in adult female Swiss-Webster mice and
was shown by Schow and Jolly (37) to inhibit NF-B
activation by inhibiting IB degradation and inhibiting the
proteolytic processing of p105. In our study, ALLN effectively
inhibited NF-B activation in cardiac tissue, prevented burn-related
TNF- secretion by cardiomyocytes, and improved postburn cardiac performance.
TNF- synthesis and secretion by several cell types such as
macrophages is dependent on NF-B activation. We have shown
previously that cardiomyocytes prepared from either adult rats or
guinea pigs secrete significant TNF- after burn injury (19,
29), and these previous studies have shown that in vitro LPS
challenge in cardiac myocytes exacerbated burn-related secretion of
inflammatory cytokines (29). Data from this present study
show that in vivo administration of ALLN immediately after burn injury
prevented both burn-mediated and LPS-mediated TNF- secretion by
cardiac myocytes, a result that was identical to the finding that
IB overexpression inhibited cardiomyocyte secretion of TNF-.
It could be argued that in vivo administration of ALLN in burned mice
altered several aspects of the postburn inflammatory cascade, downregulating the overall inflammatory response and secondarily improving cardiac performance. However, our studies in mice with IB overexpression exclusively in the heart suggested that the doses of ALLN used in our study specifically targeted TNF- secretion within the myocardium.
We attributed the improved cardiac performance after burn trauma in
ALLN-treated animals and IB overexpressing mice to the decreased
TNF- secretion by cardiomyocytes. The negative inotropic effects of
TNF- are well recognized. Kumar and colleagues (31) and
Yokoyama and colleagues (41) showed significant
cardiodepression with TNF- challenge in isolated ventricular muscle
preps or Langendorff-perfused hearts. These data are consistent with
our previous finding that TNF- added to the perfusate (Langendorff
model) of naive hearts produced significant cardiodepression
(26). That TNF- mediates, in part, the myocardial
depression seen in burn trauma, sepsis, and ischemia
reperfusion has been supported by the finding that anti-TNF-
strategies, which inhibit or neutralize TNF- provide significant
cardioprotection (9, 19, 24, 36, 39, 40). It is likely
that secretion of the inflammatory mediator TNF- by cardiac myocytes
produces local or compartmentalized levels of TNF- that far exceed
those measured in the systemic circulation. In addition, local
synthesis and accumulation of TNF- in the myocardium may not be
rapidly and effectively neutralized by soluble receptors or other
endogenous scavenging mechanisms.
While we have shown previously that NF-B activation is one signaling
event involved in the inflammatory cascade that culminates in
cardiomyocyte secretion of inflammatory cytokines (29), we clearly recognize that other transcription factors (i.e., AP-1) may
also play a role. However, the data presented in this present study
suggest that therapeutic strategies that specifically target some
aspects of the signaling cascade that regulate myocardial secretion of
TNF- may be useful in the treatment of disease and injury that are
characterized by profound cardiac contractile depression.
In summary, data from this present study show that IB
overexpression specifically in cardiomyocytes prevented burn-mediated cardiac NF-B activation, prevented cardiomyocyte secretion of TNF-, and improved burn-mediated cardiac contractile performance. Parallel pharmacological studies confirmed that the proteosome inhibitor ALLN inhibited postburn NF-B nuclear migration in the myocardium, prevented inflammatory cytokine secretion by
cardiomyocytes, and significantly improved myocardial performance.
Targeting specific aspects of the signal transduction mechanisms that
regulate inflammatory cytokine synthesis by specific cell populations
may provide novel and useful therapeutic modalities to treat patient
populations with significant cardiac contraction and relaxation deficits.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grant 2-P50-GM-21681-36.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. W. Horton, Dept. of Surgery, The Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9160 (E-mail: jureta.horton{at}utsouthwestern.edu).
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/ajpheart.00394.2001
Received 11 May 2001; accepted in final form 25 October 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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P < 0.05, significant difference in sham WT versus I
