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B during
ischemia in perfused rat heart
Department of Surgery, James H. Quillen College of Medicine, Mountain Home Veterans Affairs Center, East Tennessee State University, Johnson City, Tennessee 37614
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ABSTRACT |
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 Top
 Abstract
 Introduction
Materials and methods
Results
Discussion
References
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The transcription factor nuclear factor 
B
(NF-B) regulates multiple immediate-early gene expressions involved
in immune and inflammatory responses and cellular defenses.
Ischemia-reperfusion induces many immediate-early gene
expressions, but little is known about the NF-B activation in
myocardium during ischemia and reperfusion. This study
demonstrated that ischemia alone rapidly induced NF-B activation in the myocardium of isolated working rat hearts.
Electrophoretic mobility shift assay showed that NF-B binding
activity significantly increased in the nucleus after 5 min of
ischemia and remained elevated for up to 30 min. Western blot
analysis suggested that the levels of inhibitory IB
protein in
the cytoplasm became markedly decreased at 4, 5, 7.5, and 10 min of
ischemia but were gradually restored following 10 min of
ischemia. Reduction of IB protein in the cytoplasm by
ischemia resulted in NF-B translocation to the nucleus.
Northern blot hybridization showed that IB mRNA levels were not
significantly elevated during myocardial ischemia. Pyrrolidine
dithiocarbamate, an antioxidant, significantly inhibited the loss of
IB protein from the cytoplasm and prevented NF-B binding
activity in the nucleus. Reperfusion following short periods of
ischemia augmented NF-B binding activity in the nucleus
induced by ischemia. The results suggest that early activation
of NF-B induced by ischemia in the myocardium could be a
signal mechanism for controlling and regulating immediate-early gene
expression during ischemia-reperfusion.
inhibitory protein IB; nuclear factor B; reperfusion; antioxidant; reactive oxygen species
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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A NUMBER OF STUDIES HAVE shown that ischemia
and reperfusion induce cytokine gene expression including tumor
necrosis factor- (TNF-), interleukin (IL)-1
, IL-6, IL-8,
interferon-
, and intercellular adhesion molecule-1 (ICAM-1) in
myocardium (18, 22-24, 32, 47). These locally overexpressed
myocardial cytokines may play a critical role in the progression of
myocardial dysfunction, including ischemia-reperfusion injury,
vascular wall remodeling, heart failure, and cardiac hypertrophy (5, 7,
21, 22, 32). Recent evidence suggests that locally produced TNF could also contribute to postischemic myocardial dysfunction via direct depression of contractility and induction of myocyte apoptosis (30).
However, the molecular mechanism for controlling and regulating these
immediate-early gene expressions in myocardium during ischemia and reperfusion has not been well studied.
Nuclear factor B (NF-B) is an ubiquitous inducible transcription
factor that is primarily involved in immune, inflammatory, and stress
responses (1, 2). In the majority of cells, NF-B exists as a latent
cytoplasmic complex bound to the inhibitory IB proteins. In the
family of IB proteins, the most important appear to be IB,
IB, and the newly discovered IB
. Treatment of cells with
various inducers, including lipopolysaccharide (LPS), mitogens,
cytokines, phorbol esters, ultraviolet (UV) radiation, free radicals,
and oxidative stress, causes the IB proteins' phosphorylation,
dissociation from NF-B, and rapid degradation. The released NF-B
translocates to the nucleus, binds to cognate DNA binding sites, and
regulates inducible gene expression. Because all known stimuli of
NF-B activity (1, 2) also induce the formation of transient reactive
oxygen species (ROS) and the activation of NF-B can be blocked by
antioxidants, the ROS may serve as a common messenger mediating the
activation of NF-B. Recently, increased NF-B levels in
postischemic rat myocardium (8) and a time-dependent increase in
NF-B binding activity induced by hypoxia in cultured cardiac cells
(19) have been reported. NF-B may also be involved in the regulation
of ischemic preconditioning of the heart (29). Transfection of NF-B
decoy oligodeoxynucleotides to myocardium significantly reduced the
area of infarction (31) and improved tolerance to
ischemia-reperfusion injury in association with the inhibition
of neutrophil adherence and tissue IL-8 production (36). However, it is
unclear when NF-B is activated and how IB is regulated in
myocardium during ischemia and reperfusion.
In this report we show, for the first time to our knowledge, that short
periods of ischemia alone rapidly diminish IB levels in
the cytoplasm and induce NF-B activation in the nucleus of myocardium using an isolated perfused heart preparation, whereas reperfusion augments the NF-B binding activity. Early activation of
NF-B by ischemia in the myocardium may be responsible for the regulation of immediate-early gene expression during
ischemia-reperfusion. Modulation of NF-B activation could
provide a means of reducing myocardial ischemic injury.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Heart
perfusion. Male Sprague-Dawley rats
(300-350 g) were purchased from a licensed vendor and maintained
in the Animal Care facility of the East Tennessee State University in
accordance with the guidelines of the Principles of
Laboratory Animal Care and the Guide
for the Care and Use of Laboratory Animals. The research protocol was reviewed and approved by the East Tennessee State
University Committee on Animal Care. The rats were heparinized with
heparin sodium (2.5 mg/100 g) and anesthetized with chloral hydrate (36 mg/100 g). The hearts were removed and immediately placed into a beaker
containing cold (2°C) Krebs-Henseleit bicarbonate buffer (in mM:
119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 25 NaHCO3; pH 7.4 at 37°C)
supplemented with 10 mM glucose and equilibrated with 95%
O2-5%
CO2. Within 1 min (45.6 ± 1.4 s)
of the chest incision, the aorta was cannulated and preliminary retrograde (Langendorff) perfusion at a perfusion pressure of 70 mmHg
was begun with 37°C oxygenated Krebs-Henseleit bicarbonate buffer.
During the period of preliminary perfusion, the buffer was not
recirculated and the cannulation of the left atrium was completed. The
hearts were then switched to a working mode. After equilibration (15 min) under stable working conditions, global normothermic
ischemia was initiated by clamping the perfusion tubing for 0, 1, 2, 3, 4, 5, 7.5, 10, 15, and 30 min, respectively, with 6 or 7 hearts for each time point. The perfusion apparatus and technique were
described previously (20, 46), and the myocardial temperature was
maintained at 37°C by keeping the heart within the heart chamber
containing the perfusate. Control hearts were perfused for up to 60 min
after the preequilibration period without interruption of perfusate
flow. To examine the effects of antioxidants on NF-B activation
during ischemia, pyrrolidine dithiocarbamate (PDTC), a potent
inhibitor of NF-B activation, was added to the buffer at a
concentration of 100 µM from the beginning of perfusion. In other
experiments studying the effects of short periods of ischemia
followed by reperfusion on NF-B activation, the rat hearts were
subjected to 2, 5, and 15 min of global normothermic ischemia
followed by 5, 10, 20, and 30 min of reperfusion for each ischemic
duration. Immediately at the completion of the perfusion protocols,
ventricles were frozen with Wollenberger clamps precooled in liquid
nitrogen and pulverized under liquid nitrogen temperature. The powders
of the myocardial samples were extracted for nuclear proteins,
cytoplasmic proteins, and total cellular RNA.
Isolation of nuclear and cytoplasmic
proteins. Nuclear and cytoplasmic proteins were
isolated using a method described previously (13) with modifications
(3, 14). Briefly, ~0.1 g of pulverized myocardial sample was
homogenized in 0.8 ml of ice-cold hypotonic buffer [10 mM HEPES
pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT);
protease inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, aprotinin,
pepstatin, leupeptin (10 µg/ml each); and phosphatase inhibitors: 50 mM NaF, 30 mM -glycerophosphate, 1 mM
Na3VO4,
and 20 mM 
-nitrophenyl phosphate]. The homogenates were
centrifuged for 30 s at 2,000 rpm at 4°C to eliminate any unbroken
tissue. The supernatants were incubated on ice for 20 min, vortexed for
30 s after addition of 50 µl of 10% Nonidet P-40, and then
centrifuged for 1 min at 4°C in an Eppendorf centrifuge. Supernatants containing cytoplasmic proteins were collected and stored
at 
80°C. The pellets, after a single wash with the hypotonic buffer without Nonidet P-40, were suspended in an ice-cold hypertonic salt buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, protease inhibitors, and phosphatase
inhibitors), incubated on ice for 30 min, mixed frequently, and
centrifuged for 15 min at 4°C. The supernatants were collected as
nuclear extracts and stored at 80°C. The concentration of
total proteins in the samples was determined by the Pierce protein
assay reagent (Pierce Chemical, Rockford, IL).
To estimate possible contamination of the nuclear extracts with the cytoplasmic extracts when preparing the nuclear and cytoplasmic proteins, lactate dehydrogenase (LDH) activity was determined by a commercially available kit for the quantitative kinetic determination of LDH activity (Sigma Chemical, St. Louis, MO). Values were expressed as LDH activity units per milligram of protein. To establish that the nuclear extracts contained mainly nuclear proteins, 40 µg of nuclear protein preparations were subjected to Western blot analysis for histone H3, a nuclear protein, with anti-histone H3 antibody (Upstate Biotechnology, Lake Placid, NY).
Electrophoretic mobility shift assay.
NF-B binding activity was performed as described previously (3, 14)
in a 15-µl binding reaction mixture containing 1× binding
buffer [50 µg/ml of double-stranded poly(dI-dC), 10 mM
Tris · HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM
DTT, 1 mM MgCl2, and 10%
glycerol], 15 µg of nuclear proteins, and 35 fmol (~50,000
cpm, Cherenkov counting) of double-stranded NF-B consensus
oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3'),
which was end-labeled with
[-32P]ATP (3,000 Ci/mmol at 10 mCi/ml; Amersham Life Sciences, Arlington Heights, IL)
using T4 polynucleotide kinase (Promega, Madison, WI). The binding
reaction mixture was incubated at room temperature for 20 min and
analyzed by electrophoresis on 5% nondenaturing polyacrylamide gels.
After electrophoresis, the gels were dried by Gel-Drier (Bio-Rad
Laboratories, Hercules, CA) and exposed to Kodak X-ray films at
70 °C. The binding bands were quantified by scanning
densitometry of a Bio-Image Analysis System (Millipore Imaging System,
Ann Arbor, MI). The results for each time point from each group were
expressed as relative integrated intensity compared with the normal
heart group measured in the same batch because the integrated intensity
of group samples from different electrophoretic mobility shift assay
(EMSA) batches would be affected by the half-life of the isotope,
exposure time, and background levels.
Competition experiments were performed to assess the specificity of
NF-B binding activity determined by EMSA. Nuclear extracts from
ischemic myocardium were incubated with unlabeled double-stranded NF-B or AP-II oligonucleotides for 5 min (14). After addition of
[32P]ATP end-labeled
NF-B oligonucleotides into the binding reaction for 20 min, the
reaction mixtures were analyzed by 5% nondenaturing polyacrylamide gel
electrophoresis. Antibody supershift assays were carried out to confirm
that NF-B binding activity in the myocardium contains subunits p50
and p65. Nuclear extracts from ischemic myocardium were incubated with
[32P]ATP end-labeled,
double-stranded NF-B oligonucleotides for 20 min followed by
addition of 2 µl of the appropriate antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) specific to NF-B subunits p50 and p65
into the reaction mixtures. After incubation for 2 h at 4°C, the
protein DNA complexes were resolved by electrophoresis on 5%
nondenaturing polyacrylamide gels. Alternatively, nuclear extracts from
ischemic myocardium were incubated with the appropriate antibodies
for 1 h, and the
[32P]ATP end-labeled
NF-B oligonucleotide probes were then added into the reaction
mixtures. After incubation for 20 min, the reaction mixtures were run
on 5% nondenaturing polyacrylamide gels.
Western blot analysis of
IB in cytoplasm.
Cytoplasmic proteins (40 µg) from each sample were mixed with
2× SDS sample buffer [62 mM Tris (pH 6.8), 10% glycerol,
2% SDS, 5% -mercaptoethanol, 0.003% bromophenol blue],
heated at 95°C for 5 min, and separated by SDS-polyacrylamide gel
electrophoresis. After electrophoresis on 12.5% polyacrylamide gels,
the separated proteins were transferred from the gels onto Hybond
electrochemiluminescence membranes (Amersham) using a Bio-Rad semidry
transfer system (Bio-Rad) for 2 h. The membranes were blocked with 5%
nonfat dry milk in TBS-0.05% Tween 20 for 1 h at room temperature,
washed three times for 10 min each in TBS-0.05% Tween 20, and
incubated with a primary IB antibody (Santa Cruz Biotechnology)
in TBS-0.05% Tween 20 containing 5% nonfat dry milk for 1-2 h at
room temperature. After being washed three times for 10 min each in
TBS-0.05% Tween 20, the membranes were incubated with a second
antibody of peroxidase-conjugated goat anti-rabbit immunoglobulin G
(Sigma) for 1 h at room temperature. After washing, the membranes were
analyzed by the enhanced chemiluminescence system according to the
manufacturer's protocol (Amersham). The IB protein signal was
quantified by scanning densitometry of a Bio-Image Analysis System
(Millipore). The results from each experimental group were expressed as
relative integrated intensity compared with normal hearts measured with
the same batch.
RNA isolation and Northern blot analysis. Total cellular RNA was isolated from rat myocardium using an Ultraspec-II RNA Isolation System (Biotecx Laboratories, Houston, TX). Briefly, pulverized heart samples were homogenized with 2 ml of the Ultraspec RNA solution, and one-fifth of the homogenate volume of chloroform was added. After incubation on ice for 5 min, the lysate was centrifuged at 12,000 g for 15 min at 4°C. Total RNA in the aqueous phase was transferred to a clean tube and precipitated by adding 0.5 volume of isopropanol and 0.05 volume of RNA Tack Resin, mixed by vortex, and centrifuged at 12,000 g for 1 min. The pellets were washed twice in 1 ml of cold-75% ethanol and resuspended in an appropriate volume of dimethyl pyrocarbonate-treated water by vortex. After centrifugation, the supernatant containing purified RNA was transferred to a clean RNase-free tube. The concentration of total RNA isolated was quantified by UV spectrophotometry at 260/280 nm (16).
For Northern blot hybridization, 10 µg of total RNA was denatured for
2 min at 95°C and fractionated by electrophoresis on 1% agarose
gels (SeaKem LE Agarose; FMC Products, Rockland, ME) containing
formaldehyde in 1× MOPS buffer (20 mM MOPS, 5 mM sodium acetate,
and 10 mM EDTA). The gels were washed twice for 20 min each in
10× SSC (1.5 M NaCl, 0.15 M citrate), and the total RNA on the
agarose gels was transferred onto nylon membranes (Schleicher & Schuell, Keene, NH) by capillary blotting and subsequently cross-linked under UV light. The membranes were incubated in prehybridization buffer
(0.5 M
Na2HPO4-NaH2PO4,
pH 7.2, 7% SDS, and 100 µg/ml of denatured herring sperm DNA) for
1-2 h at 65°C (26). After prehybridization, the membranes were
hybridized at 65°C overnight with
[32P]dCTP
(Amersham)-labeled CMV-IB cDNA probe (kindly provided by Dr.
Albert S. Baldwin, University of North Carolina, Chapel Hill, NC) by a
random primer labeling system (Promega). The hybridized membranes were
washed twice in buffer containing 0.1 M
Na2HPO4-NaH2PO4, pH 7.2, and 2% SDS at room temperature for 15 min and twice more in
buffer containing 0.05 M
Na2HPO4-NaH2PO4,
pH 7.2, and 1% SDS at 65°C for 15 min each. The membranes were
exposed to Kodak X-OMAT AR film at 70°C with intensifying
screens for 48 h. The probe was removed from complementary sequences on
the membranes by washing in a solution containing 50% formamide and
2× SSPE buffer (0.3 M NaCl, 0.02 M
NaH2PO4,
0.002 M EDTA) at 65°C for 1 h. The same membranes were rehybridized
with [32P]dCTP-labeled
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA. The GAPDH mRNA
hybridization signal measurements were used as internal standards to
adjust for variations that occurred in preparation or loading of the
total RNA samples (16). The autoradiograms were quantified by scanning
densitometry of a Bio-Image Analysis System (Millipore). The results
for each time point from each group were expressed as relative
integrated intensity compared with the normal heart group measured
under identical conditions.
Statistical analysis. Results are expressed as means ± SE. For tests of significance between the different time points and normal hearts, one-way ANOVA was performed, with P < 0.05 considered to be significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Activation of NF-B by
ischemia. NF-B activation in the nuclear
extracts of myocardium was determined by EMSA after rat hearts were
subjected to various periods of ischemia. NF-B binding
activity was present at very low levels in normal and control
(nonischemic) rat myocardium but significantly increased after the
hearts were subjected to ischemia. NF-B binding activity
rapidly increased (42% at 4 min of ischemia, 66% at 5 min of
ischemia), and the increment persisted to 30 min of
ischemia (Fig.
1A).
NF-B binding activity did not significantly increase when the rat
hearts were subjected to continuous perfusion for 5, 10, and 15 min but
became markedly elevated after 30 and 60 min (Fig.
1B). The data suggest that both
early ischemia and prolonged oxygenated perfusion for more than
30 min induce NF-B activation in rat myocardium in an isolated
working heart preparation.
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To assess specific binding of NF-B in ischemic myocardium, 100-fold
excesses of unlabeled NF-B or AP-II oligonucleotides were added into
the EMSA reaction. Unlabeled NF-B oligonucleotides competed for the
binding proteins in nuclear extracts prepared from ischemic myocardium,
whereas the unrelated AP-II oligonucleotides did not (Fig.
2). To confirm that the predominant protein
complex of NF-B in the ischemic myocardium is composed of p65 and
p50 subunits, antibody supershift assays were performed with polyclonal antibodies recognizing NF-B p65 and p50 subunits. Both antibodies considerably shifted the major ischemia-induced NF-B binding complex (Fig. 2). These results confirm that the activated NF-B in
ischemic rat myocardium contains subunits p65 and p50.
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The possibility that the cytoplasmic extracts contaminated the nuclear extracts was assessed by detecting LDH activity as a marker. The levels of LDH activity in the nuclear extracts were almost 20-fold less than that in the cytoplasmic extracts (3.67 ± 1.71 U/mg nuclear protein vs. 152 ± 42 U/mg cytoplasmic protein, n = 14). To confirm that the nuclear extracts contain primarily nuclear proteins, immunoblot analysis of the nuclear extracts was performed with an antibody specific to histone H3, a nuclear protein. Histone H3 was primarily found to be in the nuclear extracts (n = 14).
Ischemia causes rapid, transient loss of
IB protein in
cytoplasm. Because ischemia rapidly induces
NF-B binding activity in the myocardium, the effects of
ischemia on the dynamics of IB protein in the cytoplasm
were investigated. Hearts were subjected to various periods of
ischemia, and the cytoplasmic extracts were assayed by Western
blot analysis with an antibody specific to IB. Figure
3A shows
that IB levels in the cytoplasm began to decrease after 3 min of
ischemia and were reduced 75.5% at 4 min and 74.4% at 5 min
of ischemia compared with that of normal hearts. After that,
the IB levels in the cytoplasm gradually increased. As shown in
Fig. 3A, IB levels at 5 min of
ischemia rose from 26.6% to 52, 75, and 89% of the normal
levels after ischemia for 10, 15, and 30 min, respectively. In
control (nonischemic) hearts, however, perfusion for 5 min did not
reduce IB levels in the cytoplasm, but the levels dropped ~15%
following 10 and 15 min of perfusion without ischemia.
Significant decrement of IB levels in the cytoplasm was only
found when the nonischemic hearts were continuously perfused for 30 and
60 min (Fig. 3B). The results showed
that although continuous perfusion for 30 min or longer can cause
consistent loss of IB protein from the cytoplasm, only 4 min of
ischemia results in significant, rapid, but transient loss of
IB protein from the cytoplasm. Because the kinetics of NF-B
binding activity in the nuclear extracts parallels the kinetics of
IB decrement in the cytoplasm, the data suggest that the
transient ischemia induced the loss of IB protein from the cytoplasm and translocation of activated NF-B to the nucleus.
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IB mRNA expression
during ischemia. Because IB protein in
the cytoplasm diminished following 4 min of ischemia but began to increase after 10 min of ischemia, IB mRNA levels in
rat myocardium were determined. Total cellular RNA was isolated from each heart, separated by electrophoresis on 1% agarose gels, and subjected to Northern blot analysis for both IB and GAPDH mRNA levels. As shown in Fig.
4A,
IB mRNA levels increased 40 and 64% after 7.5 and 10 min of
ischemia, respectively, and peaked (74%) at 15 min of
ischemia compared with that of the normal hearts. Although
these findings did not achieve statistical significance, IB mRNA
levels did not change in continuously perfused hearts during the entire
perfusion period (Fig. 4B).
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Antioxidant prevents ischemic effects.
To investigate the effects of the antioxidant PDTC on
ischemia-induced loss of IB protein from the cytoplasm
and increased NF-B activity in the nucleus, PDTC was added to the
buffer at a concentration of 100 µM from the beginning of perfusion.
The EMSA results showed that PDTC suppressed the ischemic-induced
activation of NF-B in the nuclear extracts (Fig.
5A).
Western blot analysis demonstrated that PDTC prevented the loss of
IB protein from the cytoplasm of ischemic myocardium (Fig.
5B). PDTC, however, did not alter IB mRNA levels as shown by Northern blot hybridization (Fig. 5C). The results demonstrate that
both IB decrement in the cytoplasm and NF-B activation in the
nucleus, induced by ischemia, can be prevented by this
antioxidant.
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Reperfusion augments NF-B binding
activity induced by ischemia. To investigate
the effects of reperfusion following short periods of ischemia
on NF-B binding activity, rat hearts were subjected to 2, 5, and 15 min of ischemia followed by reperfusion for 5, 10, 20, and 30 min for each ischemic duration. As shown in Fig.
6A,
IB levels in the cytoplasm did not significantly change in
myocardium subjected to 2 min of ischemia followed by reperfusion for 5 min. When the ischemic hearts were subjected to
reperfusion for 10, 20, and 30 min, the IB levels in the cytoplasm were reduced ~32-37%, whereas NF-B binding
activity in the nucleus was increased 18.8-38.8% compared with
those from normal hearts (Fig. 6B).
After 5 min of ischemia and reperfusion for 5, 10, 20, and 30 min, cytoplasmic IB remained at the same low levels as after 5 min of ischemia alone, whereas NF-B binding activity in the
nucleus was increased 65-82.7% compared with those from normal
hearts as shown in Fig. 7. Figure
8A shows
that IB levels in the cytoplasm were restored to normal levels
after ischemia for 15 min followed by reperfusion for 10, 20, and 30 min, but NF-B binding activity in the nucleus was increased
from 158 to 212% after reperfusion for 20 and 30 min compared with
that of normal hearts (Fig. 8B).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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A significant finding of this study is that ischemia alone
rapidly reduces IB levels in the cytoplasm, which results in the
activation and translocation of NF-B to the nucleus in isolated perfused heart. Inflammatory gene expression, including IL-1, TNF-,
inducible nitric oxide synthase, ICAM-1, granulocyte macrophage colony-stimulating factor, IL-6, and IL-8 are controlled by NF-B in
most types of cells. Recent studies have shown that these cytokine gene
expressions are also induced by ischemia and reperfusion in
myocardium (12, 18, 21, 24, 28, 32, 39, 47) and are suggested to be
important factors involved in the depression of cardiac functions,
mediation of remodeling, and cardiac hypertrophy (5, 7, 21, 22, 28,
32). Specific blocking of NF-B activation with NF-B decoy
oligodeoxynucleotides has been shown to improve the recovery of cardiac
function after ischemia-reperfusion insult (31, 36). The
present study suggests that early activation of NF-B by
ischemia could be a potential acute molecular mechanism for
regulating inflammatory cytokine gene expression in the heart. The
other important observation is that continuous perfusion without ischemia for more than 30 min also induced NF-B activation
in isolated perfused rat hearts, which may account for the suppressed myocardial performance during prolonged perfusion observed in our
earlier studies (20, 46). In addition, reperfusion after ischemia only augmented NF-B binding activity in the
nucleus, which was previously induced by short periods of
ischemia, suggesting that induction of increased NF-B
activity in the postischemic myocardium (8) may have been initiated
during the periods of ischemia.
PDTC, a well-known antioxidant, prevented the decrement of IB
levels in the cytoplasm and the activation of NF-B in the nucleus
induced by ischemia. The inhibition of NF-B activation by
PDTC could be due to the prevention of the loss of IB protein from the cytoplasm during ischemia, by suppression of IB
release from the latent cytoplasmic form of NF-B (38) and inhibition of IB degradation (40). Because the inhibitory effects of PDTC on
NF-B activation have been attributed to its antioxidant and
metal-chelating properties, the prevention of the activation of NF-B
in ischemic rat hearts by the antioxidant suggests the involvement of
ROS in the activation of NF-B. It has been demonstrated that the
activation of NF-B by different stimuli can be blocked by
antioxidants (1, 2, 38, 40). Antioxidants and metal chelators prevent
NF-B activation induced not only by oxidizing agents, like hydrogen
peroxide, but also by inducers unrelated to oxidizing agents, such as
inflammatory cytokines, mitogens, protein synthesis inhibitors, and
certain drugs (38). Overexpression of the antioxidative enzyme
thioredoxin can also prevent the activation of NF-B (37). These data
suggest that the activation of NF-B is controlled by ROS and the
intracellular redox state (34) and that multiple signaling pathways
could be involved in the release of ROS as a common signaling moiety.
Both hypoxia-reoxygenation and ischemia-reperfusion can induce
ROS formation and oxidative stress (11), and ROS are hypothesized to be
responsible for myocardial ischemic and reperfusional injury. Our
finding that activation of NF-B by ischemia is involved in
ROS production during ischemia is consistent with a number of
studies reporting increased ROS, including hydroxyl radicals during
myocardial ischemia in isolated hearts (15, 33, 44, 45).
Hydroxyl radicals were generated even during brief (2 min) periods of
ischemia, and the rate of production of hydroxyl radicals
during reperfusion increased in direct proportion to the duration of
ischemia (33). Superoxide and
H2O2
were also generated during ischemia before reperfusion in
isolated cardiomyocytes (45). It has been reported that hydroxyl
radicals probably constitute the ROS responsible for the induction of
NF-B (34).
IB levels in the cytoplasm of rat hearts were rapidly decreased
in response to ischemia for 4-7.5 min but gradually
restored after 10 min of ischemia. Degradation and subsequent
resynthesis of IB have commonly been observed in myeloid,
epithelial, and fibroblast cells stimulated with cytokine, phorbol
myristate acetate (PMA), and LPS (4, 6, 17). The rate of IB
degradation in cytoplasm varied among different types of cells, but
translocation of the released NF-B to the nucleus paralleled the
loss of IB protein from the cytoplasm. In U937 cells, the
restoration of IB occurred within 20-40 min, but replacement
required 1-2 h in HeLa, Jurkat, and THP-1 monocytic cells (4, 6, 10, 41). In the present study, ischemia caused rapid loss of
IB protein from the cytoplasm within 4-7.5 min, whereas
restoration began after 10 min of ischemia. Restoration of
IB in myocardium responding to ischemia was more rapid
than that of other cell types. Because IB may be involved in the
regulation of the multiple NF-B-dependent gene expressions (35, 40),
degradation of IB coupled with the activation of NF-B in the
myocardium during ischemia and consequent restoration of
IB could play important roles in the activation and regulation of
inflammatory cytokine gene expression and/or the mediation of
immediate-early gene expression in the ischemic-reperfused heart (11).
IB mRNA levels were increased ~64% after 10 min of
ischemia and peaked at 74% after 30 min of ischemia.
Although the increased IB mRNA levels did not achieve statistical
significance because of variance among the animals and limited
experimental numbers, the increased mRNA levels could account for the
restoration of IB protein in the cytoplasm after 10 min of
ischemia. It has been demonstrated that activation of NF-B
by various inducers can subsequently upregulate IB mRNA
expression (9, 25), and newly synthesized IB protein replaces the
depleted pool of IB protein in the cytoplasm. Upregulation of
IB mRNA levels by activated NF-B has been confirmed by the
presence of NF-B binding sites in the IB gene promoter (4, 5,
10, 17, 41). Recent studies have suggested the existence of an
autoregulatory feedback of NF-B/IB activation. In this model,
IB controls the NF-B activation, while activated NF-B also
in turn regulates the expression of the IB gene, thus
facilitating an autoregulatory feedback mechanism that serves to
temporarily restrict NF-B activation (6, 41). It has been reported
that NF-B activation has been detected within 15 min of LPS or PMA
stimulation of pre-B cells, whereas the levels of IB mRNA
expression increased after 15 min of stimulation and reached maximum
levels by 60 min (9). Stimulation of U937 monocytes by PMA or TNF-
for 20 min significantly increased IB mRNA expression (6), and
the new IB protein synthesis was associated with a massive
increase in IB mRNA. A recent study (27) has shown that the
IB levels were markedly diminished within 5 min of monocyte
adhesion and rapidly replaced within 20 min thereafter. The
accumulation of IB mRNA was not due to mRNA stabilization, and
the IB gene transcription rate was unchanged. Increased IB
mRNA levels were found in the nuclei, but not in the cytoplasm,
suggesting that a translation-dependent degradation mechanism may
maintain the low levels of IB mRNA in the cytoplasm. Because
immediate-early gene expression has been observed in myocardium
subjected to hypoxia, ischemia, reperfusion, hyperthermia, and
oxidative stress (11), it is possible that the autoregulatory feedback
of NF-B/IB activation in the myocardium during short periods
of ischemia may be involved in the modulation of the expression
of multiple NF-B-dependent immediate-early genes.
A full understanding of the contribution of reperfusion to myocardial
injury after ischemia is still lacking. Whether reperfusion causes further injury to ischemic tissue or simply unmasks irreversible myocardial damage previously induced by ischemia has not been resolved. To investigate the effects of reperfusion following short
periods of ischemia on NF-B activation, 2, 5, and 15 min were chosen as ischemic time points followed by 5, 10, 20, and 30 min
of reperfusion for each ischemic duration. The three ischemic time
points were chosen because IB levels in the cytoplasm did not
change during 2 min of ischemia but dropped to the lowest levels at 5 min of ischemia and were restored to 75% of normal levels after 15 min of ischemia, whereas NF-B binding
activity was not induced at 2 min of ischemia but significantly
increased after 5 min of ischemia, and the increment remained
at 15 min of ischemia. Patterns of IB levels in the
cytoplasm and NF-B binding activity in the nucleus generated by 2 min of ischemia followed by reperfusion were similar to those
of the perfusion group. Interestingly, IB levels in the cytoplasm
after reperfusion following 5 min of ischemia were continually
maintained at the same low levels as 5 min of ischemia alone
but restored to normal levels after reperfusion following 15 min of
ischemia. However, NF-B binding activity in the nucleus was
further increased even if IB levels were restored in the
cytoplasm. Recent studies have proposed that inducers such as LPS or
differentiation signals cause persistent NF-B activation by
affecting IB complexes, which act as chaperones to protect
NF-B from IB, allowing NF-B to translocate to the
nucleus (42, 43). It is possible that the increased ROS generation
during reperfusion (33) and the IB complexes may be involved in
the persistent activation of NF-B during ischemia and reperfusion.
In conclusion, the data presented suggest that short periods of
ischemia rapidly reduce IB levels in the cytoplasm and
increase NF-B binding activity in the nucleus of isolated perfused
rat hearts. Early activation of NF-B in ischemic myocardium could be
a potential acute molecular mechanism for regulating cytokine gene
expression. PDTC, an antioxidant, significantly prevents the loss of
IB protein from the cytoplasm and inhibits NF-B binding
activity in the nucleus. Reperfusion after short periods of
ischemia, however, only augments NF-B binding activity in the nucleus that was previously induced by ischemia. Because
NF-B activation is induced during the early stages of
ischemia, modulation of NF-B activation might prove to be
beneficial for preventing myocardial ischemia and reperfusion injury.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge Dr. Tuanzhu Ha, Dr. Liping Liu, and Janet Davis for technical assistance; Margaret Hatch for secretarial support; and Dr. Donald A. Ferguson, Jr., for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-54286, a Veterans Affairs Merit Review Grant, and a Cardiovascular Research Institute Grant of East Tennessee State University.
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. §1734 solely to indicate this fact.
Address for reprint requests: R. L. Kao, Dept. of Surgery, James H. Quillen College of Medicine, East Tennessee State University, PO Box 70575, Johnson City, TN 37614-0575.
Received 27 May 1998; accepted in final form 13 October 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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