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during in vivo myocardial
ischemia
Departments of 1 Surgery and 2 Internal Medicine, James H. Quillen College of Medicine, 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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We have demonstrated that in vitro brief ischemia
activates nuclear factor (NF)-
B in rat myocardium. We report in vivo
ischemia-reperfusion (I/R)-induced NF-B activation, IB
kinase -
(IKK) activity, and IB
phosphorylation and
degradation in rat myocardium. Rat hearts were subjected to occlusion
of the coronary artery for up to 45 min or occlusion for 15 min
followed by reperfusion for up to 3 h. Cytoplasmic and nuclear
proteins were isolated from ischemic and nonischemic
areas of each heart. NF-B activation was increased in the
ischemic area (680%) after 10 min of ischemia and in
the nonischemic area (350%) after 15 min of ischemia
and remained elevated during prolonged ischemia and
reperfusion. IKK activity was markedly increased in ischemic
(1,800%) and nonischemic (860%) areas, and phosphorylated
IB levels were significantly elevated in ischemic (180%)
and nonischemic (280%) areas at 5 min of ischemia and
further increased after reperfusion. IB levels were decreased in
the ischemic (45%) and nonischemic (36%) areas after
10 min of ischemia and remained low in the ischemic area during prolonged ischemia and reperfusion. The results
suggest that in vivo I/R rapidly induces IKK activity and increases
IB phosphorylation and degradation, resulting in NF-B
activation in the myocardium.
IB phosphorylation; nuclear factor-B; reperfusion
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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INFLAMMATORY
CYTOKINES, such as tumor necrosis factor- (TNF-) and
interleukin (IL)-1, are thought to be important in the pathogenesis
of myocardial ischemia-reperfusion (I/R) injury and heart
failure (4, 5, 10, 14, 19-21, 31, 42, 45). Transgenic
mice that overexpress myocardial TNF- develop cardiomyopathy associated with ventricular dilation, myocyte apoptosis,
transmural myocarditis, and biventricular fibrosis (4, 20,
21). I/R induces overexpression of inflammatory cytokine and
adhesion molecule genes in myocardium (15, 22, 23, 27, 41,
49). However, the factors that regulate inflammatory cytokine
gene expression in the myocardium during I/R have not been delineated.
Nuclear factor-B (NF-B) is a ubiquitous inducible transcription
factor that activates a number of genes, including inflammatory cytokines (1, 2, 32). We previously reported that in vitro I/R significantly induced NF-B activation in the myocardium
(26) and that the antioxidant pyrrolidine dithiocarbamate
(PDTC) (26) and adenosine (27) will prevent
the ischemia-induced NF-B binding activity in isolated rat
hearts. Other investigators have also shown that regulation of NF-B
activation is important in cardiac responses to hypoxia and
ischemia (17, 24, 38, 43, 48). Increased NF-B
binding activity, induced by hypoxia, has been reported in cultured
cardiac cells (17). Inhibition of NF-B activation by
NF-B decoy oligodeoxynucleotides significantly reduced infarct size
(38), improved the functional recovery (43),
and blocked intercellular adhesion molecule-1 (ICAM-1) upregulation by
I/R (24). NF-B may also be involved in the regulation
of ischemic preconditioning of the heart (48). All known stimuli of NF-B activity induce the formation of reactive oxygen species (ROS). Antioxidants can block NF-B activation, suggesting that ROS may serve as a common messenger mediating the
activation of NF-B (1, 2, 32).
Normally, NF-B exists in an inactive cytoplasmic form, bound to the
inhibitory proteins termed IBs. The phosphorylation and degradation
of IB proteins are key steps in NF-B activation, translocation
into the nucleus, and stimulation of gene expression (1, 2,
32). Recently, a high-molecular-mass kinase complex containing
kinase activity specific for the phosphorylation of IB
Ser32 and Ser36 and IB
Ser19 and Ser23 has been documented, and the
two main kinases in this complex, IKK and IKK, have been cloned
(9, 37, 51, 52). NF-B-activating stimuli, such as
inflammatory cytokines (TNF- and IL-1), phorbol 12-myristate
13-acetate, and lipopolysaccharide induce IKK activity (9, 37,
51, 52). Induction of IKK activity during myocardial I/R under
in vivo conditions has not been reported.
Previously, we reported (26) that brief ischemia
alone rapidly induced NF-B activation concomitantly with IB
degradation in isolated rat hearts. We report here, for the first time
to our knowledge, that in vivo ischemia rapidly induces IKK
activity with subsequent IB phosphorylation and degradation,
resulting in NF-B translocation into the nucleus and activation of
gene expression in the myocardium. Early activation of NF-B by in vivo ischemia in the myocardium may be a molecular mechanism
for regulating immediate-early gene expression in response to I/R stimulation. Modulation of the NF-B activation pathway 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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In vivo coronary artery occlusion. Male Sprague-Dawley rats (250-300 g) were purchased from a licensed vendor and maintained in the animal care facility of 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 anesthetized with chloral hydrate (360 mg/kg ip) and ventilated with room air using a rodent ventilator (Harvard Apparatus) with a volume of ~2.5 ml and a rate of 60 cycles/min. The hearts were exposed through a left thoracotomy in the fourth intercostal space. A 6-0 silk ligature was placed under the left anterior descending coronary artery (LAD) and tied using a "shoestring" knot. Myocardial ischemia was confirmed by S-T segment changes and ventricular tachycardia on the electrocardiogram. The ischemic area was readily recognizable by a cyanotic appearance and a bulging region, which was carefully noted as an anatomic landmark. The chest was compressed briefly to expel intrapleural air and closed, leaving one end of the coronary suture protruding from the chest. After completion of a desired period of occlusion, the coronary artery was reperfused by pulling on the exteriorized suture to release the knot.
To study the effect of ischemia on the NF-B activation
pathway in the myocardium, the coronary artery was occluded for 0, 5, 10, 15, 30, and 45 min for a group of hearts. To investigate the effect
of reperfusion on the activation of NF-B in the myocardium, the
hearts were subjected to 15 min of coronary occlusion followed by
reperfusion for 0, 10, 15, 30, 60, and 180 min. Each time point represents five to six hearts. At the end of each time period, the
hearts were immediately harvested and the blood was removed by rinsing
with ice-cold PBS. The right ventricle and atria were trimmed away, and
the left ventricle was divided into ischemic and
nonischemic zones, on the basis of the anatomic landmarks of a
cyanotic and bulging region, before it was frozen in liquid nitrogen
and pulverized at liquid nitrogen temperature. The powders of the
myocardial samples were extracted for nuclear and cytoplasmic proteins.
Isolation of nuclear and cytoplasmic proteins.
The detailed procedures were described previously (26,
27). Briefly, the 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, and 1 mM dithiothreitol (DTT), with
protease inhibitors and phosphatase inhibitors]. After being centrifuged for 30 s at 2,000 rpm at 4°C, the supernatants were incubated on ice for 20 min, vortexed for 30 s after the addition of 50 µl of 10% Nonidet P-40, and then centrifuged for 1 min at 10,000 rpm 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, and 1 mM DTT, with
protease inhibitors and phosphatase inhibitors), incubated on ice
for 30 min, mixed frequently, and centrifuged for 15 min at
14,000 rpm at 4°C. The supernatants were collected as nuclear extracts and stored at 80°C. The concentration of total protein in
the samples was determined by the Pierce bicinchoninic acid protein
assay reagent (Pierce Chemical). By measuring the lactate dehydrogenase
activity as an index for the cytoplasmic proteins and histone H3
proteins as a marker for the nuclear proteins in the sample
preparations, we have confirmed that cytoplasmic extracts contained
primarily cytoplasmic proteins while nuclear extracts consisted
predominantly of nuclear proteins (27).
Electrophoretic mobility shift assay.
NF-B binding activity was determined as described previously
(26, 27) in 15 µl of binding reaction mixture containing 1× binding buffer [50 µg/ml 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 of double-stranded NF-B consensus oligonucleotide
(5'-AGT TGA GGG GAC TTT CCC AGG C-3') end labeled with
[
-32P]ATP (Amersham) using T4 polynucleotide kinase
(Promega). After incubation at room temperature for 20 min, the binding
reaction mixture was analyzed by electrophoresis on 5% nondenaturing
polyacrylamide gels, and the gels were dried by Gel-Drier, scanned, and
quantified by scanning densitometry (Genomic Solutions, 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. We have determined the specific binding of
NF-B in an isolated heart by competition experiments and confirmed that the activated NF-B in the myocardium contains p65 and p50 subunits by antibody supershift assays (26, 27). To
confirm the specific NF-B binding activity in the myocardium
subjected to in vivo ischemia, competition and antibody
supershift assays were performed as described previously (26,
27).
Kinase activity assay.
Approximately 200 µg of cytoplasmic proteins from each sample were
immunoprecipitated with 2 µg of IKK antibody (Santa Cruz) at 4°C
for 1 h. After the addition of 10 µl of protein A-agarose beads
(Santa Cruz) for another 1 h at 4°C, the precipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C. The pellets were washed twice in lysis buffer (1% Nonidet P-40, 5 µg/ml
aprotinin, 250 mM NaCl, 5 µg/ml leupeptin, 50 mM HEPES, pH 7.4, 0.5 µg/ml pepstatin, 1 mM EDTA, 7.5 µg/ml bestatin, 1 mM
phenylmethylsulfonyl fluoride, and 5 µg/ml trypsin inhibitor) and
once in kinase buffer (10 mM HEPES, pH 7.4, 1 mM MnCl2, 5 mM MgCl2, 12.5 mM -glycero-2-phosphate, 50 µM
Na3VO4, 2 mM NaF, 50 µM DTT, and 10 µM ATP)
and resuspended in 15 µl of kinase buffer. Kinase reactions were
performed in the presence of 1 µg of glutathione S-transferase-IB substrate (Santa Cruz) and 5 µCi of
[-32P]ATP (6,000 Ci/mmol; Amersham) at 30°C for 30 min. The reactions were stopped by the addition of 3× Laemmli loading
buffer, and the reaction mixtures were resolved on 15% polyacrylamide
gels. After electrophoresis, the gels were dried by Gel-Drier and
exposed to Kodak X-ray films at 70°C. The phosphorylation of
substrate examined by autoradiography was quantified by scanning
densitometry (Genomic Solutions). The results from each experimental
group were expressed as integrated intensity relative to that of normal hearts measured with the same batch.
Western blot analysis.
Cytoplasmic proteins (40 µg) were mixed with 2× SDS sample buffer,
heated at 95°C for 5 min, and separated by SDS-polyacrylamide (12.5%) gel electrophoresis (26, 27). The separated
proteins were transferred onto Hybond enhanced chemiluminescence
membranes (Amersham) and then incubated with an appropriate rabbit
primary antibody [IB antibody (Santa Cruz Biotechnology) and
phosphorylated IB antibody (New England Biolabs)] in
Tris-buffered saline-0.05% Tween 20 containing 5% nonfat dry milk for
1-2 h at room temperature. After they were washed three times in
Tris-buffered saline-0.05% Tween 20, the membranes were incubated with
peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical) for 1 h at room temperature. After three washes in PBS, the conjugated
peroxidase was visualized by enhanced chemiluminescence according to
the manufacturer's instructions (Amersham). The protein signals of
IB or phosphorylated IB were quantified by scanning
densitometry (Genomic Solutions). The results from each experimental
group were expressed as relative integrated intensity compared with
normal and sham-operated hearts.
Statistical analysis. Values are means ± SE. For tests of significance between the different time points and normal hearts, one-way ANOVA was performed. P < 0.05 was 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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In vivo ischemia induces NF-B binding activity.
We previously demonstrated that ischemia alone rapidly induced
myocardial NF-B activation in isolated rat hearts (26). To investigate whether in vivo ischemia could have the same
effect on myocardial NF-B activation, rat hearts were subjected to
occlusion of the LAD for 0, 5, 10, 15, 30, and 45 min. At each time
point, nuclear proteins were isolated from the ischemic and
nonischemic zones of each heart sample and analyzed for NF-B
binding activity. Ischemia alone significantly induced NF-B
binding activity in ischemic and nonischemic zones of
the myocardium (Fig. 1). In the
ischemic zone, the nuclear NF-B binding activity was not significantly induced by 5 min of ischemia but increased
(680%) after 10 min of LAD occlusion and increased further (1,260%)
after 45 min of ischemia compared with controls. In the
nonischemic zone, the NF-B binding activity was present at
very low levels during initial ischemia but significantly
increased (64, 350, 890, and 1,130%) after 10, 15, 30, and 45 min of
ischemia compared with controls. To investigate the effect of
reperfusion on the activation of NF-B in the myocardium, rat hearts
were subjected to LAD occlusion for 15 min and reperfusion for 15, 30, 60, and 180 min. Figure 2 shows that
NF-B binding activity peaked during early reperfusion (15 and 30 min) and then gradually decreased after prolonged reperfusion (60 and
180 min) in ischemic and nonischemic zones. The data
suggest that in vivo brief ischemia is a potent stimulus for
the activation of NF-B, while reperfusion enhances the
ischemic effects in the myocardium.
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B in ischemic myocardium was
confirmed by addition of 100-fold excesses of unlabeled NF-B or activator protein (AP)-2 oligonucleotides into the electrophoretic mobility shift reaction. Unlabeled NF-B oligonucleotides competed for the binding proteins in nuclear extracts prepared from
ischemic myocardium, whereas the unrelated AP-2
oligonucleotides did not (Fig. 3). The
predominant protein complex of NF-B containing p65 and p50 subunits
in the ischemic myocardium was demonstrated by antibody
supershift assays. Both antibodies considerably shifted the major
ischemia-induced NF-B binding complex (Fig. 3). These results confirm that the activated NF-B in ischemic rat
myocardium contains subunits p65 and p50.
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Ischemia induces IKK activity.
Recent studies have demonstrated that a multisubunit IKK complex, which
contains two interactive catalytic components, IKK and IKK,
mediates specific phosphorylation of IB at Ser32 and
Ser36, resulting in NF-B activation (9, 37,
50). We reasoned that the induction of NF-B
activation by ischemia might be through the activation of IKK.
To test this hypothesis, we examined the effects of I/R on IKK
activity. We chose to examine the IKK activity, because IKK is
primarily responsible for the activation of NF-B in response to
proinflammatory stimuli, whereas IKK is essential for keratinocyte
differentiation (8, 28-30). Figure 4 shows that IKK activity was very low
in normal and sham-operated control hearts but markedly increased in
ischemic (1,800%) and nonischemic (860%) areas after
5 min of ischemia. The enhanced IKK activity persisted up to
45 min of ischemia. After slightly decreasing after 15 min of
reperfusion, the IKK activity further increased in ischemic
(3,800%) and nonischemic (4,800%) areas at 30 min of
reperfusion. The data suggest that I/R-induced NF-B activation is
associated with increasing IKK activity in the myocardium.
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Ischemia rapidly induces myocardial IB
phosphorylation and degradation.
We previously reported that in vitro brief ischemia markedly
decreased IB protein levels in the cytoplasm of isolated rat hearts and that the antioxidant PDTC (26) or adenosine
(27) prevented ischemia-induced decreases in the
cytoplasmic IB protein levels, thereby inhibiting NF-B
activation. Because IKK activity was rapidly induced by I/R (Fig.
4), we further investigated whether I/R will result in the cytoplasmic
IB phosphorylation and degradation. The levels of phosphorylated
IB (phospho-IB) and IB proteins in the cytoplasm
during in vivo myocardial I/R were examined by Western blots. Figure
5 shows that the levels of phosphorylated IB were very low in the normal and sham-operated controls but significantly increased in ischemic (180%) and
nonischemic (280%) areas at 5 min of ischemia. The
elevated phosphorylated IB persisted up to 45 min of
ischemia in ischemic and nonischemic areas.
Reperfusion further increased the levels of phosphorylated IB in
ischemic and nonischemic areas.
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B proteins in the cytoplasm were slightly decreased after 5 min
of ischemia and reduced by 45% in the ischemic zone
after 10 min of ischemia, and the decreased IB levels
persisted up to 30 min of ischemia. The IB levels
returned to control levels after 45 min of ischemia. In the
nonischemic zone, IB protein levels were also decreased 36% after 10 min of ischemia but returned to control levels by 15 min of ischemia. We also investigated the effects of
reperfusion on the levels of cytoplasmic IB protein. Figure
7 shows that the levels of IB
proteins were decreased in the ischemic zone after 30 and 60 min of reperfusion but returned to control levels after reperfusion for
180 min. There was no significant change in the IB levels in the
nonischemic zone during reperfusion (Fig. 7). The data suggest
that NF-B activation in the myocardium induced by in vivo
ischemia is concomitant with increases in IKK activity and
cytoplasmic IB phosphorylation and degradation.
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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 brief in vivo
ischemia is a potent stimulus that rapidly induces IKK
activity and increases IB phosphorylation and degradation in the
cytoplasm, resulting in NF-B translocation and activation in the
nucleus of rat myocardium. Reperfusion enhances the ischemic effects.
The activation of NF-B is thought to be critical in the stimulation
of inducible gene expression, including inflammatory cytokines
(TNF-, IL-1, IL-2, IL-6, IL-8, and interferon-) and adhesion
molecules (vascular cell adhesion molecule-1, ICAM-1, and E-selectin)
(1, 2, 32). The expression of these genes has been
demonstrated to be significantly increased after I/R in the myocardium
(15, 19, 21, 22, 27, 31, 39, 41), and these genes have
been suggested to be factors that might be involved in the depression
of cardiac function, mediation of remodeling, and cardiac hypertrophy
(4, 5, 10, 19-21, 31, 41). Specific blockade of
NF-B activation with NF-B decoy oligodeoxynucleotides has been
shown to reduce infarct size and improve recovery of cardiac function
after I/R insult, which correlated with the inhibition of inflammatory
cytokine gene expression in the myocardium (38, 43). These
data suggest a cause-and-effect relationship between NF-B activation
and myocardial I/R injury. The present study suggests that early
activation of NF-B after a brief ischemia may be a molecular
mechanism for regulating inflammatory cytokine gene expression in the myocardium.
Our results show that NF-B activation after I/R was induced first in
the ischemic area, increased in the nonischemic area, and then gradually decreased during reperfusion up to 3 h. These results were in contrast to an earlier study (6), using
proteins from whole tissue homogenates, which reported that NF-B
activation was not induced in the nonischemic area and that
NF-B activation in the ischemic area increased biphasically
with peaks at 15 min and 3 h of reperfusion. The differences may
be due to the use of different protein extracts to analyze NF-B
activation. In the present study, we used nuclear extracts for the
analysis of nuclear NF-B binding activity. The detection of NF-B
binding changes using whole tissue homogenates will not truly reflect NF-B activation and translocation into the nucleus. This is not only
because NF-B normally exists in the cytoplasm as an inactive form
but also because the levels of NF-B in the cytoplasm may increase
due to the NF-B/IB autoregulatory feedback mechanism (7,
44).
Consistent with our observation that NF-B activation was induced in
the nonischemic zone during I/R, it was recently reported that
in vivo I/R significantly increased P-selectin, E-selectin, and ICAM-1
production on endothelial cells not only in the ischemic area
but also in the nonischemic area in a murine model of
myocardial I/R (16). Also, in a rat model of myocardial
infarction, gene expression of TNF-, IL-1, and IL-6 was
significantly upregulated in the nonischemic region after
coronary occlusion (41). NF-B activation in the
nonischemic area may be responsible for the increased
expression of these genes in the nonischemic region after
coronary occlusion. Several factors may account for this observation.
First, ROS that are rapidly generated during I/R (3, 12, 40, 46,
47) may be responsible for the activation of NF-B in the
nonischemic area. A significant increase in the levels of
hydroxyl radicals in the coronary vein has been observed during
ischemia for 1-10 min (40). A
membrane-permeable free radical scavenger, tempol, has been shown to
significantly reduce infarct size/area at risk (33),
suggesting that the ROS-NF-B activation pathway plays an important
role in the myocardial I/R injury. Second, TNF-, which is preformed
in mast cells and interstitial cells in the heart (11,
35), may also contribute to activation of NF-B, because
TNF- was released when mast cells degranulate during myocardial I/R.
Finally, resident macrophages in the heart (18, 36) and
cardiac myocytes are a major source of inflammatory cytokines,
including TNF- (13, 34). The locally produced TNF-
will be a potent factor for the activation of NF-B during myocardial
I/R.
We previously demonstrated that in vitro ischemia induces
NF-B activation concomitantly with cytoplasmic IB degradation in isolated rat heart (26). Prevention of cytoplasmic
IB degradation by an antioxidant, e.g., PDTC (26),
or adenosine (27) will inhibit NF-B activation induced
by ischemia, suggesting that ischemia-induced NF-B
activation in the myocardium requires the cytoplasmic IB
phosphorylation and degradation. Recently, a multisubunit IB kinase
(IKK) has been identified to be responsible for the inducible
phosphorylation of IB at Ser32 and Ser36,
which appears to be the critical step in NF-B activation induced by
most stimuli. To understand the signaling pathway of
ischemia-induced NF-B in the myocardium, we analyzed
IKK activity and the cytoplasmic phosphorylated IB levels
during myocardial I/R. A brief ischemia markedly induced IKK
activity and increased phosphorylated IB levels, which is
consistent with the data showing that IB levels in the cytoplasm
markedly decreased after 10 min of ischemia (Figs. 5 and 6).
The results suggest that ischemia-induced IKK activity leads
to IB phosphorylation and degradation, with subsequent activation
of NF-B. We have noted that the levels of IKK activity were
similar between 10 min of ischemia and 15 min of reperfusion. This may be due to the IKK autofeedback regulation mechanisms that
reduce its activity; meanwhile, the continuous reperfusion further
stimulates the IKK activity.
IKK contains two catalytic subunits, IKK and IKK, both of which
phosphorylate IB at sites phosphorylated in vivo. We did not
analyze IKK activity, because it has been demonstrated by gene
knockout studies that IKK is primarily responsible for the activation of NF-B in response to proinflammatory stimuli, whereas IKK is essential for keratinocyte differentiation (8,
28-30). We also noted that IKK activity and the levels
of phosphorylated IB in ischemic and nonischemic
zones were markedly increased during prolonged I/R. This finding may
explain why NF-B activation persisted during myocardial I/R, even if
cytoplasmic IB proteins returned to control levels. In the
NF-B activation pathway, IKK autophosphorylation is an important
feedback autoregulatory mechanism for the restriction of NF-B
activation, since persistent NF-B activation can result in
detrimental conditions due to excessive inflammatory cytokine
production (51). In this model, once activated, IKK will
autophosphorylate its COOH-terminal region, and the conformation of the
kinase domain will then be changed, resulting in decreased IKK activity
(51). This transient IKK activation and negative-feedback mechanism is very critical in limiting IKK activity, because small decreases in IKK activity will result in large decreases in IB degradation and NF-B activation (50). A significant
finding in the present study is that persistent IKK activity during
myocardial I/R could be an important molecular mechanism that causes
overproduction of inflammatory cytokines through prolonged NF-B activation.
We have observed that 10 min of ischemia significantly
increased phosphorylated IB levels and reduced IB levels in
ischemic and nonischemic areas. NF-B activation was
significantly increased in the ischemic area and enhanced
(64%) in the nonischemic area compared with sham controls. The
data may suggest that the degree of NF-B activation could be
different in response to various stimuli. NF-B activation in the
ischemic area could be induced by ROS generated during
ischemia, while it may be caused by inflammatory cytokine
(TNF-) released from degraduated mast cells and macrophages in the
nonischemic area. In addition, cells in the ischemic
area are more stressed than those in the nonischemic area,
which may be an additional factor for rapidly increasing NF-B activation.
We have observed that the cytoplasmic IB protein levels, after
rapidly decreasing during early ischemia, gradually returned to
control levels after prolonged ischemia in isolated hearts in
vitro (26, 27). This may be due to NF-B-IB
feedback autoregulation (7, 44). In this model, IB
controls NF-B activation, while activated NF-B in turn promotes
IB gene expression (7, 25). The newly synthesized
IB rapidly replenishes the depleted pool of IB protein in
the cytoplasm to reestablish inactive cytoplasmic NF-B complexes
(7, 44). In the present study, the levels of cytoplasmic
IB protein were decreased in the ischemic zone during
ischemia (10-30 min) and after reperfusion (up to 60 min)
but nearly returned to control levels after 180 min of reperfusion. In
the nonischemic zone, the cytoplasmic IB protein levels,
however, were reduced only after 10 min of ischemia but were
restored after the prolonged ischemia and reperfusion. This may
be due to a difference in the NF-B-IB feedback autoregulation mechanism in the ischemic and nonischemic zones, and it
could be dependent on the tissue conditions during myocardial I/R. For example, there may be more damaged cells in the ischemic zone, which retards the IB protein feedback autoregulation loop.
In summary, the present study suggests that in vivo brief
ischemia rapidly induces IKK activity concomitantly with
increases in the cytoplasmic IB phosphorylation and degradation,
resulting in increasing NF-B binding activity in the nucleus of the
rat myocardium.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of General Medical Sciences Grant GM-53522 (to D. L. Williams), National Heart, Lung, and Blood Institute Grant HL-54286 and a Department of Veterans Affairs Merit Review Grant (to R. L. Kao), American Heart Association Grant 0051480B, and Research Development Committee and Cardiovascular Research Institute grants from East Tennessee State University.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Li, Dept. of Surgery, James H. Quillen College of Medicine, East Tennessee State University, PO Box 70575, Johnson City, TN 37614-0575 (E-mail: Li{at}ETSU.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.
Received 5 May 2000; accepted in final form 13 October 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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