© 1999 American Heart Association, Inc.
Rapid Communication |
Nuclear Factor-
B Plays an Essential Role in the Late Phase of Ischemic Preconditioning in Conscious Rabbits
From the Experimental Research Laboratory, Division of Cardiology, University of Louisville and Jewish Hospital Heart and Lung Institute, Louisville, Ky.
Correspondence to Roberto Bolli, MD, Division of Cardiology, 3rd Floor ACB, 550 S Jackson St, University of Louisville, Louisville, KY 40292. E-mail rbolli{at}louisville.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Abstract—Although it is recognized that late preconditioning (PC) results from upregulation of cardioprotective genes, the specific transcription factor(s) that govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nuclear factor-
B (NF-B) and to elucidate the mechanisms that
control the activation of NF-B after an ischemic stimulus in
vivo. A total of 152 chronically instrumented, conscious rabbits were
used. A sequence of six 4-minute coronary occlusion/4-minute
reperfusion cycles, which elicits late PC, induced rapid activation of
NF-B, as evidenced by a marked increase in p65 content (+164%;
Western immunoblotting) and NF-B DNA binding
activity (+306%; electrophoretic mobility shift assay) in nuclear
extracts isolated 30 minutes after the last reperfusion. These changes
were attenuated 2 hours after ischemic PC and resolved by 4
hours. Competition and supershift assays confirmed the specificity of
the NF-B DNA complex signals. The mobility of the NF-B DNA
complex was shifted by anti-p65 and anti-p50 antibodies but not by
anti–c-Rel antibodies, indicating that the subunits of NF-B
involved in gene activation after ischemic PC consist of
p65-p50 heterodimers. Pretreatment with the NF-B
inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg IP 15
minutes before ischemic PC) completely blocked the nuclear
translocation and increased DNA binding activity of NF-B. The same
dose of DDTC completely blocked the cardioprotective effects of late PC
against both myocardial stunning and myocardial infarction, indicating
that NF-B activation is essential for the development of this
phenomenon in vivo. The ischemic PC-induced activation of
NF-B was also blocked by pretreatment with
N
-nitro-L-arginine (L-NA), a
nitric oxide synthase (NOS) inhibitor,
N-2-mercaptopropionyl glycine (MPG), a reactive oxygen
species (ROS) scavenger, chelerythrine, a protein kinase C (PKC)
inhibitor, and lavendustin A, a tyrosine kinase
inhibitor (all given at doses previously shown to block
late PC), indicating that ischemic PC activates NF-B
via formation of NO and ROS and activation of PKC- and tyrosine
kinase–dependent signaling pathways. A subcellular redistribution and
increased DNA binding activity of NF-B quantitatively similar to
those induced by ischemic PC could be reproduced
pharmacologically by giving the NO donor diethylenetriamine/NO
(DETA/NO) (at a dose previously shown to elicit late PC), demonstrating
that NO in itself can activate NF-B in the heart. Taken
together, these results provide direct evidence that activation of
NF-B is a critical step in the signal transduction pathway that
underlies the development of the late phase of ischemic PC in
conscious rabbits. The finding that four different pharmacological
manipulations (L-NA, MPG, chelerythrine, and lavendustin A) produced
similar inhibition of NF-B suggests that this transcription factor
is a common downstream pathway through which multiple signals elicited
by ischemic stress (NO, ROS, PKC, tyrosine kinases) act to
induce gene expression. To our knowledge, this is the first
demonstration that NO can promote NF-B activation in the heart, a
finding that identifies a new biological function of NO and may have
important implications for various
pathophysiological conditions in which NO is
involved and for nitrate therapy.
Key Words: inducible nitric oxide synthase • myocardial ischemia/reperfusion • postischemic myocardial dysfunction • peroxynitrite • myocardial adaptation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The late phase of ischemic preconditioning (PC) is a delayed adaptive response that renders the heart relatively resistant to ischemia/reperfusion injury 12 to 24 hours after a brief ischemic stress.1 2 3 4 5 6 7 8 9 10 11 12 13 Recent studies suggest that the initial signals responsible for triggering the development of late PC after the ischemic stimulus (on day 1) involve the generation of nitric oxide (NO)5 6 and reactive oxygen species (ROS),14 15 and the subsequent activation of protein kinase C (PKC)13 16 17 18 and protein tyrosine kinases.19 20 21 In addition, mounting evidence suggests that the cardioprotective effects of late PC observed 24 hours later (on day 2) are due to upregulation of NO synthase (NOS) and, specifically, of the inducible isoform (iNOS).8 11 The facts that late PC requires more than 6 hours to become manifest9 10 and that it is blocked by cycloheximide22 indicate that it is dependent on the synthesis of new proteins. Nevertheless, there is still a major gap in our understanding of the mechanism of late PC: namely, the downstream signaling pathways that transduce the activation of cellular kinases soon after ischemia into increased expression of iNOS (and possibly after genes) 24 hours later remain enigmatic. If de novo protein synthesis is involved, it seems likely that the signaling cascade of late PC would include the activation of one or more transcription factors that direct the expression of the protein(s) responsible for cardioprotection (such as iNOS). The identity (or even the existence) of such factor(s), however, is unknown.
One of the transcription factors that could activate gene
expression in response to ischemic PC is nuclear factor-B
(NF-B). This oxidant-sensitive transcription factor plays a critical
role in the immediate-early activation of a multitude of genes encoding
signaling and defense proteins expressed in response to various
stressful situations and therefore appears to be a general mediator of
cellular responses to stress (reviewed in References 23 and 2423 24 ). The
fact that iNOS mediates late PC8 11 supports the
hypothesis that NF-B may participate in this phenomenon, since it is
well established that the 5' flanking region of the iNOS gene contains
a consensus sequence that binds to NF-B25 and that
activation of NF-B is a central mechanism controlling the induction
of iNOS in several cell types, including cardiac
myocytes.25 26 27 28 However, the role of NF-B in the
delayed myocardial adaptations to ischemic stress remains
poorly understood.
The overall goal of the present study was to test the hypothesis
that the development of late PC is mediated by NF-B and to elucidate
the cellular mechanisms that control the activation of this
transcription factor after an ischemic stimulus in vivo. Four
fundamental specific questions were addressed: (1) Does
ischemic PC activate NF-B? (2) If so, does NF-B
activation play a causative role in the development of late PC, or is
it an epiphenomenon? (3) What is the chemical signal that triggers the
activation of NF-B during ischemic PC? (4) Which cellular
kinase(s) is/are responsible for the ischemic PC-induced
activation of NF-B? To address these issues, a series of molecular
analyses were combined with functional studies in a
well-characterized rabbit model of late
PC.5 6 13 15 17 18 20 21 22 29 All experiments were
performed in conscious animals to obviate the confounding effects of
factors associated with open-chest preparations, such as
anesthesia, surgical trauma, fluctuations in temperature,
elevated catecholamine levels, excessive ROS formation,
release of cytokines, which could interfere with myocardial
stunning and infarction,30 31 32 33 34 35 36 with PC,37 38 39 40
and/or with NF-B.23 24 The results demonstrate, for the
first time, that NF-B plays a necessary role in the development of
the late phase of ischemic PC and that its activation is
modulated by NO, ROS, PKC, and tyrosine kinases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Louisville School of Medicine (Louisville, Ky) and with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health, Publication No. 86-23).
Experimental Preparation
The experimental preparation has been described in detail
previously.5 6 7 8 10 11 13 18 29 Briefly, New Zealand White
male rabbits (2.2±0.1 kg; Myrtles Rabbitry, Thompson Station,
Tenn) were instrumented under sterile conditions with a balloon
occluder around a major branch of the left coronary artery, a
10-MHz pulsed Doppler ultrasonic crystal41 in the
center of the region to be rendered ischemic, and bipolar ECG
leads on the chest wall. All rabbits were allowed to recover for a
minimum of 10 days after surgery. Throughout the experiments, rabbits
were kept in a cage in a quiet, dimly lit room. No antiarrhythmic
agents were given at any time.
Experimental Protocol
The study consisted of three successive phases (A, B, and C)
(Figures 1 through 3
).
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Phase A: Effect of Ischemic PC on NF-B
Rabbits were assigned to 7 groups (Figure 1
). Group I
(control) did not receive any treatment and did not undergo
coronary artery occlusion. The rabbits were given heparin (1000
U IV), after which they were anesthetized with sodium
pentobarbital (50 mg/kg IV) and euthanized with a bolus of KCl. The
heart was immediately excised, and myocardial samples (
0.5 g) were
rapidly removed from the anterior and posterior left
ventricular (LV) wall and stored in liquid nitrogen until
used. Groups II, III, IV, and V underwent a sequence of six 4-minute
coronary occlusions interspersed with 4 minutes of reperfusion
and were euthanized at 30, 60, 120, or 240 minutes after the last
reperfusion, respectively. Myocardial samples were rapidly removed from
the ischemic-reperfused region (whose boundaries had been
marked with sutures at the time of instrumentation) and from the
nonischemic region (posterior LV wall), frozen in liquid
nitrogen, and stored at -140°C until used. In group VI
(diethyldithiocarbamate [DDTC] treatment), rabbits underwent 6
coronary occlusion/reperfusion cycles and received DDTC (150
mg/kg IP) 15 minutes before the first coronary occlusion. The
rabbits were euthanized 30 minutes after the sixth reperfusion. DDTC
(diethyldithiocarbamic acid, sodium salt; Sigma Chemical Co) was
dissolved in normal saline (total volume injected, 10 mL). In group VII
(DDTC pretreatment), rabbits received the same dose of DDTC without
undergoing occlusion/reperfusion cycles and were euthanized at a
corresponding time (89 minutes after DDTC). In both groups VI and VII,
myocardial samples were obtained as described above.
Phase B: Effect of DDTC on Ischemic PC
Studies of Myocardial Stunning
The experimental protocol consisted of 2 or 3 consecutive days
of coronary occlusions (days 1, 2, and 3, respectively). On
each day, the rabbits were subjected to a sequence of six 4-minute
occlusion/4-minute reperfusion cycles (Figure 2). Rabbits were
assigned to 3 groups: group VIII (control), group IX (DDTC treatment),
and group X (DDTC pretreatment). On day 1, group IX underwent 6
occlusion/reperfusion cycles and received DDTC at the same dose that
was used in group VI in phase A (150 mg/kg IP 15 minutes before the
first occlusion), whereas group VIII received no treatment. On day 1,
group X received the same dose of DDTC as group IX without undergoing
coronary occlusion/reperfusion; these rabbits then underwent a
sequence of 6 occlusion/reperfusion cycles on days 2 and 3.
Studies of Myocardial Infarction
On day 2, all rabbits were subjected to a 30-minute
coronary artery occlusion followed by 3 days of reperfusion
(Figure 2). Diazepam was administered 20 minutes before the
onset of ischemia (4 mg/kg IP) to relieve the stress caused by
the coronary occlusion. Rabbits were assigned to 4 groups
(Figure 2). Group XI (control group) underwent the 30-minute
occlusion with no PC and no drug pretreatment on day 1. Group XII (PC
group) was preconditioned with a sequence of six 4-minute
coronary occlusion/4-minute reperfusion cycles 24 hours before
the 30-minute coronary occlusion (day 1). Group XIII (DDTC
treatment) underwent the same protocol as group XII except that the
rabbits received DDTC (150 mg/kg IP) 15 minutes before the first
coronary occlusion on day 1 (this is the same dose that was
used in group VI in phase A). Group XIV (DDTC pretreatment) underwent
the same protocol as group XIII except that the rabbits were not
preconditioned.
Phase C: Effects of L-NA, MPG, Chelerythrine, Lavendustin A, and
DETA-NO on NF-B
Rabbits were assigned to 6 groups (groups XV through XX) (Figure 3). Five of these groups (groups XV through XIX) underwent a
sequence of six 4-minute coronary occlusion/4-minute
reperfusion cycles. Group XV
(N-nitro-L-arginine
[L-NA] group) received an intravenous infusion of L-NA at
a rate of 1.3 mg · kg-1 ·
min-1 for 10 minutes, starting 20 minutes before
and ending 10 minutes before the first coronary occlusion
(total dose, 13 mg/kg; total volume infused, 20 mL). Group XVI
(N-2-mercaptopropionyl glycine [MPG] group) received a
continuous intravenous infusion of MPG (0.42 mg ·
kg-1 · min-1),
beginning 60 minutes before the first coronary occlusion and
ending 30 minutes after the sixth reperfusion (total dose, 56 mg/kg;
total volume infused, 5.4 mL/kg). Group XVII (chelerythrine group)
received an intravenous bolus of chelerythrine (5 mg/kg) 5
minutes before the first coronary occlusion. Group XVIII
(lavendustin A group) received an intravenous bolus of
lavendustin A (1 mg/kg) 10 minutes before the first coronary
occlusion. L-NA (Sigma) was dissolved in normal saline. MPG (Sigma) was
dissolved in sterile water, and the pH was adjusted to 7.5 using
0.1 mmol/L NaOH. Chelerythrine chloride (Research Biomedicals
International) was dissolved in 0.5 mL/kg of DMSO+0.5 mL/kg of normal
saline. Lavendustin A (CalBiochem Co) was dissolved in a 10% (vol/vol)
solution of DMSO in normal saline.
Because both chelerythrine and lavendustin A were dissolved in DMSO, an
additional group of rabbits (group XIX [DMSO control group]) was
studied to examine any possible influence of DMSO itself on NF-B.
Five minutes before the first coronary occlusion, these rabbits
received an intravenous bolus of the vehicle used in group
XVII for chelerythrine (0.5 mL/kg of DMSO+0.5 mL/kg of saline) (Figure 3). (The dose of DMSO used in group XVII to dissolve
chelerythrine [0.5 mL/kg] was higher than that used in group XVIII to
dissolve lavendustin A [0.1 mL/kg]; consequently, group XIX received
the dose used in group XVII). Group XX (diethylenetriamine/NO
[DETA/NO] group) received 4 consecutive intravenous
boluses of DETA/NO (0.1 mg/kg each) every 25 minutes (total dose, 0.4
mg/kg) (Figure 3). DETA/NO (Alexis Corp) was dissolved in PBS
immediately before the infusion; to remove oxygen, the PBS solution was
bubbled with nitrogen for at least 30 minutes before dissolving
DETA/NO.29 All rabbits were euthanized 30 minutes after
the sixth reperfusion (groups XV through XIX) or 30 minutes after the
last bolus of DETA/NO (group XX).
Preparation of Cytosolic Proteins
Cytosolic proteins were prepared using a modification of the
method described by Balligand et al26 and Garcia-Cardena
et al.42 Samples were powdered and homogenized
in homogenization buffer containing 25 mmol/L
Tris-HCl [pH 7.4], 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 1
mmol/L PMSF, 1 mmol/L DTT, 25 µg/mL leupeptin, 25 mmol/L
NaF, and 1 mmol/L
Na3VO4. The
homogenates were centrifuged at 14 000g
for 15 minutes, and the resulting supernatants were collected as
cytosolic proteins for Western blot analysis. Protein contents
were determined by a Bio-Rad protein assay kit.
Preparation of Nuclear Extracts
Nuclear extracts were prepared using a modification of the
method described by Dignam et al.43 The samples were
homogenized in buffer A (10 mmol/L HEPES [pH 7.9],
1.5 mmol/L MgCl2, 10 mmol/L KCl, 1
mmol/L DTT, 25 µg/mL leupeptin, and 1 mmol/L PMSF). After a
10-minute incubation on ice, the samples were centrifuged at
1850g for 10 minutes at 4°C. The pellets were dissolved in
buffer B (buffer A+0.1% Triton X-100), incubated on ice for 10
minutes, and then centrifuged as above. The crude nuclear
pellets were washed once with buffer A and resuspended in buffer C
(20 mmol/L HEPES [pH 7.9], 25% glycerol (vol/vol), 0.42 mol/L
NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L
EDTA, 0.5 mmol/L DTT, and 1 mmol/L PMSF) for 30 minutes at
4°C. Nuclear proteins were recovered after
centrifugation at 25 000g for 30 minutes.
The resulting clear supernatants were dialyzed against 100 volumes of
buffer D (20 mmol/L HEPES [pH 7.9], 4% glycerol, 50 mmol/L
NaCl, 0.5 mmol/L EDTA, 1 mmol/L MgCl2,
0.5 mmol/L PMSF, and 0.5 mmol/L DTT) for 6 hours at 4°C.
The dialysates were centrifuged again at 25 000g
for 30 minutes. The resulting supernatants were designated as the
nuclear protein extracts. The purity of the nuclear extracts was
examined using lactate dehydrogenase (LDH) as a cytosolic marker, and
it was found that 1.8% of total myocardial tissue LDH was present
in the nuclear fraction.
Western Immunoblotting Analysis
The subcellular distribution of the p65 subunit of NF-B was
assessed using standard SDS-PAGE Western immmunoblotting techniques.
Briefly, 70 µg of cytosolic proteins and 60 µg of nuclear proteins
were electrophoresed on a 12% denaturing SDS gel for 2 to 3 hours.
After electrophoresis, the proteins on the gel were electrophoretically
transferred to nitrocellulose membranes (Bio-Rad) overnight at 4°C.
Gel transfer efficiency was carefully recorded by making
photocopies of membranes dyed with reversible Ponceau
staining17 ; gel retention was determined by Coomassie blue
staining as previously described.17 The membranes were
incubated in 5% nonfat dry milk in TBST buffer (10 mmol/L
Tris-HCl [pH 7.2], 0.15 mol/L NaCl, and 0.05% Tween-20) followed by
incubation with specific polyclonal anti–NF-B p65 antibodies (Santa
Cruz Biotechnology, Inc). After extensive rinsing with TBST buffer, the
blots were incubated with HRP-conjugated anti-rabbit secondary
antibodies and developed with the use of an enhanced chemiluminescence
system (ECL kit, Amersham). After washing with TBST buffer, the blots
were air-dried and exposed to Kodak films in x-ray cassettes with
intensifying screens. The p65 signals detected by
immunoblotting and the corresponding records of
Ponceau stains of nitrocellulose membranes were quantitated using an
image scanning densitometer (Personal PI, Molecular Dynamics). As
elaborated previously,17 despite a careful attempt to
achieve equal protein loading in all lanes of the gel, the total
amounts of proteins transferred from each lane to the nitrocellulose
membranes during blotting are rarely identical. Therefore, given the
critical importance of quantitating p65 as accurately as possible, each
p65 signal was normalized to the corresponding Ponceau stain signal
determined by densitometric analysis of the Ponceau stain
record, as previously described.17 To ensure
consistency in the data analysis, the cytosolic and
nuclear fractions of the same sample were run on the same gel (Figure 4).
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Electrophoretic Mobility Shift Assay (EMSA)
A double-stranded 22-mer oligonucleotide with
the sequence 5'-AGT TGA GGG GAC TTT
CCC AGG C-3' (Promega Corp) was end-labeled using
[
-32P]ATP (3000 Ci/mmol, Amersham) and T4
polynucleotide kinase according to the manufacturer's
protocol. This oligonucleotide has the consensus
sequence for NF-B binding, as indicated by underlines. After the
labeling reaction, the 32P-labeled
oligonucleotide was purified with a G-25
Sephadex column (Pharmacia). The binding reactions were
performed in a final volume of 10 µL that contained nuclear proteins
(8 µg), 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 1
mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5
mmol/L DTT, 4% glycerol (vol/vol), and 1 µg Poly(dI-dC) ·
Poly(dI-dC) (Sigma).44 After a 10-minute preincubation at
4°C, the labeled probe (50 000 to 60 000 cpm) was added to the
mixture and incubated for an additional 20 minutes at 22°C. After the
binding reactions, the DNA protein complexes were separated on 4%
nondenaturing polyacrylamide gels in 0.5x Tris borate-EDTA (TBE)
buffer. Gels were vacuum-dried and exposed to x-ray film at -70°C
using an intensifying screen. Specific band intensities were quantified
by an image scanning densitometer, as described above.
Gel Supershift and Competition Assays
Gel supershift assays and competition assays were performed to
ensure that the signal was specific for NF-B. For the supershift
assays, 0.3 µg of either anti-p65 or anti-p50 antibodies (Santa Cruz
Biotechnology, Inc) was added, separately or together, to the reaction
mixtures immediately after addition of radiolabeled probe. For the
competition assays, increasing fold molar excess of unlabeled NF-B
oligonucleotide was added into separate reaction
mixtures.
Measurements of Regional Myocardial Function, Region at Risk, and
Infarct Size
Regional myocardial function was assessed as systolic
thickening fraction using the pulsed Doppler probe, as previously
described.41 The total deficit of systolic wall
thickening (WTh) (an integrative assessment of the overall severity of
myocardial stunning) was calculated by measuring the area comprised
between the systolic WTh-versus-time line and the baseline
(100% line) during the 5-hour recovery phase after the sixth
reperfusion.5 7 8 10 13 18 29 The size of the
ischemic-reperfused region (region at risk) and the size of the
infarct were determined by postmortem perfusion followed by staining
with triphenyltetrazolium chloride, as
previously detailed.6 11 18 29
Statistical Analysis
Data are reported as mean±SEM. In phases A and C, differences
among groups with respect to p65 cytosolic content, p65 nuclear
content, and NF-B DNA binding activity were analyzed using
one-way ANOVA. If the ANOVA showed an overall difference, post hoc
contrasts were performed with Student t tests for either
paired or unpaired data, and the resulting probability values were
adjusted according to the Bonferroni correction.45 In
phase B, data were analyzed by either one-way or two-way
repeated-measures (time and group) ANOVA followed by paired or unpaired
Student t tests, as appropriate, with the Bonferroni
correction.45
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 152 conscious rabbits were used in the present study.
Phase A: Effect of Ischemic PC on NF-B
The goals of phase A were to determine (1) whether
ischemic PC is associated with activation of NF-B and (2)
whether such activation can be blocked by DDTC in vivo. Five rabbits
were assigned to each of the groups studied (groups I through VII). All
of the rabbits completed the protocol successfully.
Effect of Ischemic PC on the Subcellular Distribution
of p65
Figure 4A shows a representative Western
blot analysis of the p65 content in the cytosolic and nuclear
fractions prepared from a control tissue sample (group I) and from
samples taken at serial times after the ischemic PC protocol
(groups II through V). In the control sample, almost all of the p65
protein was found in the cytosolic fraction. After 6
occlusion/reperfusion cycles, however, the content of p65 protein
increased in the nuclear fraction and decreased in the cytosolic
fraction (Figure 4A). Quantitative analysis showed that
the cytosolic content of p65 protein was 83% of
control at 30 minutes, 79% at 60 minutes, and 80% at 120 minutes
(P<0.05 versus control at all time points) and returned to
control levels at 240 minutes (Figure 4B). The nuclear p65
content, on the other hand, increased 2.6-fold versus control at 30
minutes (P<0.05) and 1.7-fold (P<0.05) at 60
minutes after ischemic PC and returned to control levels at 120
minutes (Figure 4C). Thus, the kinetics of the increase in p65
in the nucleus mirrored the kinetics of its decrease in the cytosol,
indicating that the ischemic PC protocol induced a rapid
translocation of this subunit of NF-B from the cytosolic to the
nuclear compartment.
Specificity of NF-B DNA Binding
To determine whether the translocated NF-B was active, we next
tested whether the increased NF-B in the isolated nuclear fraction
could specifically bind to B DNA motifs. Nuclear extracts were
prepared from tissue samples obtained 30 minutes after ischemic
PC (when the translocation was maximal [Figure 4C]) and
analyzed by an EMSA using a 32P-labeled
oligonucleotide probe with a high-affinity B binding
site. As illustrated in Figure 5A, a
shifted band of NF-B DNA complex was observed in nuclear extracts
from postischemic tissues (lane 2) but not in the reaction
medium alone (lane 1). Competition assays were performed to confirm
that the signal was specific for the putative NF-B DNA complex. When
increasing folds of unlabeled NF-B oligonucleotide
(2.5-, 5-, and 10-fold molar excess) were preincubated with the nuclear
extracts before the addition of the labeled probe, the NF-B DNA
complex signal disappeared progressively (Figure 5A, lanes 3
through 5). In contrast, when a 10-fold molar excess of unlabeled
oligonucleotides containing the consensus sequence for
either AP-1 or CREB was incubated with the reaction mixture, the
NF-B DNA complex signal was unaffected (data not shown).
Furthermore, supershift assays demonstrated that when specific anti-p65
or anti-p50 antibodies were added to the EMSA reaction mixture, the
mobility of the NF-B DNA complex was shifted (Figure 5B). A
combination of anti-p65 and anti-p50 antibodies caused further gel
retardation and almost abolished the NF-B DNA band (Figure 5B).
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The results of these competition studies and supershift
analyses clearly demonstrate the presence of NF-B–specific
DNA binding in nuclear extracts of preconditioned
myocardium. Additionally, the results of the supershift
analysis with specific anti-p65 and anti-p50 antibodies
indicate that both the p65 and the p50 subunits of NF-B are involved
in the increased NF-B DNA binding activity after ischemic
PC. This finding, coupled with the results of the Western blot
analysis showing a time-dependent translocation of p65 from the
cytosolic to the nuclear compartment (Figure 4), supports the
conclusion that the subunits of NF-B proteins involved in the
activation of B motifs consist of p65-p50 heterodimers. In contrast
to anti-p65 and anti-p50 antibodies, anti–c-Rel antibodies failed to
shift the mobility of the NF-B DNA complex (data not shown),
suggesting that the c-Rel subunit does not participate in the
activation of B motifs during ischemic PC in this model.
Effect of Ischemic PC on the NF-B DNA Binding
Activity
As illustrated in Figure 6A and 6B,
the NF-B DNA binding activity detected in nuclear extracts increased
4.1-fold at 30 minutes after ischemic PC (group II
[P<0.05 versus control]), remained elevated at 60 minutes
(group III; 1.7-fold [P<0.05]) and at 120 minutes (group
IV; 1.9-fold [P<0.05]), and returned to values not
significantly different from control by 240 minutes (group V). Thus,
ischemic PC was associated with a marked increase in NF-B
DNA binding activity in the nuclear fraction, which resolved within 4
hours from the PC stimulus. In nuclear extracts prepared from the
nonischemic region (posterior LV wall), the NF-B DNA binding
activity did not change appreciably after ischemic PC (Figure 6B).
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Effect of DDTC on NF-B
Administration of DDTC 15 minutes before the 6
occlusion/reperfusion cycles (group VI) prevented the ischemic
PC-induced decrease in p65 in the cytosolic fraction (Figure 7A) and its increase in the nuclear
fraction (Figure 7B), as well as the increase in NF-B DNA
binding activity in the nuclear fraction (Figure 8A and 8B). Thus, this dose of DDTC
effectively abrogated the activation of NF-B elicited by
ischemic PC in conscious rabbits.
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Phase B: Effect of DDTC on Ischemic PC
Having found that ischemic PC activates
NF-B and that this activation can be blocked by DDTC, in phase B, we
determined whether DDTC blocks late PC against stunning and infarction.
The aim of these studies was to establish the functional significance
of NF-B activation during ischemic PC.
Studies of Myocardial Stunning
Exclusions and Postmortem Analysis
Of the 26 rabbits instrumented for the studies of myocardial
stunning, 10 were assigned to the control group (group VIII), 10 to the
DDTC-treated group (group IX), and 6 to the DDTC-pretreated group
(group X). One rabbit in group VIII died of ventricular
fibrillation (during the second occlusion on day 1). One rabbit in
group IX died 4 hours after the sixth reperfusion on day 2. Three
rabbits were excluded because of balloon occluder malfunction (1 in
each group). Therefore, 8 rabbits completed the protocol in group VIII,
8 in group IX, and 5 in group X. The size of the occluded-reperfused
vascular bed was similar in the 3 groups: 0.75±0.06 g (17.4±1.6% of
LV weight) in group VIII, 0.93±0.13 g (18.2±2.1%) in group IX, and
0.80±0.10 g (15.9±1.5%) in group X. Tissue staining with
triphenyltetrazolium chloride confirmed the
absence of infarction in all animals.
Regional Myocardial Function
There were no significant differences among the 3 groups with
respect to heart rate (data not shown) or baseline systolic
thickening fraction (see Figure 9
legend).
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Group VIII (Control Group).
On day 1, thickening fraction remained significantly
(P<0.05) depressed for 4 hours after the sixth reperfusion
and returned to values not significantly different from preocclusion
values by 5 hours (Figure 9). Thus, the sequence of six 4-minute
occlusion/4-minute reperfusion cycles resulted in severe myocardial
stunning that lasted, on average, 4 hours. On days 2 and 3, however,
the recovery of WTh was improved compared with day 1 (Figure 9).
The total deficit of WTh after the sixth reperfusion was 48% and 47%
less on days 2 and 3, respectively, compared with day 1
(P<0.05 for both) (Figure 10). Thus, as
expected,5 7 8 10 13 18 myocardial stunning was
attenuated markedly, and to a similar extent, on days 2 and 3 compared
with day 1 (late PC against stunning).
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Group IX (DDTC-Treated Group).
Unlike the results obtained in control rabbits, in DDTC-treated
rabbits, the recovery of WTh during the 5-hour reperfusion period was
not improved on day 2 compared with day 1 (Figure 9), so that
the total deficit of WTh did not differ significantly between the 2
days (Figure 10). On day 3, the recovery of WTh was markedly
improved and the deficit of WTh markedly reduced compared with day 2
(Figures 9 and 10). Thus, administration of DDTC on day
1 completely blocked the development of late PC on day 2; when DDTC was
not administered (day 2), a full PC effect became apparent on day
3.
Group X (DDTC-Pretreated Group). On day 2, both the recovery
of WTh (Figure 9) and the total deficit of WTh (Figure 10) were similar to the corresponding values measured on day 1
in the control group. Thus, administration of DDTC did not exacerbate
the severity of myocardial stunning 24 hours later, indicating that the
absence of late PC against stunning observed on day 2 in group IX
cannot be ascribed to a delayed deleterious action of DDTC on
myocardial contractility.
Studies of Myocardial Infarction
Exclusions
Of the 55 rabbits instrumented for the studies of myocardial
infarction, 21 were excluded for the reasons specified in the legend to
Figure 11.
|
Region at Risk and Infarct Size
There were no appreciable differences among groups XI, XII, XIII,
and XIV with respect to heart rate or baseline systolic
thickening fraction (data not shown). The weight of the region at risk
did not differ among the 4 groups (0.73±0.13 g [16.3±2.2% of LV
weight]) in group XI; 0.72±0.08 g [16.0±2.0%] in group XII;
0.77±0.08 g [17.5±1.4%] in group XIII; and 0.92±0.16 g
[21.5±2.8%] in group XIV). The average infarct size was 45%
smaller in group XII (ischemic PC group) compared with group XI
(control group) (P<0.05 [Figure 11]), indicating
a late PC effect against myocardial infarction. In contrast, in rabbits
treated with DDTC (group XIII), infarct size was similar to that
measured in controls (Figure 11), indicating that DDTC
abrogated the late PC effect against infarction. In group XIV, infarct
size did not differ significantly from that in controls (Figure 11), indicating that pretreatment with DDTC did not affect the
extent of cell death in nonpreconditioned
myocardium. Analysis of covariance
demonstrated that for any given size of the region at risk, the
resulting infarction was greater in preconditioned rabbits treated with
DDTC than in untreated preconditioned rabbits (data not shown). Thus,
administration of DDTC on day 1 completely blocked the development of
late PC against infarction on day 2, and this effect cannot be ascribed
to delayed deleterious actions of DDTC on infarct size. In keeping with
the infarct size data, the recovery of systolic WTh after the
30-minute occlusion was significantly improved in group XII versus
group XI (3.2±5.7% of baseline versus -15.8±5.1%, respectively, at
72 hours [P<0.05]), and this improvement was abolished by
DDTC (-17.6±3.9% of baseline at 72 hours).
Phase C: Effects of L-NA, MPG, Chelerythrine, Lavendustin A, and
DETA/NO on NF-B
Having found that inhibition of NF-B blocks late PC, in phase
C, we sought to elucidate the cellular mechanisms whereby
ischemia induces activation of NF-B in vivo.
Exclusions
Of the 36 rabbits instrumented for phase C, 6 were assigned to
group XV (L-NA treatment), 6 to group XVI (MPG treatment), 5 to group
XVII (chelerythrine treatment), 5 to group XVIII (lavendustin A
treatment), 6 to group XIX (DMSO control group), and 5 to group XX
(DETA/NO treatment). Two rabbits could not be used because of
malfunction of the balloon occluder (one in group XV and one in group
XIX). One rabbit in group XVI was excluded because of
ventricular fibrillation. Therefore, a total of 5 rabbits
completed the protocol in each group. Two additional rabbits were used
as controls (in addition to the 5 control rabbits in group I), and 1
rabbit was added to the PC group (in addition to the 5 rabbits in group
II).
Subcellular Distribution of p65 and NF-B DNA Binding
Activity
Administration of L-NA, MPG, chelerythrine, or lavendustin A
before the 6 occlusion/reperfusion cycles prevented the
ischemic PC-induced decrease in p65 in the cytosolic fraction
(Figure 7A) and its increase in the nuclear fraction (Figure 7B), as well as the ischemic PC-induced increase in
NF-B DNA binding activity in the nuclear fraction (Figure 8A and 8B). Thus, in the conscious rabbit, the same doses of
L-NA,5 6 MPG,15 chelerythrine,13
and lavendustin A20 that abrogate the cardioprotective
effects of late PC also abrogate the activation of NF-B elicited by
ischemic PC. DMSO (the vehicle used for chelerythrine and
lavendustin A), in itself, had no significant effect (group XIX)
(Figures 7 and 8).
Administration of DETA/NO without ischemia/reperfusion (group
XX) caused a decrease in p65 in the cytosolic fraction (Figure 7A), an increase in p65 in the nuclear fraction (Figure 7B), and an increase in NF-B DNA binding activity in the
nuclear fraction (Figure 8A and 8B), all of which were
comparable to those elicited by ischemic PC in group II. Thus,
the same dose of DETA/NO that induces cardioprotective effects similar
to those of the late phase of ischemic PC29 also
induces a subcellular redistribution and activation of NF-B similar
to that induced by ischemic PC.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is generally agreed that the development of the late phase of ischemic PC involves a cascade of signaling events that culminates in the upregulation of cardioprotective genes.12 46 Although the early events of this cascade have been partially deciphered (eg, formation of NO and ROS,5 6 14 15 29 stimulation of adenosine A1 receptors,3 activation of PKC and tyrosine kinases13 16 17 18 19 20 21 ), little or nothing is known regarding the distal events beyond kinase activation. In particular, the identity of the transcription factor(s) downstream of PKC and tyrosine kinases remains unknown and represents an important unresolved problem.
Salient Findings
The present study provides significant new insights into this
issue. The results reported herein demonstrate that, in the conscious
rabbit, a sequence of six 4-minute occlusion/4-minute reperfusion
cycles, which induces late PC against both myocardial stunning and
myocardial infarction, induces rapid activation of NF-B, as
evidenced by its nuclear translocation and increased DNA binding
activity. Pretreatment with the NF-B inhibitor DDTC
blocks the nuclear translocation and increased DNA binding activity of
NF-B and, at the same time, blocks the cardioprotective effects of
late PC against both myocardial stunning and myocardial infarction,
indicating that activation of NF-B is an essential mechanism whereby
brief ischemia results in delayed cardioprotection. The
ischemic PC-induced activation of NF-B is also blocked by
pretreatment with the NOS inhibitor L-NA, the ROS scavenger
MPG, the PKC inhibitor chelerythrine, and the tyrosine
kinase inhibitor lavendustin A (all given at doses
previously shown to block late PC5 6 13 15 20 ),
demonstrating that the cellular mechanism whereby ischemic PC
activates NF-B involves the formation of NO and ROS and the
activation of PKC- and tyrosine kinase–dependent pathways. The
multiplicity of the signals that modulate NF-B suggests that this is
a common downstream effector in the genesis of late PC. Finally, a
subcellular redistribution and increased DNA binding activity of
NF-B quantitatively similar to those induced by ischemic PC
can be reproduced pharmacologically with the administration of the NO
donor DETA/NO (at doses previously shown to elicit a late PC
effect29 ), demonstrating that NO in itself, in the absence
of the other cellular perturbations associated with ischemia,
can induce NF-B activation in the heart. This last finding reveals
an NO-dependent signaling pathway that was heretofore unrecognized.
Taken together, these results provide direct evidence, for the first
time, that activation of NF-B is a critical step in the signal
transduction pathway that underlies the development of the late phase
of ischemic PC. To our knowledge, this is also the first study
to suggest that NO, ROS, and PKC play an important role in modulating
the response of NF-B to a brief ischemic stress.
Functional Significance of NF-B Activation After Ischemic
PC
In phase B, the functional significance of NF-B activation in
late PC was interrogated using DDTC. DDTC was selected for several
reasons. Dithiocarbamates, such as DDTC, have been shown to be potent
inhibitors of NF-B activation (and iNOS induction) in
various cell types,47 48 49 including cardiac
myocytes.50 51 DDTC is effective at micromolar
concentrations and is considerably more potent than other
compounds.47 Furthermore, dithiocarbamates prevent
activation of NF-B by many different stimuli and in different cell
lines but do not interfere with other transcription factors, such as
AP-1, TRE, oct-1, CRE, and Sp1.47 We found that abrogation
of NF-B activation with DDTC resulted in abrogation of protection 24
hours later, demonstrating that the nuclear translocation and increased
DNA binding activity of NF-B observed after ischemic PC are
not merely an epiphenomenon but rather are an obligatory component of
the signaling cascade that underlies late PC. The fact that inhibition
of NF-B activation resulted in loss of late PC in rabbits subjected
to six 4-minute occlusion/4-minute reperfusion cycles (group IX) as
well as in rabbits subjected to a 30-minute occlusion (group XIII)
indicates that this transcription factor plays a critical role not only
in the delayed protection against mild reversible injury associated
with brief ischemia (myocardial stunning) but also in the
delayed protection against severe irreversible injury associated with
sustained ischemia (myocardial infarction). Therefore,
mobilization of NF-B appears to be involved in the acquisition of
tolerance to ischemia/reperfusion injury in general.
Although NF-B plays an essential role in late PC, little is known
regarding the cellular mechanisms whereby a brief ischemic
stress leads to the nuclear translocation and DNA binding of this
protein. Accordingly, in phase C, we tested the hypothesis that NO,
ROS, PKC, and tyrosine kinases are involved in ischemic
PC-induced NF-B activation.
Role of NO in Ischemic PC-Induced Activation of
NF-B
To test the hypothesis that NO formation is an important mechanism
whereby ischemic PC induces NF-B activation, we examined the
effects of L-NA, a nonselective inhibitor of all three
isoforms of NOS,27 given at the same dose that has
previously been shown to abrogate late PC in this
model.5 6 L-NA completely prevented NF-B activation
(Figures 7 and 8), demonstrating that NO formation is
necessary for ischemic PC to mobilize this transcription
factor. These results are corroborated by those obtained when the NO
donor DETA/NO was administered in the absence of ischemia.
DETA/NO induced nuclear translocation of p65 (Figure 7) and
increased the nuclear NF-B DNA binding activity (Figure 8) in
a manner similar to that observed after ischemia, indicating
that NO in itself can trigger NF-B activation, without the need for
any concomitant stimuli. Thus, increased availability of NO is not only
necessary, but also sufficient to activate NF-B in the heart
of conscious animals. The dose of DETA/NO we used is the same that has
previously been found to induce late PC against both myocardial
stunning and infarction in conscious rabbits.29
Taken together, the results obtained with L-NA and DETA/NO point to a
central role of NO in modulating NF-B in the setting of myocardial
ischemia/reperfusion. To our knowledge, this is the first
demonstration that NO can promote NF-B activation in the heart. This
finding identifies a new biological function of NO and a new mechanism
in the signaling cascade of ischemic PC. Because both NO and
NF-B participate in numerous biological
processes,23 24 27 28 this finding has potentially wide
implications for many cardiovascular conditions besides
myocardial ischemia. Furthermore, the notion that NO donors
(eg, nitroglycerin) induce NF-B activation could
have significant implications for nitrate therapy.
The finding that NO activates NF-B in the heart may appear
surprising, since the opposite effect has been observed in isolated
vascular (noncoronary) endothelial
cells52 53 and in purified preparations of recombinant
NF-B,54 in which NO has been shown to inhibit NF-B.
Because of the many obvious differences (eg, species, cell types,
stimulus for NF-B, and in vitro versus in vivo setting), a direct
comparison is not feasible. Nevertheless, the discordance between these
prior results52 53 54 and the present observations
raises the possibility that the effect of NO on NF-B may differ in
different cell types (eg, endothelial cells versus
cardiac myocytes), particularly in view of the fact that NO has been
reported to activate NF-B in T cells.55 An
alternative hypothesis is that the modulation of NF-B by NO may be
concentration dependent, as suggested by the finding that, in murine
endothelial cells, NO enhances NF-B activation at
low levels but inhibits it at high levels.56 Additional
studies will be necessary to elucidate the cell-type specificity and
concentration dependence of the effects of NO on NF-B.
Role of ROS in Ischemic PC-Induced Activation of
NF-B
NO and ·O2- react
very rapidly to form peroxynitrite (ONOO-),
which then can generate hydroxyl radical (·OH) or another oxidant
with similar reactivity.57 To test the hypothesis that
formation of NO- and
·O2--derived oxidants
(eg, ONOO- and/or ·OH) is an important
mechanism whereby ischemic PC activates NF-B, we
examined whether MPG, a cell-permeant antioxidant, interferes with
NF-B activation. Because MPG reacts avidly with both
ONOO- and ·OH by virtue of its thiol
group,58 and because it blocks DETA/NO-induced late
PC,29 we reasoned that it would be a useful tool to
interrogate the role of NO- and
·O2--derived reactive
species in the activation of NF-B. We used the same dose of MPG that
has been shown to abrogate late PC after an ischemic stimulus
in this model.15 Our results demonstrate that MPG
completely eliminated both the nuclear translocation (Figure 7)
and the increased DNA binding activity (Figure 8) of NF-B
after ischemic PC, indicating that MPG-sensitive oxidants (such
as ONOO- and/or ·OH) play an important
role in ischemic PC-induced activation of NF-B. Accordingly,
on the basis of the present results and of previous
studies,5 6 14 15 29 we propose that brief myocardial
ischemia mobilizes NF-B via generation of NO and
·O2- and subsequent
formation of secondary reactive species, most likely
ONOO- and/or ·OH.
Role of PKC and Tyrosine Kinases in Ischemic PC-Induced
Activation of NF-B
To interrogate the role of PKC in NF-B activation, we examined
the effects of chelerythrine, given at a dose that prevents
translocation of PKC as well as late PC in this
model.13 18 Chelerythrine is a very potent
inhibitor of PKC (IC50 0.7
µmol/L) and reportedly has very high selectivity for PKC compared
with PKA (250:1),
Ca2+/calmodulin-dependent protein
kinase (150:1), and tyrosine kinases (150:1).59 The
finding that chelerythrine completely blocked NF-B activation after
ischemic PC (Figures 7 and 8) indicates that this
phenomenon occurs via a PKC-dependent signaling pathway. To our
knowledge, this is the first evidence that PKC controls the activity of
NF-B during myocardial ischemia.
To test the hypothesis that tyrosine kinases may also be involved in
NF-B activation, we examined the effects of lavendustin A, given at
a dose that was previously found to abrogate late PC in this conscious
rabbit model.20 Lavendustin A is a potent and extremely
selective inhibitor of tyrosine kinases
(IC50 for epidermal growth factor receptor
tyrosine kinase=0.011 µmol/L60 ;
IC50 for pp60src=0.5
µmol/L61 ). Even at concentrations of 100 µmol/L,
lavendustin A does not inhibit PKC, PKA, or
Ca2+/calmodulin-dependent protein
kinase.60 61 The finding that lavendustin A abrogated the
nuclear translocation (Figure 7) as well as the increased DNA
binding activity (Figure 8) of NF-B after ischemic PC
indicates that tyrosine kinases are important modulators of the
activation of NF-B in this setting.
Possible Role of NF-B as the Common Distal Pathway of Late
PC
One of the most striking findings of our study is the remarkable
consistency with which inhibition of NF-B resulted in
abrogation of protection. Every time NF-B activation was blocked,
late PC was also blocked. Five different agents were tested (DDTC,
L-NA, MPG, chelerythrine, and lavendustin A). These agents are totally
unrelated to one another and target different components of the signal
transduction cascade of late PC. They were chosen to interrogate the
signaling elements currently known to be involved in late PC in
conscious rabbits (ie, NO, ROS, PKC, and tyrosine
kinases).5 6 13 15 16 17 18 19 20 21 29 All five agents inhibited
NF-B activation (Figures 7 and 8), and all five agents
consistently blocked the cardioprotective effects of late PC,
as documented by our present findings (Figures 10 and 11) and by previous studies.5 6 13 15 18 20
Therefore, the present study not only demonstrates an essential
role of NF-B in late PC but also strongly suggests that this
transcription regulatory protein is a common downstream pathway through
which multiple signals elicited by ischemic stress (NO, ROS,
PKC, and tyrosine kinases) act to induce gene transcription. Because in
the setting of late PC the activation of PKC is NO
dependent18 and the activation of tyrosine kinases is PKC
dependent,21 62 these signals appear to operate in series,
forming a cascade that culminates in NF-B activation.
Previous Studies of NF-B in Myocardial Ischemia
No previous study has examined the role of NF-B in the late
phase of either ischemic PC or NO donor–induced PC. Two prior
investigations have addressed the effect of myocardial
ischemia/reperfusion on NF-B in vivo.63 64 In
the first study, Chandrasekar and Freeman63 reported in
open-chest rats that a 15-minute coronary occlusion followed by
reperfusion induced an increase in NF-B DNA binding activity and p65
protein levels in total tissue homogenates. Unlike our
findings, a 5-fold increase in p65 content was reported at 15 minutes
of reperfusion.63 In a subsequent study from the same
group,64 DDTC was found to inhibit the activation of
NF-B and the concomitant induction of iNOS. An important difference
between these studies and the present investigation is that we
analyzed NF-B selectively in nuclear extracts rather than in
the total tissue homogenate. Because of this, and because
of the numerous differences between the studies (eg, species,
ischemic protocols, and time points examined), it is difficult
to compare our results with those of Chandrasekar et
al.63 64 Maulik et al65 have recently
reported in isolated rat hearts that ischemic PC (4 cycles of
5-minute ischemia/10-minute reperfusion) activated
NF-B, and that this phenomenon was inhibited by the tyrosine kinase
inhibitor genistein and the p38 mitogen-activated
protein kinase (MAPK) inhibitor SB 203580, suggesting that
NF-B activation is regulated by both tyrosine kinase activity and
p38 MAPK activity.
Conclusions
In summary, the present findings identify a new, important
component of the signal transduction cascade of late PC. Our results
demonstrate that activation of NF-B is required for the development
of late PC against both myocardial stunning and myocardial infarction
in conscious animals, and that this signaling event is controlled by
NO, ROS, PKC, and tyrosine kinases. Thus, the initial signals elicited
by a brief ischemic stress are transduced into protective
changes in gene expression via an NF-B–dependent mechanism. On the
basis of these results and of previous
studies,5 6 13 15 16 17 18 19 20 21 29 we propose a
pathophysiological paradigm in which brief
ischemia causes increased formation of NO and
·O2-, which then leads
to the sequential activation of PKC, tyrosine kinases, NF-B, and
iNOS. Because of the ubiquitous roles of NO, ROS, PKC, tyrosine
kinases, and NF-B, the present results have implications not
only for ischemic PC but also for a variety of other
pathophysiological conditions in which NF-B is
involved. Furthermore, the rapid activation of NF-B observed after
injection of the NO donor DETA/NO may have significant implications for
nitrate therapy.
|
Acknowledgments |
|---|
This study was supported in part by National Institutes of Health (NIH) grants R01 HL-43151 and HL-55757 (to R.B.), by NIH grant R01 GM-48473 (to Y.X.), and by the Medical Research Grant Program of the Jewish Hospital Research Foundation, Louisville, Ky. We gratefully acknowledge Gregg Shirk and Wen-Jian Wu for expert technical assistance and Trudy Keith for expert secretarial assistance.
Received January 6, 1999; accepted March 17, 1999.
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E. Kodani, K. Shinmura, Y.-T. Xuan, H. Takano, J. A. Auchampach, X.-L. Tang, and R. Bolli Cyclooxygenase-2 does not mediate late preconditioning induced by activation of adenosine A1 or A3 receptors Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H959 - H968. [Abstract] [Full Text] [PDF]
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 Y.-T. Xuan, Y. Guo, H. Han, Y. Zhu, and R. Bolli An essential role of the JAK-STAT pathway in ischemic preconditioning PNAS, July 31, 2001; 98(16): 9050 - 9055. [Abstract] [Full Text] [PDF]
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 N. Blondeau, C. Widmann, M. Lazdunski, and C. Heurteaux Activation of the Nuclear Factor-{kappa}B Is a Key Event in Brain Tolerance J. Neurosci., July 1, 2001; 21(13): 4668 - 4677. [Abstract] [Full Text] [PDF]
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 K. Nandagopal, T. M. Dawson, and V. L. Dawson Critical Role for Nitric Oxide Signaling in Cardiac and Neuronal Ischemic Preconditioning and Tolerance J. Pharmacol. Exp. Ther., April 12, 2001; 297(2): 474 - 478. [Abstract] [Full Text]
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T. M. Vondriska, J. B. Klein, and P. Ping Use of functional proteomics to investigate PKC{epsilon}-mediated cardioprotection: the signaling module hypothesis Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1434 - H1441. [Abstract] [Full Text] [PDF]
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C. Li, R. L. Kao, T. Ha, J. Kelley, I. W. Browder, and D. L. Williams Early activation of IKK{beta} during in vivo myocardial ischemia Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1264 - H1271. [Abstract] [Full Text] [PDF]
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 P. Bauer, T. Welbourne, T. Shigematsu, J. Russell, and D. N. Granger Endothelial expression of selectins during endotoxin preconditioning Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2000; 279(6): R2015 - R2021. [Abstract] [Full Text] [PDF]
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A. Lochner, E. Marais, S. Genade, and J. A. Moolman Nitric oxide: a trigger for classic preconditioning? Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2752 - H2765. [Abstract] [Full Text] [PDF]
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R. Bolli The Late Phase of Preconditioning Circ. Res., November 24, 2000; 87(11): 972 - 983. [Abstract] [Full Text] [PDF]
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H. Takano, X.-L. Tang, and R. Bolli Differential role of KATP channels in late preconditioning against myocardial stunning and infarction in rabbits Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2350 - H2359. [Abstract] [Full Text] [PDF]
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Y.-T. Xuan, X.-L. Tang, Y. Qiu, S. Banerjee, H. Takano, H. Han, and R. Bolli Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2360 - H2371. [Abstract] [Full Text] [PDF]
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H. Takano, X.-L. Tang, E. Kodani, and R. Bolli Late preconditioning enhances recovery of myocardial function after infarction in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2372 - H2381. [Abstract] [Full Text] [PDF]
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R. C. X. Li, P. Ping, J. Zhang, W. B. Wead, X. Cao, J. Gao, Y. Zheng, S. Huang, J. Han, and R. Bolli PKCepsilon modulates NF-kappa B and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1679 - H1689. [Abstract] [Full Text] [PDF]
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K. Shinmura, X.-L. Tang, Y. Wang, Y.-T. Xuan, S.-Q. Liu, H. Takano, A. Bhatnagar, and R. Bolli Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits PNAS, August 29, 2000; 97(18): 10197 - 10202. [Abstract] [Full Text] [PDF]
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J. W. Mockridge, A. Punn, D. S. Latchman, M. S. Marber, and R. J. Heads PKC-dependent delayed metabolic preconditioning is independent of transient MAPK activation Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H492 - H501. [Abstract] [Full Text] [PDF]
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G. Valen, G. K Hansson, A. Dumitrescu, and J. Vaage Unstable angina activates myocardial heat shock protein 72, endothelial nitric oxide synthase, and transcription factors NF{kappa}B and AP-1 Cardiovasc Res, July 1, 2000; 47(1): 49 - 56. [Abstract] [Full Text] [PDF]
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J. Sadoshima Cytokine Actions of Angiotensin II Circ. Res., June 23, 2000; 86(12): 1187 - 1189. [Full Text] [PDF]
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A. Dana, A. K. Jonassen, N. Yamashita, and D. M. Yellon Adenosine A1 Receptor Activation Induces Delayed Preconditioning in Rats Mediated by Manganese Superoxide Dismutase Circulation, June 20, 2000; 101(24): 2841 - 2848. [Abstract] [Full Text] [PDF]
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J. Liu, I. Ginis, M. Spatz, and J. M. Hallenbeck Hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF-alpha and ceramide Am J Physiol Cell Physiol, January 1, 2000; 278(1): C144 - C153. [Abstract] [Full Text] [PDF]
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B. Dawn, Y.-T. Xuan, Y. Qiu, H. Takano, X.-L. Tang, P. Ping, S. Banerjee, M. Hill, and R. Bolli Bifunctional Role of Protein Tyrosine Kinases in Late Preconditioning Against Myocardial Stunning in Conscious Rabbits Circ. Res., December 3, 1999; 85(12): 1154 - 1163. [Abstract] [Full Text] [PDF]
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K. Shinmura, X.-L. Tang, H. Takano, M. Hill, and R. Bolli Nitric oxide donors attenuate myocardial stunning in conscious rabbits Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2495 - H2503. [Abstract] [Full Text] [PDF]
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Y. Guo, W. K. Jones, Y.-T. Xuan, X.-L. Tang, W. Bao, W.-J. Wu, H. Han, V. E. Laubach, P. Ping, Z. Yang, et al. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene PNAS, September 28, 1999; 96(20): 11507 - 11512. [Abstract] [Full Text] [PDF]
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P. Ping, J. Zhang, Y.-T. Zheng, R. C. X. Li, B. Dawn, X.-L. Tang, H. Takano, Z. Balafanova, and R. Bolli Demonstration of Selective Protein Kinase C–Dependent Activation of Src and Lck Tyrosine Kinases During Ischemic Preconditioning in Conscious Rabbits Circ. Res., September 17, 1999; 85(6): 542 - 550. [Abstract] [Full Text] [PDF]
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A. Rizvi, X.-L. Tang, Y. Qiu, Y.-T. Xuan, H. Takano, A. K. Jadoon, and R. Bolli Increased protein synthesis is necessary for the development of late preconditioning against myocardial stunning Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H874 - H884. [Abstract] [Full Text] [PDF]
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T. C. Zhao, M. M. Taher, K. C. Valerie, and R. C. Kukreja p38 Triggers Late Preconditioning Elicited by Anisomycin in Heart: Involvement of NF-{kappa}B and iNOS Circ. Res., November 9, 2001; 89(10): 915 - 922. [Abstract] [Full Text] [PDF]
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K. Shinmura, Y.-T. Xuan, X.-L. Tang, E. Kodani, H. Han, Y. Zhu, and R. Bolli Inducible Nitric Oxide Synthase Modulates Cyclooxygenase-2 Activity in the Heart of Conscious Rabbits During the Late Phase of Ischemic Preconditioning Circ. Res., March 22, 2002; 90(5): 602 - 608. [Abstract] [Full Text] [PDF]
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