Circulation Research. 1999;85:1164-1172
(Circulation Research. 1999;85:1164.)
© 1999 American Heart Association, Inc.
Differential Regulation of p90 Ribosomal S6 Kinase and Big MitogenActivated Protein Kinase 1 by Ischemia/Reperfusion and Oxidative Stress in Perfused Guinea Pig Hearts
Yasuchika Takeishi,
Jun-ichi Abe,
Jiing-Dwan Lee,
Hisaaki Kawakatsu,
Richard A. Walsh,
Bradford C. Berk
From the Department of Medicine (Y.T., R.A.W.), Case Western Reserve
University, Cleveland, Ohio; Center for Cardiovascular Research (J.-i.A.,
B.C.B.), University of Rochester, Rochester, NY; Department of Immunology
(J.-D.L.), The Scripps Research Institute, La Jolla, Calif; and Lung Biology
Center (H.K.), University of California, San Francisco.
Correspondence to Jun-ichi Abe, MD, PhD, Cardiology Unit, Box 679, 601 Elmwood Ave, Rochester, NY 14642. E-mail jun-ichi_abe{at}urmc.rochester.edu
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Abstract
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AbstractReactive oxygen species
(ROS) activate members
of the Src kinase and
mitogen-activated protein kinase superfamily,
including big
mitogen-activated protein kinase 1 (BMK1) and
extracellular
signal-regulated kinases (ERK1/2). A potentially
important downstream
effector of ERK1/2 is p90 ribosomal S6
kinase (p90RSK), which plays an
important role in cell growth
through the activation of several
transcription factors, as
well as the Na
+/H
+
exchanger. Previously, we showed that Src
regulates BMK1 via a
redox-sensitive signaling pathway. Because
ROS are generated during
ischemia and reperfusion after ischemia,
we assessed
the effects of these stimuli (H
2O
2,
ischemia, and
reperfusion) in the activation of ERK1/2, p90RSK,
Src, and BMK1
in perfused guinea pig hearts.
H
2O
2 (100 µmol/L) significantly
activated
all kinases. Ischemia alone stimulated
p90RSK, Src, and BMK1
but not ERK1/2. These results suggest that p90RSK
activation
through ischemia occurs via a pathway other than
ERK1/2. A role
of Src in ischemia-mediated BMK1 activation was
demonstrated
through inhibition with the Src inhibitor
4-amino-5-(4-chlorophenyl)-7-(
t-butyl)pyrazolo[3,4-
d]pyrimidine.
Reperfusion
after ischemia stimulated both p90RSK and ERK1/2.
In contrast,
although ROS increase during reperfusion after
ischemia, the
activities of both BMK1 and its upstream
regulator, Src, were
markedly attenuated by reperfusion after
ischemia. The activation
of C-terminal Src kinase during
ischemia but not during reperfusion
suggests that the
attenuation of Src and BMK1 activity by reperfusion
was not regulated
by C-terminal Src kinase activity. The antioxidant
N-2-mercaptopropionylglycine
completely inhibited ERK1/2
and p90RSK activation by reperfusion
but only partially inhibited
ischemia-induced Src and BMK1 activation.
The present study
is the first to show the coregulation of Src
and BMK1 by reperfusion
after ischemia, which we propose to
occur via a novel,
ROS-independent pathway.
Key Words: transduction oxidative stress myocardium ischemia
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Introduction
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In ischemic myocardial disease, the heart is
exposed to numerous
cell stresses, including the increased
production of reactive
oxygen species (ROS), ionic imbalances,
osmotic stress, mechanical
stress, and metabolic
deprivation.
1 2 ROS are generated in
the heart
during ischemia (and reperfusion after ischemia) via
several
mechanisms, including mitochondrial uncoupling, xanthine
oxidase,
cytochrome P-450 mono-oxygenase, and NADH/NADPH
oxidase. ROS
stimulate intracellular signal events similar to those
activated
by growth factors and cytokines. ROS
stimulate small G proteins
and kinases such as Src and
mitogen-activated protein kinases
(MAPKs) that lead to the
activation of transcription factors
both in vitro and in the perfused
heart.
3 4 The subsequent
changes in gene expression are
likely to account for many of
the changes in cell function induced by
ROS.
Four subfamilies of MAPKs that are sensitive to ROS have been
identified: extracellular signal-regulated protein kinase (ERK1/2),
c-Jun NH2 terminal kinase (JNK), p38 kinase, and
big MAP kinase 1 (BMK1, or ERK5).1 Each subfamily may be
regulated via different signal transduction pathways and modulate
specific cell functions.5 ERK1/2 is activated by
an upstream kinase (MAPK kinase 1, or MEK1) via dual
phosphorylation of the TEY motif, whereas JNK and p38
kinases are activated by MEK4/7 and MEK3/6 via
phosphorylation of the TPY and TGY motifs,
respectively. BMK1 is a recently identified MAPK family member that
shares the TEY activation motif with ERK1/2 but is activated by
MAPK kinase 5 (MEK5). We have shown that BMK1 is very strongly
activated by ROS and that ROS-mediated BMK1 activation requires
Src in cultured fibroblasts.6 7
Increased oxidative stress due to increased ROS generation, a relative
deficit in the endogenous antioxidant reserve, or both can
cause cardiac contractile depression.8 Importantly, Dhalla
et al9 reported that oxidative stress is one of the
contributing factors in the transition from compensated
hypertrophy to decompensated heart failure. It is
established that ischemia and reperfusion produce ROS in the
heart,2 10 but the signal transduction mechanisms via
which ischemia and reperfusion alter cardiac function remain
unclear.
There is a growing body of evidence for a key role of the plasma
membrane Na+/H+ exchanger
isoform 1 (NHE-1) in the pathophysiology of cardiac ischemia
and reperfusion.11 Several investigators have shown that
H2O2 stimulates both ERK1/2
and NHE-1 in neonatal cardiac myocytes.12 Because
phosphorylation regulates NHE-1 exchanger
activity13 14 and the inhibition of ERK1/2 decreases NHE-1
activity,14 it appears likely that the ERK1/2 pathway
regulates NHE-1. Recently, we showed that p90 ribosomal S6 kinase
(p90RSK), one of the downstream regulators of ERK1/2, is a
serum-stimulated NHE-1 kinase.15 However, we have
recently found that p90RSK is strongly activated by ROS in an
ERK1/2-independent manner in cultured fibroblasts and Jurkat T
cells,16 suggesting that other MAPKs may be required, such
as BMK1. In fact, we demonstrated that ROS-mediated BMK1 activation
requires Src.7 Thus, to define the relative roles of BMK1
and p90RSK in ischemia and ischemia/reperfusion, we
compared the effects of
H2O2 and
ischemia/reperfusion on BMK1 and p90RSK activity in the
perfused heart. We found that ischemia activates p90RSK
and BMK1 but, surprisingly, not ERK1/2. On reperfusion after
ischemia, both p90RSK and ERK1/2 are activated, but
BMK1 was inhibited by ischemia/reperfusion. The antioxidant
N-2-mercaptopropionyl glycine (MPG) completely inhibited
ERK1/2 and p90RSK activation by reperfusion but only partially
inhibited ischemia-induced Src and BMK1 activation. These
results demonstrate a new ischemia-sensitive mechanism
responsible for the activation of Src and BMK1 that is not mediated by
ROS.
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Materials and Methods
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Heart Perfusion
Adult male Charles River guinea pigs were anesthetized
with
54 mg/kg ketamine IP, 1.8 mg/kg acepromazine IP, and 10.9
mg/kg
xylazine IP and heparinized. Hearts were quickly excised and
perfused
according to the Langendorff method with a modified
Krebs-Henseleit
buffer containing (in mmol/L) NaCl 113.8, KCl 4.7,
MgSO
4 1.1,
KH
2PO
4 0.12,
NaHCO
3 23.6, CaCl
2 2.5,
mannitol 6.0, and glucose 11.0,
as previously reported.
17
The solution was saturated with 95%
O
2/5%
CO
2 (pH 7.4) at 37°C. A saline-filled latex
balloon
attached to a 3F micromanometer catheter
(Millar Instruments)
was inserted into the left ventricle through the
mitral valve
for pressure measurements. The balloon was inflated to
achieve
10 mm Hg initial minimum diastolic pressure
and was kept isovolumic
during the perfusion. The coronary flow
rate was adjusted to
10 mL · min
-1
· g net heart wt
-1 and was kept
constant
throughout the experiment. Atrial pacing was carried
out at 200 bpm,
except for the period of global ischemia and
the first 3
minutes of reperfusion thereafter.
Experimental Protocols
All hearts were allowed to equilibrate for
20 minutes before
the protocols were begun. The animals were assigned to protocols as
summarized in Figure 1
. Three guinea pigs
perfused with Krebs-Henseleit buffer alone were used as control
animals. The guinea pig hearts were subjected to 10, 20, 30, or 40
minutes of ischemia alone or to 10 or 20 minutes of reperfusion
after 20 minutes of ischemia (n=3 for each group).
Ischemia was induced through the suspension of circulation of
the perfusion pump. The guinea pig heart is very sensitive to
ischemia, and ischemia for >30 minutes prevented
recovery of the heart from ischemia. Therefore, we used a
20-minute ischemic period for the reperfusion studies. Three
guinea pig hearts were perfused with 100 µmol/L
H2O2 for 20 minutes. After
the equilibration period, 300 µmol/L MPG was added to the
perfusate and infused for 20 minutes before ischemia
(n=3) and ischemia/reperfusion (n=3). The dose and perfusion
time with MPG were chosen on the basis of previous
reports.18 19 A dose of 10 µmol/L
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
(PP2; Calbiochem), a selective inhibitor of the Src family
of tyrosine kinases, was infused for 20 minutes before ischemia
(n=3).

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Figure 1. Experimental study protocol. Open boxes
represent periods of global ischemia. Kinase assays
were performed at times indicated by arrows.
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Immunoprecipitation and Western Blot Analysis
After the completion of perfusion, the left ventricle was
quickly freeze-clamped with a liquid nitrogenprecooled Wollenberger
clamp, powdered in liquid nitrogen, and stored at
-80°C.20 After treatment, heart powders were
homogenized with 4 vol of lysis buffer (50 mmol/L
sodium pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 5
mmol/L EDTA, 5 mmol/L EGTA, 100 µmol/L
Na3VO4, 10 mmol/L
HEPES, pH 7.4, 1% Triton X-100, 0.1% SDS, 500 µmol/L PMSF, and
10 µg/mL leupeptin). The heart homogenates were
centrifuged at 14 000g (4°C for 30 minutes), and
protein concentration was determined with the use of the Bradford
protein assay (Bio-Rad). For immunoprecipitation, cell lysates were
incubated with rabbit anti-BMK1 or C-terminal Src kinase (Csk)
(Santa Cruz Biotechnology) antibody for 12 hours at 4°C and then
incubated with 20 µL of protein A/Sepharose CL-4B (Pharmacia Biotech)
for 1 hour on a roller system at 4°C. The beads were washed 2 times
with 1 mL lysis buffer, 2 times with 1 mL LiCl wash buffer (500
mmol/L LiCl, 100 mmol/L Tris-Cl, pH 7.6, 0.1% Triton X-100, and
1 mmol/L DTT), and 2 times with 1 mL wash buffer (20
mmol/L HEPES, pH 7.2, 2 mmol/L EGTA, 10 mmol/L
MgCl2, 1 mmol/L DTT, and 0.1% Triton
X-100). For Western blot analysis, cell lysates or
immunoprecipitates were subjected to SDS-PAGE and proteins were
transferred to nitrocellulose membranes (Hybond-ECL; Amersham) as
previously described.6 The membrane was blocked for 1
hour at room temperature with a commercial blocking buffer from GIBCO
BRL. The blots were then incubated for 4 hours at room temperature with
anti-BMK1, anti-Src (Santa Cruz), anti-Csk (Santa Cruz), or
activated-Src antibody clone 28,21 followed by
incubation for 1 hour with secondary antibodies (horseradish peroxidase
conjugated). For ERK1/2 activation, the blots were incubated for 12
hours with anti-phosphospecific ERK1/2 (New England Biolabs) or
nonspecific ERK1 and ERK2 antibodies (Santa Cruz). Immunoreactive bands
were visualized with the use of enhanced chemiluminescence (ECL Kit;
Amersham International).
p90RSK and BMK1 Kinase Assays
p90RSK kinase activity was measured through the use of GST-NHE-1
phosphorylation and BMK1 kinase activity was measured
through the use of autophosphorylation as described
previously with slight modifications.6 For
analysis of whole cell extracts, heart powders were
homogenized with 3 vol of lysis buffer and
centrifuged at 14 000g (4°C for 30 minutes), and
protein concentrations were determined. p90RSK or BMK1 were
immunoprecipitated through the incubation of 1000 µg protein from
each sample with 3 µL of the rabbit polyclonal anti-p90RSK (Santa
Cruz) antibody and anti-BMK1 antibody for 3 hours, the addition of 40
µL of a 1:1 slurry of protein A/Sepharose (Pharmacia Biotech) beads
to the extract/antibody mixture, and then incubation for 1 hour at
4°C. The beads were washed 2 times with 1 mL lysis buffer, 2 times
with 1 mL LiCl wash buffer, and 2 times in 1 mL modified buffer A
(20 mmol/L HEPES, pH 7.2, 2 mmol/L EGTA, 10 mmol/L
MgCl2, 1 mmol/L DTT, and 0.1% Triton
X-100). Immunoprecipitated p90RSK and BMK1 were resuspended in
25 mmol/L HEPES, pH 7.4, 10 mmol/L
MgCl2, and 10 mmol/L
MnCl2, and the kinase reaction was initiated by
the addition of 200 pmol GST-NHE-1 (625-747), 15 µmol/L
ATP, and 0.5 mCi/mL [
-32P]ATP. After the
reaction proceeded for 20 minutes at 30°C, it was terminated by the
addition of Laemmlis sample buffer. BMK1 kinase activity of the
immunoprecipitate was measured at 30°C for 20 minutes in a reaction
mixture (40 µL) containing 15 µmol/L ATP,10 mmol/L
MgCl2, 10 mmol/L
MnCl2, and 3 µCi of
[
-32P]ATP. Proteins were analyzed
with 10% SDS-PAGE, followed by autoradiography. NHE-1
phosphorylation and BMK1
autophosphorylation were determined through
densitometry of bands at the correct molecular weights in the linear
range of film exposure with the use of a scanner and NIH Image
1.54.
Csk Activity Assay
Immunoprecipitated Csk kinase activity was measured through the
phosphorylation of poly(E4Y) with
acid precipitation onto filter paper.22 The
phosphorylation reactions were performed in a volume of
50 µL at 30°C for 30 minutes. The standard
phosphorylation reaction contained 3 µCi of
[
-32P]ATP, 1 mg/mL
poly(E4Y), 6 mmol/L
MgCl2, 75 mmol/L HEPES-NaOH (pH 8.0), 5%
glycerol, 0.005% Triton X-100, and 0.05% 2-mercaptoethanol. At
the end of the reaction time, 35 µL of the reaction mixture was
spotted onto p81 Whatman filter paper, which was washed in 5% TCA at
65°C (3 times for 10 minutes each). The radioactivity incorporated
into poly(E4Y) was determined through liquid
scintillation counting.
Materials
All materials were obtained from Sigma Chemical Co, except where
indicated. H2O2 was
obtained from Fisher Scientific.
Statistical Analysis
Data are reported as mean±SD. Statistical analysis was
performed with the StatView 4.0 package (Abacus Concepts). Differences
were analyzed with 1- or 2-way repeated measures ANOVA as
appropriate, followed by Scheffés correction.
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Results
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Activation of ERK1/2 and p90RSK by Ischemia and
Ischemia/Reperfusion
To determine whether ERK1/2 and p90RSK are activated in
the
perfused heart in response to ischemia and
ischemia/reperfusion,
we performed Western blotting with
anti-phosphospecific ERK1/2
and an immune complex kinase assay with
GST-NHE-1 as substrate,
respectively. ERK1/2 was not activated
by ischemia alone, as
shown by other investigators, even
at early time points
23 24 (Figures 2A

and 2C

). There was a small but
significant activation
of ERK1/2 by ischemia/reperfusion
(2.5±1.1-fold increase
with 20-minute ischemia and 10-minute
reperfusion; Figures 2B
and 2C

). In contrast, p90RSK was
activated rapidly and transiently
by ischemia alone
(4.3±1.3-fold increase), and activity
rapidly declined within 20
minutes (Figures 3A

and 3C

). Reperfusion
after
20-minute ischemia also activated p90RSK
(5.6±1.5-fold
increase with 20-minute ischemia and 10-minute
reperfusion)
and was sustained for 20 minutes after reperfusion
(Figures
3B

and 3C

). Although reperfusion after ischemia
activated both
ERK1/2 and p90RSK in the perfused heart, only
p90RSKwas activated
by ischemia alone (Figures 2

and 3

). These results suggest that
in addition to ERK1/2, there
is an alternative pathway for the
activation of p90RSK.

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Figure 2. Activation of ERK1/2 in hearts exposed to
ischemia and ischemia/reperfusion. Hearts were perfused
under control conditions (-), or subjected to
ischemia (Ischem 10, 20, 30, or 40 minutes) (A) or
ischemia (Ischem 10 or 20 minutes) and
ischemia/reperfusion (Ischem 20 minutes; Reperf 10 or 20
minutes) (B). Same conditions and protocols were used for all
experiments as indicated. A and B, ERK1/2 activity in whole extracts
was measured through Western blot analysis with a
phosphospecific ERK1/2 antibody. No difference in amount of
ERK1/2 was observed in lysates from any heart samples on Western blot
analysis with anti-ERK1/2 (data not shown). C, Densitometric
analysis of ERK1/2 activation. Results were normalized for all
experiments by arbitrarily setting densitometry of control heart
samples (time=0) at 1.0 (values are mean±SD, n=3;
*P<0.05). IB indicates immunoblotting.
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Figure 3. Activation of p90RSK in hearts exposed to
ischemia and ischemia/reperfusion. Perfusion protocols
and data analysis were performed as described in legend for
Figure 2 . A and B, p90RSK activity was measured through use of
an in vitro kinase assay with GST-NHE-1 (625-747) as substrate. C,
Densitometric analysis of p90RSK activation. Results were
normalized for all experiments by arbitrarily setting densitometry of
control heart samples (time=0) at 1.0 (values are mean±SD, n=3;
*P<0.05).
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Regulation of BMK1 and Src by Ischemia and
Ischemia/Reperfusion
We showed previously that ROS activated BMK1 in vascular
smooth muscle cells, endothelial cells, and
fibroblasts.6 7 25 Because there is significant release of
ROS on reperfusion after ischemia, we determined the activation
of BMK1 in response to ischemia/reperfusion in the perfused
heart. Ischemia alone stimulated BMK1, which was maximal at 30
minutes (5.8±1.5-fold increase) and sustained for 40 minutes after
ischemia (Figures 4A
and 4C
).
However, in contrast to ERK1/2 and p90RSK, BMK1 activity was
significantly inhibited by reperfusion after ischemia (Figures 4B
and 4C
). No difference in the amount of BMK1 was observed in
BMK1 immunoprecipitated from ischemia- and
ischemia/reperfusiontreated heart samples on Western blot
analysis with anti-BMK1 antibody (Figures 4A
and 4B
,
bottom).

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Figure 4. Activation of BMK1 in hearts exposed to
ischemia and ischemia/reperfusion. Perfusion protocols
and data analysis were performed as described in legend
for Figure 2 . BMK1 activity was analyzed through
autophosphorylation in an immune complex kinase assay.
BMK1 protein level was assayed through Western blot analysis
with anti-BMK1 antibody. A and B, Representative
autoradiogram showing BMK1 kinase activity (top) and
Western blot analysis showing BMK1 protein levels (bottom). C,
Densitometric analysis of BMK1 kinase activity (values are
mean±SD, n=3; *P<0.05). IB indicates
immunoblotting.
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To determine the role of Src in the BMK1 signaling pathway
activated by ischemia and ischemia/reperfusion,
we investigated the effect of ischemia/reperfusion on Src
activation in the perfused heart. Src has been shown to be
activated by ROS in Jurkat T cells, fibroblasts, and
endothelial cells.1 7 25 In addition, we
demonstrated previously that the activation of BMK1 by
H2O2 in fibroblasts is
dependent on Src.7 Src activity was measured through
Western blotting with Src antibody clone 28, which recognizes the
activated form of Src.21 This assay was validated
in a comparison of clone 28 immunoreactivity with Src activity measured
on the basis of 32P incorporation into soluble
enolase. There was a good correlation between the 2 techniques as
previously described.25 Ischemia alone stimulated
Src activation, which was maximal at 30 minutes (6.7±1.8-fold
increase) and sustained for 40 minutes after ischemia (Figures 5A
and 5C
). Similar to BMK1 activity, Src
activity was markedly inhibited by ischemia/reperfusion
(Figures 5B
and 5C
). No difference in the amount of Src was
observed in lysates from ischemia- and
ischemia/reperfusiontreated heart samples on Western blot
analysis with anti-Src (Figures 5A
and 5B
, bottom) These
results suggest that Src and BMK1 are coregulated by ischemia
and reperfusion in the perfused heart.

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Figure 5. Activation of Src in hearts exposed to
ischemia and ischemia/reperfusion. Perfusion protocols
and data analysis were performed as described in legend
for Figure 2 . A and B, Src kinase activity in whole extracts was
measured through Western blot analysis with Src antibody clone
28, which recognizes activated form of Src (top). No difference
in amount of Src was observed in lysates from any of heart samples on
Western blot analysis with anti-Src antibody (Santa Cruz)
(bottom). C, Densitometric analysis of Src activation (values
are mean±SD, n=3; *P<0.05). IB indicates
immunoblotting.
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To confirm the role of Src as an upstream signaling mediator of BMK1
activation by ischemia, we studied the effect of the
Src-specific inhibitor PP2 on ischemia-induced BMK1
activity (Figure 6
). PP2 interacts
specifically with Src family kinases and is a competitive
inhibitor of ATP.26 As shown in Figure 6
, 10
µmol/L PP2 completely inhibited
ischemia-induced BMK1 activation. These results support an
important role for Src in ischemia-induced activation of
BMK1.

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Figure 6. Src-specific inhibitor PP2 inhibits
BMK1 activation during ischemia. Hearts were perfused with
10 µmol/L PP2 or vehicle for 20 minutes and then perfused under
control conditions or subjected to ischemia (20 minutes). BMK1
activity was analyzed through
autophosphorylation in an immune complex kinase
assay. BMK1 protein level was assayed through Western blot
analysis with anti-BMK1 antibody. A,
Representative autoradiogram showing
BMK1 kinase activity (top) and Western blot analysis showing
BMK1 protein levels (bottom). B, Densitometric analysis of BMK1
kinase activity (values are mean±SD, n=3; **P<0.01).
IB indicates immunoblotting.
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Figure 10. Comparison of Src, BMK1, ERK1/2, and p90RSK
activation through ischemia and ischemia/reperfusion.
Perfusion protocols and data analysis were performed as
described in legend for Figure 2 . To more clearly show
comparison, we did not include SDs.
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Role of ROS in Activation of ERK1/2, p90RSK, Src, and BMK1 in
Perfused Hearts
We4 6 7 and others27 28 29 30 31 have shown that
cellular stresses, including ROS, activate Src, ERK1/2, p90RSK,
and BMK1 kinases in several cell lines. All 4 kinases showed
significant activation when the hearts were perfused with
H2O2 (100 µmol/L)
for 20 minutes (ERK1/2 4.2-fold increase, p90RSK 3.4-fold increase, Src
3.8-fold increase, BMK1 3.1-fold increase) (Figure 7
).
H2O2 decreased left
ventricular developed pressure and increased left
ventricular end-diastolic pressure in the heart
as described previously (Table
).32 33 These
results suggested that these kinases could also be activated by
endogenously generated ROS in hearts. Importantly, the
control activities of these kinases were not inhibited by
H2O2 stimulation, which is
in contrast to the inhibition of Src and BMK1 by reperfusion in the
perfused heart.

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Figure 7. Activation of ERK1/2, p90RSK, Src, and BMK1 in
hearts exposed to oxidative stress. Hearts were perfused under
control conditions (-) or perfused with
H2O2 (100 µmol/L for 20 minutes). A,
ERK1/2 activity in whole extracts was measured through Western blot
analysis with a phosphospecific ERK1/2 antibody. No
difference in amount of ERK1/2 was observed in lysates from any of
heart samples on Western blot analysis with anti-ERK1/2 (data
not shown). B, p90RSK activity was measured through use of an in vitro
kinase assay with GST-NHE-1 (625-747) as substrate. C, Src kinase
activity in whole extracts was measured through Western blot
analysis with Src antibody clone 28. No difference in amount of
Src was observed in lysates from any of heart samples on Western blot
analysis with anti-Src antibody (Santa Cruz) (data not shown).
D, Representative autoradiogram showing
BMK1 kinase activity. No difference in amount of BMK1 was observed in
immunoprecipitates from any of heart samples with anti-BMK1 antibody
(data not shown). Data shown are representative of
experiments repeated 2 times with different heart samples. IB indicates
immunoblotting.
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To define the role of ROS in BMK1 and p90RSK activation, we pretreated
the hearts with the antioxidant MPG, as described
previously,18 19 and determined the effect of MPG on
ERK1/2 and p90RSK activation during reperfusion and on Src and BMK1
activation during ischemia. We found that MPG completely
inhibited ischemia/reperfusioninduced ERK1/2 and p90RSK
activation (Figures 8A
and 8B
) but only
partially inhibited Src and BMK1 activation during ischemia
(Figures 8C
and 8D
). These data support a major role for ROS in
the stimulation of ERK1/2 and p90RSK after ischemia/reperfusion
but only a partial role in ischemia-induced Src and BMK1
activation. These results suggest that there is an alternative pathway
to ROS for Src and BMK1 activation by ischemia.

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Figure 8. Effects of MPG on ERK1/2 and p90RSK activation
through ischemia/reperfusion and Src and BMK1 activation
through ischemia. Hearts were perfused with 300
µmol/L MPG or vehicle for 20 minutes and then perfused under
control conditions, subjected to ischemia/reperfusion (20
minutes/20 minutes) (A and B), or subjected to ischemia (20
minutes) (C and D). ERK1/2, p90RSK (A and B), Src, and BMK1 (C and D)
were assayed as described in Materials and Methods. B and D,
Densitometric analysis of ERK1/2 and p90RSK (B) and Src and
BMK1 kinase activity (D). Results were normalized for all experiments
by arbitrarily setting densitometry of control heart samples (time=0)
at 1.0 (values are mean±SD, n=3; *P<0.05). IB
indicates immunoblotting.
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Regulation of Csk by Ischemia and
Ischemia/Reperfusion
The protein tyrosine kinase Csk phosphorylates Src
family kinases on a tyrosine residue located near the carboxyl
terminus.34 This phosphorylation
downregulates Src kinase, suggesting that Csk may inhibit Src during
reperfusion (Figure 5
). Therefore, we determined Csk activity
during ischemia and reperfusion through the use of an in vitro
kinase assay with poly(E4Y) as a
substrate.22 Ischemia rapidly activated
Csk activity, and the maximal activation occurred at 10 minutes
(7.4±1.6) (Figure 9
). Unexpectedly,
reperfusion after ischemia did not induce Csk activation
(Figure 9
). No difference in the amount of Csk was observed in
lysates from any heart samples on Western blot analysis of Csk
antibody (data not shown).

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Figure 9. Activation of Csk in hearts exposed to
ischemia and ischemia/reperfusion. Perfusion protocols
and data analysis were performed as described in legend for
Figure 2 . Csk activity was analyzed through use of an
immune complex kinase assay with poly(E4Y) as a substrate.
No difference in amount of Csk was observed in lysates from any of
heart samples on Western blot analysis with anti-Csk antibody
(Santa Cruz) (data not shown). Results were normalized for all
experiments by arbitrarily setting incorporated radioactivity of
control heart samples (time=0) at 1.0 (values are mean±SD, n=3;
*P<0.05).
|
|
 |
Discussion
|
|---|
The major finding of this study was that Src and BMK1 are
coregulated
by ischemia and ischemia/reperfusion in the
perfused heart,
at least in part, via a pathway other than
H
2O
2 (Figures 7

and
10

). Data to support this conclusion
include the findings that
ERK1/2 and p90RSK, as well as Src and BMK1,
were activated when
hearts were perfused by
H
2O
2 (Figure 7

). In
contrast, global
ischemia stimulated p90RSK, but not ERK1/2, in
the perfused
heart (Figures 2

and 3

). On reperfusion of
ischemic hearts,
both ERK1/2 and p90RSK activities were
increased (Figures 2
and 3

). These results indicate that
in addition to ERK1/2, there
is an alternative pathway via which
ischemia activates p90RSK.
Global ischemia also
stimulates Src and BMK1 activities, but
the activities of Src and BMK1,
in contrast to those of ERK1/2
and p90RSK, are markedly attenuated by
ischemia/reperfusion
(Figures 4

and 5

).
Furthermore, we found that the antioxidant
MPG completely inhibited
ischemia/reperfusioninduced
ERK1/2-p90RSK activation but only
partially inhibited Src and
BMK1 activation during ischemia.
These findings demonstrate
important differences in MAPK signal
transduction pathways activated
via ischemia and
ischemia/reperfusion in the heart.
We summarize the data for ischemia- and
ischemia/reperfusionmediated signal transduction leading to
the activation of BMK1 and p90RSK in the heart based on the present
study (Figure 10
); the key features of these findings are (1)
differential activation of BMK1 and ERK1/2 by ischemia and (2)
opposing effects of reperfusion on the Src and BMK1 pathway versus the
ERK1/2 and p90RSK pathway. Both Src and BMK1 are redox-sensitive
kinases, and Src is required for BMK1 activation by
H2O2 in
fibroblasts.1 We also have shown through the use of
cultured fibroblasts and Jurkat T cells that p90RSK, an important
downstream effector of ERK1/2, is activated by ROS in an
ERK1/2-independent manner.16 In the present study, we
show for the first time that
H2O2 activates
p90RSK, Src, and BMK1 in the perfused heart (Figure 7
). The fact
that ischemia alone activates p90RSK but not ERK1/2
(Figure 10
) in the present study suggests that besides
ERK1/2, there is an alternative pathway by which to activate
p90RSK. A candidate for this pathway is Fyn, which we have shown to
regulate H2O2-mediated
p90RSK activation in fibroblasts.16 Because we do not have
a specific Fyn antibody for the analysis of cardiac myocytes,
we could not include Fyn activation data in the present study.
We have shown that p90RSK phosphorylates
Ser703 of NHE-1, and this
phosphorylation is required for growth factor
stimulation of Na+/H+
exchange.15 Bugge and Ytrehus35 and
others36 37 suggested that the inhibition of NHE-1 by
HOE642 provided cardioprotective and antiarrhythmic effects in hearts
exposed to ischemia and reperfusion. The ultimate biological
effects of activation of p90RSK may depend on the duration and extent
of this kinase activation. Future studies will be required to determine
the role of p90RSK in ischemia and reperfusionmediated signal
transduction (especially NHE-1 activity) in ischemic heart.
It was recognized some years ago that there was a significant release
of ROS and increase in
H2O2,38
OH· ,39 and
O2-·40 41 in
ischemia and in reperfusion occurring after ischemia.
Therefore, the effect of ischemia/reperfusion of inhibition of
Src-BMK1 activity was an unexpected finding. One of the candidate
inhibitors was Csk,34 42 which has been found
to phosphorylate the carboxyl-terminal tyrosine
(Tyr527) of c-Src, thereby suppressing c-Src kinase
activity. However, we found that ischemia/reperfusion did not
increase Csk activity, although Csk was activated by
ischemia. These results suggest 2 possible mechanisms to
regulate Src activity during reperfusion: (1) there is another unknown
Src Tyr527 tyrosine kinase other than Csk or (2)
ischemia/reperfusion inhibits an Src phosphatase that
dephosphorylates Src Tyr527.
Future studies will be required to characterize the relative roles of
these mediators.
In contrast to the present study, Knight and Buxton43
and Shimizu et al44 reported ischemia-induced
ERK1/2 activation. There are several differences in the
ischemia procedures of the present study compared with
those of these groups. First, Knight and Buxton43 and
Shimizu et al44 used rats instead of guinea pig. Second,
Knight and Buxton43 reported ERK1/2 activation during
ischemia, but they used fractionated homogenates
and assayed via the incorporation of 32P into
myelin basic protein peptide. Therefore, it is possible that another
"MBP kinase," like p38 kinase or BMK1, may have contaminated the
fractions that were used. Finally, in contrast to the Langendorff model
of the present study, Shimizu et al44 reported ERK1/2
activation in a coronary artery ligation model.
The biological consequences of Src and BMK1 activation by
ischemia and inhibition by ischemia/reperfusion in the
heart are poorly understood. Kato et al45 reported that
BMK1 activity is required for epidermal growth factormediated cell
proliferation and cell cycle progression. In contrast to p38 and JNK,
the activation of BMK1 is not apoptotic.45 46 BMK1
has been recently reported to phosphorylate MEF2C, which in
turn stimulates c-Jun expression.46 Thus, BMK1 may mediate
cellular responses through the regulation of expression of the
essential early response gene c-jun. Indeed,
increased expression of c-Jun occurs during ischemia in
isolated hearts and during hypoxia in cultured
myocytes.47 48 BMK1-dependent activation of c-Jun may
therefore contribute to the survival pathway in ischemic
myocardium.
In summary, we have shown that p90RSK, Src, and BMK1 are
activated by ischemia. The fact that
ischemia/reperfusion activated p90RSK but "shut
off" Src and BMK1 suggests that these 2 redox-sensitive kinase
pathways serve different intracellular functions with respect to
reperfusion.
 |
Acknowledgments
|
|---|
This study was supported by National Institutes of Health Grants
HL-44721
and HL-49192 (to Dr Berk) and Grant HL-52318 (to Dr Walsh).
The
authors wish to thank Drs C. Yan, H. Ueba, M. Okuda, and H.
Umemori
for their invaluable assistance and critical reading
of the
manuscript.
 |
Footnotes
|
|---|
This manuscript was sent to Eugene Braunwald, Consulting Editor,
for review by expert referees, editorial decision, and final
disposition.
Received March 10, 1999;
accepted September 21, 1999.
 |
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