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INTRODUCTION |
The heart is subjected to episodes of ischemia followed by
reperfusion in a number of situations, including angina, myocardial infarction, and cardiac surgery, and these stresses can result in cell
injury and death. Part of the cellular response to ischemia/reperfusion is activation of several members of the mitogen-activated protein kinase (MAPK)1 family. In
many different cell types, p38 MAPK and c-Jun NH2-terminal kinase (JNK) family members are activated predominantly by cellular stresses or inflammatory signals, e.g. hyperosmolarity,
chemical or heat stress, endotoxin, and cytokines (1-4), whereas the
extracellular signal-regulated kinases (ERKs) are activated by
mitogenic stimuli (5).
In the isolated perfused rat heart, p38 MAPK is activated by global
ischemia, and activation is maintained during reperfusion (6, 7). In
the same model, neither ERKs nor JNKs are activated by ischemia,
whereas reperfusion after ischemia activates JNK (6-8). Different
studies have shown activation (8) or lack of activation (6) of ERKs on
reperfusion, possibly the result of different assay methods. More
recently, it was observed that although JNK1 (also termed JNK46) is not
activated by ischemia, this stress results in translocation of JNK1 to
the nucleus, where it is then phosphorylated and activated on
reperfusion (9). Ischemia and reperfusion also activate members of the
MAPK family in kidney and liver differentially (7, 10, 11). However it
is not clear from these studies whether activation of these kinases is
part of the protective response of the cell or if these signals mediate
the cellular damage and death caused by ischemia or
ischemia/reperfusion. Evidence suggests that myocardial ischemic cell
death occurs by both apoptosis and necrosis (12, 13). From the timing
of p38 MAPK activation during ischemia and initiation of apoptosis, Yin
et al. (7) speculate that activation of p38 MAPK initiates
the signal for apoptotic cell death. Indeed, p38 MAPK activation has
been implicated in mediating apoptosis in several cell types (14-16).
Recent studies in neonatal rat cardiac myocytes support a role for the
isoform of p38 MAPK in mediating apoptosis; overexpression of
activated MAPK kinase 3b, which phosphorylates and activates p38 MAPK,
induces apoptosis that is increased by coexpression of p38
and is
decreased by expression of a dominant negative form of this isoform
(17).
In contrast, a separate study demonstrates that activation of p38 MAPK
can prevent apoptosis in neonatal rat cardiac myocytes (18).
Furthermore, others have proposed that p38 MAPK activation mediates a
phenomenon termed preconditioning, which confers cardiac protection from ischemia. Preconditioning is a highly effective method
of protecting the heart from ischemic damage by subjecting it to
sublethal periods of ischemia before the prolonged ischemia (6, 8, 19,
20). A protective function for p38 MAPK is supported by a recent study
in which the role of p38 MAPK in preconditioning was examined in
isolated rabbit cardiac myocytes (21). Pretreatment with anisomycin, an
activator of p38 MAPK, protects isolated rabbit cardiac myocytes
against ischemia-induced cell fragility, leading to the suggestion that
p38 MAPK protects the heart against ischemia. The addition of SB
203580, a selective inhibitor of p38 MAPK (22, 23), during a
preconditioning treatment abolishes the protective effect of
preconditioning, supporting the initial observation (21). Similar
results, showing that SB 203580 inhibits the protection afforded by
ischemic preconditioning against myocardial infarction, were obtained
using isolated rat hearts (24).
Most previous ischemia studies have investigated MAPK activation in
whole heart, which contains a large proportion of non-myocyte cells,
mainly fibroblasts and endothelial cells. In the present study, we used
primary cultures of neonatal rat cardiac myocytes and confirmed that
p38 MAPK is activated in a model of ischemia which uses a glucose-free
hypoxic incubation. We report that activation of p38 MAPK occurred in
two distinct phases and that inhibition of p38 MAPK during the second
phase protected cardiac myocytes from ischemic injury. These results
are consistent with the hypothesis that sustained p38 MAPK mediates
ischemia-induced cell injury and death in neonatal rat cardiac myocytes.
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EXPERIMENTAL PROCEDURES |
Reagents--
All antibodies were used according to
manufacturers' protocols. Anisomycin (Sigma) was dissolved in dimethyl
sulfoxide (Me2SO) at 5 mg/ml and used to give a final
Me2SO concentration less than 0.01%. SB 203580 (Calbiochem) was dissolved in Me2SO at 10 mM and used to give final a Me2SO concentration less than
0.1%.
Culture of Ventricular Myocytes--
Primary cultures of
ventricular myocytes from 1-day-old Sprague-Dawley rats were performed
by gentle, serial trypsinization, as described previously (25) with
modifications (26). A preplating step was included to reduce the number
of contaminating non-myocytes. Myocytes were plated at 800 cells/mm2 in 35- or 60-mm dishes (Falcon). Myocytes
represented 90-95% of total adhering cells. Division of non-myocytes
was prevented by the addition of 0.1 mM bromodeoxyuridine
to medium for the first 4 days of culture. Cells were maintained at
37 °C in a 1% CO2 incubator in M-199 medium (Life
Technologies, Inc.) containing 10% fetal bovine serum (Hyclone), 50 units/ml penicillin, and 80 µM vitamin B12
for the first 4 days. Vitamin C (80 µM) was present from
day 2. On day 4, myocytes were placed in defined (10 µg/ml insulin,
10 µg/ml transferrin, 80 µM vitamin C, 50 units/ml penicillin, and 80 µM vitamin B12) M-199
medium. Myocytes exhibited a spontaneous contraction rate of
approximately 250-300 beats/min, and cultures with a slower
contraction rate were not used. The higher rate of contraction
versus that seen by Simpson and Savion (25) is partly the
result of the increased density of cultures used here. Experiments were
performed on days 5 and 6 of culture.
Induction of Ischemia--
Ischemia was induced in a humidified
37 °C incubator within an air-tight Plexiglas glove box (Anaerobic
Systems) maintained with 0.2-0.5% O2, 1%
CO2, and the balance N2. Medium (defined minimal essential medium and Hank's balanced salt solution without glucose) was equilibrated to low O2 within the glove box
for at least 90 min before commencing experiments. Inside the glove
box, cells were washed twice with warm, preequilibrated medium before the addition of incubation medium (1.5 ml/35-mm dish). O2
was measured using an electronic gas analyzer OXOR®II or
Fyrite® (both from Bacharach).
Lactate Dehydrogenase (LDH) Assay--
After ischemic or
normoxic treatments, incubation medium was stored at 4 °C, and the
same volume of cold buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) was added to the cells. The cells were scraped and
lysed by trituration. Lysates were centrifuged at 4 °C at 16,000 × g for 15 min, and the supernatant was stored
at 4 °C. LDH activity was measured from both medium (released LDH)
and cell lysate (retained LDH) using a spectrophotometric assay
(Sigma). Results were expressed as released LDH activity as a percent
of total (released plus retained) LDH activity.
Analysis of the Phosphorylation State of p38 MAPK, ERK2, and
JNK--
After treatment, cells were placed on ice, and the incubation
medium was aspirated and discarded. Cells were washed once with cold
phosphate-buffered saline. Laemmli loading buffer at 2 × concentration (2% SDS, 20% glycerol, 0.04 mg/ml bromphenol blue, 0.12 M Tris-HCl, pH 6.8, 0.28 M
-mercaptoethanol)
was added (150 µl/35-mm dish). Cells were scraped and lysed by
trituration. Samples were frozen in dry ice/ethanol then transferred
immediately to
80 °C. Prior to electrophoresis, samples were
heated to 95 °C for 5 min. Electrophoresis was performed with
approximately 20 µg of protein/sample on 10% low ratio bisacrylamide
(100:1, acrylamide:bisacrylamide). After Western blotting, filters were
probed sequentially with dual phospho-p38 MAPK (Thr180
Tyr182), total p38 MAPK (both from New England Biolabs), or
ERK2 antiserum, and immunoreactivity was detected by enhanced
chemiluminescence. ERK2 antiserum (DC3; obtained from Dr. J. E.
Ferrell, Stanford University) was raised against Xenopus
ERK2 (27) and recognizes both non-phosphorylated and phosphorylated
forms of ERK2. Antiserum was used at a dilution of 1/500. Blots were
stripped by incubation in 62.5 mM Tris-HCl, pH 6.8, 100 mM
-mercaptoethanol, 2% SDS for 30 min at 50 °C
followed by two washes with phosphate-buffered saline and 0.05% Tween,
then blocking. Activation of p38 MAPK requires phosphorylation on both
Thr180 and Tyr182, which is specifically
recognized by the antibody used, and therefore activation is expressed
as the ratio of dual phospho-p38 MAPK to total p38 MAPK
immunoreactivity, which allows correction for differences in protein
loading. Because dual phospho-p38 immunoreactivity was usually
undetectable in control cells, results were normalized to the ratio of
the 10-min ischemia sample.
Phosphorylation of JNK was assayed as above, except that 50 µg of
cell lysate protein was electrophoresed, and filters were probed with
anti-active JNK (Promega).
Assay of p38 MAPK Activity--
The p38 MAP kinase assay kit
from New England Biolabs was used with a few modifications. Sodium
orthovanadate (2 mM), 1 mM phenylmethylsulfonyl
fluoride, 25 µg/ml aprotinin, 25 µg/ml leupeptin, and 20 µg/ml
soybean trypsin inhibitor were added fresh to lysis buffer, and the
cells were lysed by trituration. p38 MAPK was immunoprecipitated, its
catalytic activity determined using the in vitro kinase
assay to phosphorylate recombinant activating transcription factor-2
(ATF2), and the reaction mixture separated by SDS-PAGE. Western blots
were probed with the ATF2 antibody provided in the kit (specific for
phospho-Thr71), and immunoreactivity was detected by
enhanced chemiluminescence. Filters were stripped as above and reprobed
with anti-p38 MAPK. The ratio of phospho-ATF2 to p38 MAPK
immunoreactivity was determined for each sample, and then results were
expressed as fold activation over control.
Immunoprecipitation of Dual Phosphorylated p38 MAPK--
Cardiac
myocytes (one 100-mm dish/treatment) were treated, and then the
incubation medium was aspirated and discarded. Cells were washed once
with cold phosphate-buffered saline and then scraped into 800 µl of
lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 1 mM EGTA, 1% Triton X-100, 2 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 25 µg/ml leupeptin, and 20 µg/ml soybean trypsin
inhibitor) and lysed by trituration. Samples were extracted on ice for
15 min, and then cell debris was removed by centrifugation at
15,000 × g for 10 min. A sample of supernatant was
retained for electrophoresis. 20 µl of dual phospho-p38 MAPK
(Thr180 Tyr182) antibody (New England Biolabs)
was added to the remainder of the supernatant, and samples were rotated
overnight at 4 °C. 60 µl of a 50% slurry of protein A-Sepharose
CL-4B (Amersham Pharmacia Biotech) was added, and the samples were
rotated for 2 h at 4 °C. The immunoprecipitates were
washed four times with phosphate-buffered saline, electrophoresed on
10% low ratio bisacrylamide SDS-PAGE, then subjected to Western blot
analysis with anti-p38 MAPK, anti-dual phospho-p38 MAPK
(Thr180 Tyr182), or anti-p38
(Santa Cruz Biotechnology).
Densitometric Analysis--
Autoradiographs were scanned using
an ArcusII flatbed scanner (AGFA) with FotoLookPS 2.07.2, and the band
density was analyzed by NIH ImageTM.
ATF2 Phosphorylation--
Preparation of nuclear proteins from
cardiac myocytes was performed exactly as described by Clerk and Sugden
(28). Approximately 50 µg of nuclear protein was electrophoresed on
8% SDS-PAGE, and Western blot analysis was performed. ATF2 was
detected using phosphorylation state-independent antibody from Santa
Cruz Biotechnology.
Cell Viability Assay--
Cell death from ischemic or normoxic
incubations was assessed using two dyes that distinguish between live
and dead cells. Calcein acetoxymethyl ester (calcein AM; Molecular
Probes) and propidium iodide (PI) were added to the incubation medium
at final concentrations of 2 µM and 1 µg/ml,
respectively, and dishes incubated at 37 °C for 15 min (ischemic
samples were maintained under ischemic conditions during this
incubation). Cells were viewed using a Zeiss microscope and a 40 × objective and were scored as live (green cytosolic fluorescence from
calcein AM) or dead (red nuclear fluorescence from PI).
Isolation of DNA and Agarose Gel Electrophoresis--
After
ischemic or normoxic incubation, cells were scraped into incubation
medium to allow retention of any floating cells and were harvested by
centrifugation. DNA was prepared by standard techniques (29). Identical
amounts of DNA (2 µg) were electrophoresed through 1.8% agarose and
DNA visualized on a UV transilluminator.
Detection of CPP32 Immunoreactivity--
Samples were treated
exactly as for analysis of p38 MAPK immunoreactivity except that
samples were submitted to 12% SDS-PAGE, and filters were probed with
anti-CPP32 (H-277) from Santa Cruz Biotechnology.
Statistical Analysis--
Data were compared using Student's
t test for observations between two samples with unequal
variance, with one-tailed distribution. A p value of less
than 0.05 was considered significant.
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RESULTS |
Activation of MAPKs and Stress-activated Kinases by
Ischemia--
Activation of MAPKs in primary neonatal rat cardiac
myocytes in response to simulated ischemia was investigated. The model used combines two properties of ischemia: decreased energy source, because incubations are performed in the absence of glucose, and hypoxia, with oxygen levels between 0.2 and 0.5%. p38 MAPK activation was estimated by Western blot analysis using an antibody that specifically recognizes the dual phosphorylated (on residues
Thr180 and Tyr182), active form of the enzyme.
Antibody recognizing p38 MAPK regardless of its phosphorylation state
was used to normalize for differences in protein loading. This antibody
is specific for the
isoform of p38 MAPK, and the level detected
remained constant throughout ischemia. Dual phosphorylation of p38 MAPK
was observed within 10 min of ischemia, remained maximal until 30 min,
then decreased but remained above basal until 180 min of ischemia (Fig.
1, A and B). At
later time points, dual phosphorylation increased again with a peak at
240 min and remained high for 420 min (Fig. 1, A and
B).

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Fig. 1.
Ischemia results in phosphorylation and
activation of p38 MAPK. Panel A, cardiac
myocytes were incubated in ischemic conditions for 10-420 min or were
harvested without any treatment (control). Cell lysates were prepared
as described under "Experimental Procedures," and approximately 30 µg of protein was separated by 10% SDS-low bis ratio PAGE. The same
filter was probed sequentially with antibodies against p38 MAPK or
phospho-p38 MAPK. This result is representative of between three and
six experiments for different time points (for graphic representation
of combined results, see panel B). Also shown is a 15-min
incubation with 0.2 µM anisomycin or 10 min with 100 nM 4 -phorbol 12-myristate 13-acetate treatment. Note
that in other experiments, anisomycin resulted in higher
phosphorylation of p38 MAPK than seen here, giving about the same level
as 10 min of ischemia. Panel B, the ratio of
phospho-p38 MAPK to p38 MAPK immunoreactivity was determined from
Western blot analysis as represented in panel A. The ratios
were then normalized to 1 for the 10-min ischemia sample. The data
represent between three and six experiments, each performed with a
different preparation of cells and are plotted as the mean (±S.E.).
All ischemia time points are significantly different from normoxia
(p < 0.05). Panel C, cardiac
myocytes were fed with fresh medium and then incubated in normoxic
conditions for 10-420 min, ischemic conditions for 10 min, or were
harvested without any treatment (control). Samples were then treated as
described for panel A. Panel D, the
ratio of phospho-p38 MAPK to p38 MAPK immunoreactivity was determined
from Western blot analysis as represented in panel C. The
ratios were then normalized to 1 for the 10-min ischemia sample such
that panels B and D have the same scale. Data
represent between three and five experiments, each performed with a
different preparation of cells, and are plotted as the mean (±S.E.).
Panel E, cardiac myocytes underwent an ischemic
incubation for 20 min or were treated with 0.2 µM
anisomycin for 30 min or were harvested without treatment (control).
Cells were lysed and p38 MAPK activity measured as described under
"Experimental Procedures." The same Western blot was probed
sequentially with anti-phospho-ATF2 and anti-p38 MAPK. The ratio of
activation was calculated as phospho-ATF2:p38 MAPK immunoreactivity and
was normalized to 1 for the control sample. This result is
representative of three experiments, each with a different preparation
of cells.
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We and others (30) have observed that when cardiac myocytes are fed
with fresh medium, they cease contracting for a period of time. In our
study, cells stopped contracting for approximately 20 min, then
spontaneous contraction recovered gradually to a normal rate within
60-90 min. Because this corresponds with the timing of the first phase
of p38 MAPK activation, and a change of medium is required to induce
ischemic conditions, we examined p38 phosphorylation levels after
changing incubation medium and maintaining cells under normoxic
conditions. Transient phosphorylation of p38 MAPK was observed after
simply feeding fresh medium (Fig. 1, C and D).
Cardiac myocytes can be maintained healthily in culture for up to 8 days with multiple changes of medium. Although this does not show that
the initial ischemic p38 MAPK activation and that induced by
changing medium are equivalent, these results do demonstrate that
transient activation of p38 MAPK can occur without long term harmful
effects to cardiac myocytes.
To confirm that dual phosphorylation of p38 MAPK during ischemia truly
reflected activation, p38 MAPK was immunoprecipitated and used in an
in vitro kinase assay with recombinant ATF2 as a substrate.
We observed more than a 6-fold increase in the phosphorylation of ATF2
over basal after 20 min of ischemia (Fig. 1E) and more than
a 4-fold increase after 25 or 30 min of ischemia (n = 1, data not shown). A similar activation ratio was obtained by
incubating cells with anisomycin, an activator of p38 MAPK (Fig.
1E). Thus, p38 MAPK is indeed activated during ischemia.
To determine if ischemia-induced phosphorylation is unique to p38 MAPK,
we examined phosphorylation of other MAPKs. Phosphorylation of ERK2
(p42 MAPK) was estimated using a gel electrophoresis mobility shift
assay with an antibody that detects both inactive (non-phosphorylated) and active (dual phosphorylated) ERK2. The reduced mobility form of
ERK2, indicating phosphorylation as seen with 4
-phorbol 12-myristate 13-acetate treatment, was not observed at any time during prolonged ischemia (Fig. 2A). Similarly,
probing with anti-active JNK, which detects the dual phosphorylated
active forms of both JNK1 and JNK2, showed little or no activation of
JNK in 10-240 min of ischemia (Fig. 2B) and no activation
in 300, 360, or 420 min of ischemia (n = 1, data
not shown).

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Fig. 2.
Ischemia does not result in phosphorylation
of ERK2 or JNKs. Panel A, as for Fig.
1A, except that filters were probed with ERK2 antiserum.
4 -Phorbol 12-myristate 13-acetate and anisomycin treatment are as
described for Fig. 1A. Panel B, as for
Fig. 1A, except that 50 µg of protein was loaded, and the
filter was probed with anti-active JNK. As a positive control, cells
were treated with 0.2 µM anisomycin for 30 min. This
filter was coincubated with anti-p38 MAPK, showing equal protein
loading. This result is representative of three experiments, except
that in one experiment a small amount of active JNK was detected in the
30-min ischemia sample; however, this represented only approximately
10% of active JNK seen with anisomycin treatment.
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Taken together, our results indicate that changing medium transiently
activates p38 MAPK, whereas ischemia results in a transient, then
sustained activation of p38 MAPK, but not ERK2 or JNK, in primary
cultures of neonatal rat cardiac myocytes; these results are consistent
with previous studies performed in whole heart (6-8).
Significance of p38 MAPK Activation in Ischemia--
Release of
the cytosolic enzyme LDH, caused by cell membrane leakage, was used to
assess cell damage resulting from ischemia. Under the conditions used
in this study, 7-9 h of ischemic incubation resulted in the release of
between 45 and 60% of cellular LDH, whereas 7-9 h of normoxic
incubation resulted in release of less than 6% of total LDH. To
determine if the activation of p38 MAPK protects myocytes from ischemic
stress or mediates the damage from ischemia, we first used anisomycin.
Anisomycin is a protein synthesis inhibitor that activates MAPK family
members and has been shown to protect myocytes from ischemia (21). This
result has been used to implicate p38 MAPK in the protective mechanism (21). To confirm that anisomycin was protective in our model, cells
were pretreated with anisomycin and then subjected to ischemia, either
in the presence or absence of additional anisomycin. Fig. 3 demonstrates that anisomycin protected
myocytes from ischemia-induced injury; LDH release was reduced
significantly when anisomycin was present either during the
pretreatment only or during both the pretreatment and the prolonged
ischemia.

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Fig. 3.
Anisomycin protects cardiac myocytes from
ischemic injury. Cardiac myocytes were pretreated with 0.2 µM anisomycin or 0.1% Me2SO vehicle, then
incubated in ischemia for 7-9 h in the presence (n = 2, duplicates) or absence (n = 3, duplicates) of 0.2 µM anisomycin, as indicated. LDH activity from medium and
cell lysate was measured as described under "Experimental
Procedures." Results are expressed as LDH released into the medium as
a percent of the total LDH activity and then normalized to 100% for
vehicle-treated samples. Data are plotted as the mean (±S.E.).
*p < 0.05; **p < 0.025 versus vehicle-treated sample. The two right
columns are not significantly different from each
other.
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Anisomycin is a nonspecific reagent, and therefore these data do not
prove that p38 MAPK activation is the mechanism by which anisomycin
protects myocytes. More direct evidence for this would require
inhibition of the anisomycin-induced protection by a selective p38 MAPK
inhibitor. Therefore, we next used an inhibitor of p38 MAPK, SB 203580, which has been shown to inhibit p38 MAPK selectively over other MAPK
family members and several other kinases (23, 31). Surprisingly, we
found that the presence of SB 203580 during ischemia resulted in a
significant dose-dependent decrease in LDH release from
myocytes (Fig. 4A). These data
indicate that inhibition of p38 MAPK during ischemia protects myocytes
from ischemic damage, whereas previous results have suggested that activation of p38 MAPK protects myocytes.

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Fig. 4.
p38 MAPK inhibitor protects neonatal cardiac
myocytes from ischemic injury. Panel A, cardiac
myocytes were incubated in ischemia or normoxia in the presence of 2, 5, or 10 µM SB 203580 or 0.1% Me2SO vehicle.
SB 203580 or Me2SO was added at the start of the
incubation. After 7-9 h, LDH activity was measured from medium and
cell lysate as described under "Experimental Procedures." Results
were expressed as LDH released into the medium as a percent of the
total LDH activity and then normalized to 100% for vehicle-treated
ischemia samples. The number of experiments, performed in duplicate, is
indicated above the bars. *p < 0.05, **p < 0.0001 versus vehicle-treated sample.
Panel B, as in panel A, except that
for the left three bars, 10 µM SB 203580 (SB) was added 45 min after the start of ischemic incubation
or at the start of ischemia for comparison. For the right
panel, 10 µM SB 203580 or 0.1%
Me2SO vehicle was added at the start of ischemia. After 45 min, the incubation medium was removed from all dishes, and the cells were washed twice with fresh
medium, preequilibrated to hypoxic conditions. Cells were then
incubated with vehicle or 10 µM SB 203580 for the
remainder of ischemia, as indicated. For both panels, data represent
the mean (±S.E.) from three experiments performed in duplicate.
*p < 0.001 versus vehicle-treated sample.
ns indicates that the difference is not significant.
Panel C, cardiac myocytes were incubated in
ischemia or normoxia in the presence of 10 µM SB 203580 or 0.1% Me2SO vehicle for 7-8 h, as indicated. Cells were
stained with calcein AM and PI and then scored as live (green cytosolic
fluorescence) or dead (red nuclear fluorescence). Data represent the
percent of viable cells and are plotted as the mean (±S.E.) from three
experiments. In each experiment, more than 800 cells were counted for
each treatment. *p < 0.05.
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As mentioned previously, activation of p38 MAPK was shown to occur in
two phases in these cells (Fig. 1, A and B). To
determine if inhibition of p38 MAPK during only one, or both phases,
was necessary for the protection seen with SB 203580, the p38 MAPK inhibitor was added at different times during ischemia, and the effect
on injury was examined. Adding the inhibitor to cells 45 min after the
start of ischemia (which is after the first peak of p38 MAPK
phosphorylation, Fig. 1B) gave a level of protection not
significantly different from that seen when the inhibitor was present
from the start of ischemia (Fig. 4B). SB 203580 is a
reversible inhibitor; isolated p38 MAPK can be washed free of inhibitor
and be fully active (23, 31). Therefore, to examine inhibition during
the first phase only, cells were incubated with SB 203580 and then
washed free of inhibitor after 45 min of ischemia. Using this protocol,
we found that the presence of the inhibitor during only the first 45 min resulted in cell damage similar to that seen in vehicle-treated
cells (Fig. 4B). Thus, SB 203580 induced protection of
myocytes from ischemia when present during only the second sustained
phase of p38 MAPK activation, but not when present during only the
first, transient phase of p38 MAPK activation. This suggests that
sustained p38 MAPK activation results in cell damage.
LDH release is a commonly used marker for cell damage, but to confirm
that decreased LDH release reflected a protection from cell death, we
used a cell viability assay. Live cells are distinguished by the
conversion of calcein AM to fluorescent calcein by intracellular esterases, but the intact membrane excludes PI. Dead cells are distinguished by entry of PI through damaged membrane and fluorescence of PI on binding to nucleic acid. We demonstrated that coincubation of
myocytes with 10 µM SB 203580 during ischemia
significantly decreased cell death at two time points (Fig.
4C). It is important to note that cell death is delayed but
not prevented; after 8 h of ischemia, cell death is reduced
significantly by the presence of the inhibitor, but it is increased
from basal cell death under normoxic conditions. Thus, inhibition of
p38 MAPK delays cell injury and death resulting from ischemia.
Because it appeared that the consequence of the first and second phases
of p38 MAPK activation differed, we attempted to determine whether the
same or different isoforms of p38 MAPK are activated during these two
different phases. By immunoprecipitation of dual phospho-p38 MAPK
(Thr180 Tyr182; this antibody detects both
activated
and
isoforms of p38 MAPK) at different times during
ischemia, we determined that p38
is activated in both phases during
ischemia (Fig. 5). p38
was only weakly
detected in cell lysates, and therefore we could not determine if
p38
was also present in any of the immunoprecipitates.

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Fig. 5.
p38 is
phosphorylated during both phases of ischemia. Cardiac myocytes
underwent ischemic incubation for 30, 240, or 300 min or were harvested
without any treatment (0). Dual phospho-p38 MAPK was immunoprecipitated
(IP), and approximately 5% of cell lysate before
immunoprecipitation and all of the immunoprecipitated material were
electrophoresed on 10% SDS- low bis ratio PAGE as described under
"Experimental Procedures." The filter was probed with anti-p38
(Santa Cruz Biotechnology; SC), anti-p38 (from New England
Biolabs; NEB), which is also specific for p38 , or
anti-dual phospho-p38 MAPK. This result is representative of two
experiments performed with different preparations of cells.
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Ischemia Results in Apoptosis in Neonatal Rat Cardiac
Myocytes--
Staining with PI as described above in the cell
viability assay allows visualization of nuclei and could potentially be
used to distinguish morphologically between apoptotic and necrotic cells. Apoptotic cells generally show condensed, fragmented nuclei, whereas necrotic cells have normal nuclei. However, after ischemia in
this cell type, many nuclei appeared slightly condensed but not
fragmented and therefore were difficult to classify clearly as
apoptotic or necrotic.
Previous studies have documented both apoptosis and necrosis induced by
myocardial infarction (12, 13). To examine if our model of ischemia
induces apoptosis in cardiac myocytes, cells were subjected to a DNA
laddering assay to assess the extent of DNA fragmentation. Identical
amounts of DNA, from cells treated under either normoxic or ischemic
conditions for 8 h, were visualized after agarose gel
electrophoresis. In control samples incubated under normoxic
conditions, a low level of basal DNA fragmentation was observed (Fig.
6A). However, in the ischemic
sample, there was a decrease in high molecular weight DNA and a
corresponding increase in low molecular weight DNA when compared with
the normoxic sample (Fig. 6A). The low molecular weight DNA
both in normoxia and ischemia showed the hallmark intranucleosomal
laddering of apoptosis. Therefore, although it was not possible to
quantitate apoptosis versus necrosis, we clearly
demonstrated that ischemia induces apoptosis in neonatal cardiac
myocytes. These data do not rule out the possibility that some cells
undergo necrosis during ischemia.

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Fig. 6.
Ischemia induces apoptosis in neonatal rat
cardiac myocytes, and SB 203580 inhibits activation of caspase-3.
Panel A, DNA was prepared from cells incubated in
normoxia (C) or ischemia (I) for 8 h. DNA (2 µg) was electrophoresed on 1.8% agarose and visualized using
ethidium bromide. The positions of molecular weight standards (in base
pairs) are indicated on the left of the panel.
This result is representative of three independent experiments.
Panel B, cardiac myocytes were treated with 10 µM SB 203580 (SB) or with vehicle
(V) and then were incubated in normoxic conditions for
7 h or ischemic conditions for 6 or 7 h. Preparation of cell
lysates and electrophoresis are as described under "Experimental
Procedures." Filters were probed with anti-CPP32 (caspase-3) and then
anti-p38 MAPK to normalize for protein loading. Caspase-3
immunoreactivity was expressed relative to p38 MAPK immunoreactivity,
and results were normalized to 100% for vehicle-treated control
sample. This result is representative of three experiments, each with a
different preparation of cells.
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SB 203580 Inhibits Activation of Caspase-3--
To confirm that
inhibition of p38 MAPK by SB 203580 delayed apoptosis, we examined the
activation state of caspase-3 (also known as CPP32, Yama, or apopain).
This enzyme has been identified as a key protease during the early
stages of apoptosis and is activated by degradation of the 32-kDa
proenzyme to approximately 17- and 12-kDa subunits that heterodimerize
to give active enzyme (32). Using Western blot analysis, we determined
that caspase-3 degradation is delayed in cardiac myocytes treated with
SB 203580 compared with vehicle-treated cells (Fig. 6B).
Therefore, apoptosis is delayed by inhibition of p38 MAPK.
SB 203580 Blocks Phosphorylation of p38 MAPK Substrate--
To
confirm that SB 203580 inhibits p38 MAPK in this system, we examined
phosphorylation of an endogenous p38 MAPK substrate, ATF2. p38 MAPK
phosphorylates ATF2 on Thr69 and Thr71,
resulting in an electrophoretic mobility shift (3, 28). Phosphorylation
of these residues is essential for increased transcriptional activity
of ATF2 (33). ATF2 is also phosphorylated on these residues by JNK
(33), but, because JNK is not activated by ischemia in these cells
(Fig. 2B), p38 MAPK is expected to be a major ATF2 kinase
under ischemic conditions.
In nuclear extracts from control cells, maintained under normoxic
conditions, the majority of ATF2 migrates at 69 kDa (Fig. 7, band 1), with a minor amount migrating
with a reduced electrophoretic mobility (Fig. 7, band 2). Incubation
under ischemic conditions resulted in ATF2 phosphorylation seen by an
increase in intensity of band 2 and the appearance of an additional
reduced mobility band (Fig. 7, band 3). When 10 µM SB
203580 was present during the ischemic incubation, these
phosphorylation events were partially inhibited; band 3 is no longer
detectable, and bands 1 and 2 are increased in intensity compared with
ischemia only. The partial inhibition of ATF2 phosphorylation may
indicate incomplete inhibition of p38 MAPK or that another ATF2 kinase,
which is not inhibited by SB 203580, is activated by ischemia. These
data demonstrate that the p38 MAPK substrate, ATF2, is phosphorylated
during ischemia and that the phosphorylation events are sensitive to
inhibition by SB 203580.

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Fig. 7.
SB 203580 partially reverses ischemia-induced
phosphorylation of ATF2. A nuclear fraction (50 µg of protein)
from cardiac myocytes incubated under normoxia (C), ischemia
(I), or ischemia plus 10 µM SB 203580 (I+SB) for 60 min was electrophoresed on 8% SDS-PAGE and
detected with anti-ATF2. An upward mobility shift (to bands 2 and 3)
was used as an indicator of ATF2 phosphorylation (3, 28). This result
is representative of three independent experiments.
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DISCUSSION |
In response to ischemia, cells activate biochemical pathways that
allow adaptation to this stressful environment. However, on prolonged
ischemia, these protective mechanisms may not be sufficient to maintain
normal cellular function, and cell injury and death follow. Therefore,
signal transduction pathways activated during ischemia may be either
protective or part of the signal that leads to cell death. In this
study, we demonstrated that ischemia induces both a transient and
sustained activation of p38 MAPK in neonatal rat cardiac myocytes. In
contrast, ERK2 and JNK are not activated during ischemia. By using the
specific p38 MAPK inhibitor SB 203580, we demonstrated that sustained
activation of p38 MAPK is deleterious to the cells and at least partly
mediates apoptosis.
At least four members of the p38 MAPK family have been identified, and
it is probable that different isoforms have specific physiological
functions (1, 22, 34-37). The isoforms
(also termed p38, CSBP, or
RK) and
are both expressed in heart tissue. Although these isoforms
share approximately 74% sequence identity, they have been suggested to
have opposing functions in cardiac myocytes (17). Expression of an
activated mutant of MAPK kinase 3b, an upstream activator of both
and
isoforms of p38 MAPK, results both in a hypertrophic response
and to apoptosis in neonatal rat cardiac myocytes (17). By using
coexpression of individual activated p38 MAPK isoforms or of dominant
negative inhibitory fragments, the same study showed that apoptosis
appears to be mediated by the
isoform, whereas the hypertrophic
response is mediated by the
isoform. In addition, suppression of
p38
using the dominant negative fragment results in an increase in
cell death, suggesting that this isoform can function to promote cell survival. In another study, overexpression of MAPK kinase 6, a different selective activator of p38 MAPK, protects neonatal cardiac myocytes against apoptosis (18). Thus, in cardiac myocytes it appears
that p38
protects against, whereas p38
promotes, apoptotic cell death.
In our study, we observed that transient activation of p38
does not
have a deleterious effect on the cell, whereas sustained activation of
p38
induces apoptosis. However, we could not rule out activation of
p38
or other p38 MAPK isoforms during one or both phases of p38 MAPK
activation seen during ischemia. Therefore, it is possible that the two
phases differ not only in duration, but also in the balance of isoforms
activated. A short period of ischemia is not detrimental to cardiac
myocytes and paradoxically protects against subsequent prolonged
ischemia, as discussed in the Introduction. In models similar to that
used in our study, preconditioning neonatal rat cardiac myocytes with
25 or 30 min of ischemic incubation followed by 30 min of normoxic
recovery was shown to protect cultured neonatal cardiac myocytes from
ischemia (30, 38). Thus, from the data presented here, preconditioning would be expected to result in only the first phase of p38
activation, and previous studies have shown that p38 MAPK is necessary
for the protective effect of preconditioning (21, 24). It is intriguing that p38 MAPK appears to play a role both in protection of myocytes against injury and in mediating cellular apoptosis, and we propose that
transient versus sustained activation of p38 MAPK, possibly in combination with activation of different isoforms, determines these
different cellular effects. The most likely explanation is that the
transient p38 MAPK activation that occurs on initiation of ischemia
represents an adaptive response of the cell. Cardiac myocytes clearly
can adapt to ischemic stress as shown by the fact that they can be
protected by preconditioning. This hypothesis provides an explanation
for the apparent discrepancy between this study, where SB 203580 protects against ischemia, and the previous studies in which SB 203580 inhibits preconditioning protection (21, 24).
Differential cellular effects of transient versus prolonged
activation have been demonstrated previously for MAPK family members, for ERK2 in primary rat hepatocytes (39), and for JNK in rat mesangial
cells (40). In addition, transient p38 MAPK activation is not
sufficient to induce neuronal differentiation in rat pheochromocytoma PC12 cells, whereas sustained activation is sufficient (41). Interestingly, a transient activation of the ERK/MAPK pathway is
additionally required to allow diffentiation (41). In a similar way, it
is likely that other proteins activated or inactivated at different
times during ischemia regulate or act in combination with p38 MAPK. For
example, the prolonged phase of p38 MAPK activation may be caused by
inactivation of specific regulatory phosphatases or, alternatively,
degradation of an antiapoptotic protein may be required before p38
MAPK-mediated apoptosis can proceed. These possibilities can now be explored.
Although adult and neonatal cardiac myocytes can differ in their
responses, it is unlikely that the protection seen here with SB 203580 would not translate to adult cells. A study performed in isolated adult
rat hearts demonstrates that the presence of SB 203580 during ischemia
preserves cardiac function during ischemia and improves postischemic
recovery of cardiac function (42). Therefore, it is predicted that SB
203580, or a novel selective inhibitor of p38
, will prove useful to
protect against damage from ischemic episodes in adult animals, both by
decreasing cell death and by improving cardiac function.
We cannot rule out the possibility that SB 203580 targets another
kinase, in addition to p38 MAPK, which mediates cell death. Selectivity
has been demonstrated against other kinases (22, 23), but it has
recently been reported that SB 203580 inhibits cardiac JNK2-related
isoforms, albeit with a higher IC50 than for p38 MAPK (43).
It is unlikely that inhibition of JNK explains the protection reported
here because we and others (6, 7) have shown that JNK2 is not activated
by ischemia.
In conclusion, our results strongly support a role for sustained p38
MAPK activation in mediating apoptosis induced by ischemia. This study
used an experimental design that does not require overexpression of the
proteins involved but rather inhibition of endogenous kinase. In
addition, we suggest that transient activation can have very different
cellular consequences from sustained activation of p38 MAPK in cardiac myocytes.