Download Involvement of mitogen-activated protein kinases in adriamycin

Am J Physiol Heart Circ Physiol 288: H1925–H1930, 2005;
doi:10.1152/ajpheart.01054.2004.
Involvement of mitogen-activated protein kinases in adriamycin-induced
cardiomyopathy
H. Lou, I. Danelisen, and P. K. Singal
Institute of Cardiovascular Sciences, Department of Physiology, St. Boniface General
Hospital Research Centre, Faculty of Medicine, University of Manitoba, Winnipeg, Canada
Submitted 13 October 2004; accepted in final form 1 December 2004
dilated cardiomyopathy; oxidative stress and apoptosis
ADRIAMYCIN (ADR), an anthracycline antibiotic, has been
widely used in the treatment of a variety of tumors and
carcinomas; however, its clinical application is limited by its
irreversible cardiac side effect of cardiomyopathy and congestive heart failure (13, 22). The characteristic features of ADRinduced cardiomyopathy (AIC) include dose dependency, refractoriness to inotropic support, dilation of the membrane
tubular system, myofibril drop out, and lack of inflammatory
cytokine upregulation (13, 17, 22). Although the mechanism of
AIC is multifactorial, increased oxidative stress due to overproduction of free radicals and antioxidant-deficit play an
Address for reprint requests and other correspondence: P. K. Singal, Institute
of Cardiovascular Sciences, St. Boniface General Hospital Research Centre,
Rm. 3022, 351 Tache Ave., Winnipeg, Manitoba R2H 2A6, Canada (E-mail:
[email protected]).
http://www.ajpheart.org
important role (16, 23). In addition, cardiac cell apoptosis has
been suggested to be associated with AIC (2, 11). However, the
role of oxidative stress in the intracellular signaling pathways
and cardiomyocyte apoptosis in AIC remains unknown.
Mitogen-activated protein kinase (MAPK) signaling pathways are the primary intermediator of induction of apoptosis
by oxidative stress. There are three major MAPK families,
including extracellular signal-regulated kinases (ERKs), p38,
and c-Jun NH2-terminal kinases (JNKs). In the cardiovascular
system, ERK1/2 are potently and rapidly activated by growth
factors and hypertrophic agents thereby mediating cell survival
as well as offer cytoprotection (24, 26). In contrast, JNKs and
p38-MAPKs are activated by cellular stresses, including oxidative stress, and are thought to correlate with cardiomyocyte
apoptosis and cardiac pathologies (12, 21, 28).
The current study investigated the role of ERK1/2, p38, and
JNKs MAPKs in the early stage of AIC and heart failure in
rats. Because the antioxidant probucol can completely prevent
AIC by reducing oxidative stress, modulatory effects of probucol on ADR-induced changes in MAPKs were investigated.
Pro- and anti-apoptotic proteins caspase-3, Bax, and Bcl-xl
were also examined to define the significance of MAPKs and
cardiomyocyte apoptotic signaling in AIC.
MATERIALS AND METHODS
Animal groups and treatment. ADR-induced cardiomyopathy and
heart failure were produced according to the established protocol (23).
Male Sprague-Dawley rats (250 ⫾ 10 g body wt) were divided into
four groups: control, ADR, ADR ⫹ probucol, and probucol. ADR was
administered in six equal doses (2.5 mg 䡠 kg⫺1 䡠 dose⫺1 ip), on alternate
days, over a period of 2 wk for a cumulative dose of 15 mg/kg.
Probucol was given in 12 doses for a period of 2 wk before the
administration of ADR and 2 wk during the administration of ADR
(cumulative dose 120 mg/kg ip). After the final injection, rats in the
early stage of cardiomyopathy were anesthetized with an intraperitoneal injection of a ketamine (90 mg/kg) and xylazine (10 mg/kg)
mixture and euthanized at 1, 2, 4, and 24 h. Rats for the late-stage
studies were observed for body weight, general appearance, behavior,
and mortality for 3 wk. At the end of the 3-wk posttreatment period,
animals were assessed hemodynamically, and the heart, lung, and
liver tissues were collected for the further studies. All animal treatment procedures were according to the guidelines of the Animal Care
Committee of the University of Manitoba.
Cardiac function and hemodynamic study. After the 3 wk of the last
injection, rats were anesthetized by inhalation of isoflurane and
oxygen. The right carotid artery was cannulated with a microtip
pressure transducer (model SPR-249; Millar Instruments, Houston,
TX) to monitor the blood pressure heart function. With the use of the
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society
H1925
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 16, 2017
Lou, H., I. Danelisen, and P. K. Singal. Involvement of mitogenactivated protein kinases in adriamycin-induced cardiomyopathy.
Am J Physiol Heart Circ Physiol 288: H1925–H1930, 2005; doi:
10.1152/ajpheart.01054.2004.—The current study investigated the
phosphorylation of mitogen-activated protein kinases (MAPKs) as
well as pro- and anti-apoptotic proteins in adriamycin (ADR)-induced
cardiomyopathy (AIC) and heart failure in rats. Modulatory effects of
antioxidant probucol on the activation of MAPKs were also examined.
Male rats were administered with ADR (15 mg/kg body wt ip, over 2
wk) with and without probucol (120 mg/kg body wt for 4 wk ip).
Hearts from these animals were studied at 1- to 24-h as well as at 3-wk
posttreatment durations. In the 3-wk group, ADR depressed cardiac
function, increased left ventricular end-diastolic pressure (LVEDP),
and caused dyspnea and mortality. These changes were prevented by
probucol. Phosphorylation of extracellular signal-regulated kinase
(ERK)1/2, in the early stage of AIC, showed a biphasic response, with
a maximum increase to 513% seen at 4 h, followed by a decrease to
66.8% at 3 wk after the last injection of ADR. Phosphorylation of p38
and c-Jun NH2-terminal kinases (JNKs) showed a steady increase
through 2, 4, and 24 h and 3 wk (116% to 148%). In gene microarray
analysis at 3 wk (heart failure stage), mRNA expression for both
ERK1/2 and p38 kinases was decreased, whereas JNK mRNA was
undetectable. Probucol completely prevented these MAPK changes.
Activation of caspase-3 as well as the increase in the ratio of Bax to
Bcl-xl were seen at early time points (1–24 h) as well as in the heart
failure stage (3 wk). It is suggested that a transient increase in ERK1/2
at a shorter interval indicate an early adaptive response, and failure of
this response corresponded with heart failure. In contrast, a gradual
and persistent increase in p38 and JNK MAPKs as well as in
caspase-3 and the Bax-to-Bcl-xl ratio may contribute in the initiation
of apoptosis and progression of heart failure. Because probucol
modulated changes in cellular signaling pathways and cardiac function, it is likely that oxidative stress plays a key role in AIC and heart
failure.
H1926
MAP KINASES AND HEART FAILURE
Table 1. Effects of probucol on adriamycin-induced changes in the late stage (3 wk) group
Parameter
Control
ADR
ADR⫹Pro
Pro
Body weight, g
LVSP, mmHg
LVEDP, mmHg
⫹dP/dt, mmHg/s
⫺dP/dt, mmHg/s
ASP, mmHg
ADP, mmHg
Liver, wet-to-dry wt ratio
Lung, wet-to-dry wt ratio
Heart-to-body wt ratio
Mortality, %
529.5⫾19.8
121.7⫾6.2
3.10⫾0.54
10,931⫾311
12,245⫾96
120.3⫾1.3
87.6⫾1.7
3.14⫾0.04
4.93⫾0.02
2.52⫾0.07
0
335.9⫾18.0*
96.5⫾0.7*
13.2⫾1.51*
8,631⫾114*
8,797⫾251*
93.2⫾1.6*
62.3⫾2.7*
3.47⫾0.10*
5.18⫾0.04*
2.13⫾0.09*
30
406.8⫾15.9
108.5⫾2.8
3.4⫾0.76
10,469⫾298
11,800⫾198
107.5⫾2.1
84.2⫾29
3.18⫾0.08
4.97⫾0.02
2.54⫾0.06
0
526.9⫾16.8
123.6⫾4.6
2.9⫾1.3
11,235⫾146
12,685⫾306
117.6⫾1.8
90.6⫾1.9
3.12⫾0.05
4.91⫾0.05
2.48⫾0.04
0
software AcqKnowledge for Windows 3.0 (Biopac Systems, Goleta,
CA), left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), the maximum rate of isovolumic pressure
development (⫹dP/dtmax), the maximum rate of isovolumic pressure
decay (⫺dP/dtmax), aortic systolic pressure (ASP), and aortic diastolic
pressure (ADP) were recorded.
Tissue weights and sample collection. The livers and lungs were
weighed, chopped into small pieces, and dried in an oven at 65°C until
a stable weight was recorded. The liver and lung wet-to-dry weight
ratios were obtained (23). The hearts were quickly removed, and the
ventricles were weighed and cut into two pieces. One-half of the heart
was stored in RNA-Later solution (from Ambion) for gene-chip
analysis, and the other half was kept in liquid nitrogen for other
studies.
Affymetrix gene-chip probe array analyses. Total RNA was isolated (TriReagent kits) and purified (QIAGEN kits). Two mRNA
samples each from the control, ADR, and ADR ⫹ probucol groups
were sent to The Centre for Applied Genomics (Hospital for Sick
Children) for rat genome array (U34A) analyses.
Western blot analysis. Phosphorylated and total ERK1/2, p38, and
JNKs MAPKs were examined by Western blot analysis, using PhosphoPlus MAPKs antibody kits (Cell Signaling Technology). For
protein isolation, the frozen heart tissues were powdered in liquid
nitrogen and then suspended in cell lysis buffer [20 mM Tris (pH 7.5),
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium
pyrophosphate, 1 mM ␤-glycerolphosphate, 1 mM Na3VO4, and 1
␮g/ml leupeptin]. The samples were homogenized and sonicated. The
lysates were centrifuged at 14,000 rpm for 10 min, and the supernatant
was transferred into a new tube and stored at 80°C. The quantity of the
protein in the samples was measured by a dye-binding assay (Bio-Rad
Laboratories) using bovine serum albumin as a standard. The isolated
protein was subjected to SDS-PAGE and transferred to 0.45-␮m
nitrocellulose membranes. The detection of membrane-bound proteins
was performed with BM chemiluminescence blotting substrate (POD)
kit (from Roche). The bands were visualized with Fluor S-MultiImager MAX system (Bio-Rad) and quantified by image analysis software (Quantity One, Bio-Rad).
Pro- and anti-apoptotic proteins, caspase-3, Bax, and Bcl-xl were
examined with their specific antibodies (bought from Cell Signaling
Technology).
Statistical analysis. Data were expressed as means ⫾ SE. Group
means were compared by one-way ANOVA, and the Bonferroni’s test
was used to identify differences between groups. The accepted level
of significance was P ⬍ 0.05.
RESULTS
General observations, body weight, tissue weight, and hemodynamics. At the earlier time points (1–24 h), animals in all
of the groups did not show any dyspnea or ascites, and no
mortality was seen. During the 3-wk posttreatment period, rats
in the ADR group developed a progressively enlarged abdomen, ascites, and dyspnea and showed 30% mortality related to
Table 2. DNA microarray analysis for gene expression of MAPK signaling cascade proteins in the late-stage (3 wk) group
Control
Accession No.
AJ00542
M61177
M64300
M64301
U73142
L27112
L27129
X96488
L27128
L04485
L14936
U48596
U23438
ADR ⫹ Pro
ADR
Gene Description
Signal
Detection
Signal
Detection
Signal
Detection
BMK1/ERK5 protein
ERK1
ERK2
ERK3
p38 MAPK
SAP kinase ␣II mRNA
SAP kinase ␥-isoform
mRNA for SAP kinase-3
SAP kinase ␤-isoform
MAP kinase
MKK2
MEKK1
MKP-2
18.4
22.5
101.5
278.6
305.5
9.1
26.8
36.2
4
188.1
95.2
53.2
6.2
A
A
P
P
P
A
A
A
A
P
A
P
A
10.6
108.3
63
238.1
117.8
22.1
24.8
32.9
14.4
281.7
85
16.8
4.9
A
A
P
P
P
A
A
A
A
P
A
A
A
16.4
124
93.9
188.2
269.5
12.2
17.9
40.6
8.8
309.8
174.6
46.8
14.1
A
A
P
P
P
A
A
A
A
P
A
P
A
A, absent; P, present. Average of two independent assays. ERK, extracellular signal-regulated kinase 1; MAPK, mitogen-activated protein kinase; SAP,
stress-activated protein kinase; MKK2, MAPK kinase; MEKK1, MAPK kinase kinase; MKP-2, MAPK phosphatase; ADR, adriamycin; Pro, Probucol.
AJP-Heart Circ Physiol • VOL
288 • APRIL 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 16, 2017
Data are means ⫾ SE of 5– 6 rats in each group. Because of the high mortality in the adriamycin (ADR) group, the starting number of animals was higher.
ADR, adriamycin; Pro, Probucol; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; ⫹dP/dt, rate of pressure development;
⫺dP/dt rate of pressure decay; ASP, aortic systolic pressure; ADP, aortic diastolic pressure. *P ⬍ 0.05 compared with control group.
MAP KINASES AND HEART FAILURE
AJP-Heart Circ Physiol • VOL
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 16, 2017
heart failure. The ascites was hemorrhagic and ranged 50 –140
ml. The livers of ADR-treated rats were enlarged and appeared
dark in color. There was a significant increase in the wet-to-dry
weight ratio in the liver and lung (Table 1). During the 2-wk
treatment period, animals in the ADR and ADR ⫹ probucol
groups showed a significant decrease in heart and body
weights. But there was a gain in body weight in the posttreatment duration. However, this weight gain never reached the
weights of animals in the control group.
In the 3-wk group, aortic systolic pressure (ASP), aortic
diastolic pressure (ADP), and left ventricular peak systolic
pressure (LVSP) were significantly lower in the ADR group.
However, LVEDP was elevated more than fourfold in the ADR
group. ⫾dP/dt values were depressed in the ADR group.
Probucol prevented these changes as well as heart failure
(Table 1).
Myocardial MAPKs mRNA expression in ADR-induced
heart failure. Gene expression of myocardial ERK1/2, p38,
and JNKs kinases and their tiered cascade proteins at the heart
failure stage was analyzed using DNA microarrays analysis,
and the results are shown in Table 2. ERK2 and p38 mRNA
expression was decreased in the failing hearts in the ADR
group. JNK gene expression was undetectable in both ADR
and control groups. Expression of MEKK1, an ERK1/2 kinases
upstream stimulator, was also decreased in the ADR group.
Probucol modulated these decreases in mRNA levels of ERK2
and p38 and MEKK1 kinases.
Phosphorylation of different MAPKs in the early stage of
AIC. Phosphorylation of ERK1/2, p38, and JNK kinases in the
myocardium at 1, 2, 4, and 24 h after the last injection of ADR
was studied, and the data are shown in Fig. 1, A–C. Phosphorylation of ERK1/2, in the early stage of AIC, showed a
biphasic response (Fig. 1A). There was a 167% increase at 1 h,
209% at 2 h, and a 513% peak in the 4-h group. At 24 h, it
decreased to 197% of control. Phosphorylation of p38 showed
a steady increase through 2, 4, and 24 h (119%, 138%, and
148%, respectively), as shown in Fig. 1B. A gradual increase
phosphorylation of JNK was also seen through 1, 2, 4, and 24 h
(116%, 124%, 127%, and 148%, respecitvely), as shown in
Fig. 1C.
Phosphorylation of different MAPKs in ADR-induced heart
failure. After 3 wk of posttreatment, phosphorylation of
ERK1/2, p38, and JNK kinases was examined in the hearts
from the control, ADR, ADR ⫹ probucol, and probucol
groups, and the data are shown in Fig. 2, A–C. Phosphorylation
of ERK1/2 showed a significant reduction by 66.8% of the
control, and probucol prevented this decrease (Fig. 2A). In
contrast, as shown in Fig. 2, B and C, phosphorylation of both
p38 and JNK showed a significant increase to about 148% and
147%, respectively, in the ADR group compared with the
control group. Probucol in the ADR ⫹ probucol group prevented this ADR-induced activation of p38 and JNKs kinases.
Caspase-3 and Bax/Bcl-xl in AIC. Full length as well as
cleaved caspase-3 proteins were examined in both early and
late stages of AIC by Western blot analysis, and these data are
shown in Fig. 3. The activity of caspase-3 was expressed as the
ratio of cleaved caspase-3 over full length of caspase-3. When
compared with the ratio in the control heart set at 100%,
caspase-3 was activated in both the early and late stages of
H1927
Fig. 1. A–C: phosphorylation of extracellular signal-regulated kinase
(ERK1/2) (A), p38 (B), and c-Jun NH2 terminal kinase (JNK, C) mitogenactivated protein (MAP) kinases in adriamycin (ADR)-induced early cardiomyopathy (at 1 to 24 h). UP, myocardial phosphorylated MAP kinase was
measured by Western blot analysis with a phospho-specific MAP kinase
antibody. Total amounts of MAP kinases were examined from the same
stripped membranes for each kinase with the kinase-specific antibodies. LP,
densitometric analysis of MAP kinases activities. Values are the ratio of
phospho-specific MAP kinases to total MAP kinases. Results were normalized
for all experiments by a random setting of the densitometer of the control heart
samples as 100%. Data in the LP are means ⫾ SE from 4 hearts in independent
experiments. *P ⬍ 0.05 vs. control.
288 • APRIL 2005 •
www.ajpheart.org
H1928
MAP KINASES AND HEART FAILURE
AIC, and its activity was increased in a time-dependent
manner.
Pro- as well as anti-apoptotic proteins Bax and Bcl-xl were
detected in both early and late stages of AIC using Western
blot analysis, and the data are shown in Fig. 4. The ratio of Bax
over Bcl-xl showed a significant increase starting at 4 h after
the last injection of ADR and it continued to rise for 3 wk
(Fig. 4).
DISCUSSION
The current study is the first to investigate the MAPK
signaling pathways and pro- and anti-apoptotic proteins in both
early and late stages of AIC. The major findings are the
following: 1) ADR treatment activated all three MAPKs at
early time points in the evolution of cardiomyopathy; 2)
phosphorylation of ERK1/2 was upregulated at early time
points, followed by a decline in the heart failure stage; 3)
phosphorylation of P38 and JNK MAPKs was elevated at all
time points during the progression of heart failure; 4) antioxidant probucol modulated the phosphorylation of all three
MAPKs, indicating the activation of MAPKs through oxidative
stress; and 5) myocardial proapoptotic proteins were activated
in both early and late stages of AIC, which were shown as the
activation of caspase-3 and increase in the ratio of Bax to
Bcl-xl.
MAPKs in ADR-induced heart failure. Myocardial MAPKs
have been examined extensively in heart failure patients; however, there is a significant variance in the information reported.
In this regard, activities of all three MAPKs were reported to
be elevated in human patients with heart failure due to ischeFig. 2. A–C: effects of ADR with and without probucol (Pro) on the activity
of MAPKs, ERK1/2 (A), p38 (B), and JNKs (C) in ADR-induced heart failure.
UP, myocardial phosphorylated MAP kinases were measured by Western blot
analysis with phospho-specific MAPKs antibody. Total amount of MAP
kinases were examined from the same stripped membrane for each MAP
kinase with the kinase-specific antibodies. LP, densitometric analysis of MAP
kinase activation. Values are the ratio of phospho-specific MAP kinases to total
MAP kinases. Results were normalized for all experiments by a random setting
of the densitometer of the control heart samples as 100%. Data in the LP are
means ⫾ SE from 4 hearts in independent experiments. *P ⬍ 0.05 vs. control;
#P ⬍ 0.05 vs. ADR.
AJP-Heart Circ Physiol • VOL
288 • APRIL 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 16, 2017
Fig. 3. Time course (1 h, 2 h, 4 h, 24 h, and 3 wk) of activation of caspase-3
in AIC. UP, myocardial caspase-3 was measured by Western blot analysis with
cleaved caspase-3 antibody, and the same stripped membrane was examined
with caspase-3 antibody. LP, densitometry analysis of caspase-3 activation.
Values are the ratio of cleaved caspase-3 to full length of caspase-3. Results
were normalized by setting the densitometer of the control samples as 100%.
MAP KINASES AND HEART FAILURE
mic cardiomyopathy and idiopathic dilated cardiomyopathy
(8). In another study, only the activities of JNK and p38 were
reported to be increased, whereas ERK1/2 had no change in
heart failure due to ischemic heart disease (7). In contrast,
decreased activity of p38 was reported in the failing myocardium from ischemic and idiopathic dilated cardiomyopathy
(14). These are a few examples from many suggesting that
MAPKs may be differentially regulated. Thus an unique balance among MAPKs pathways may exist, depending on the
stage of heart failure as well as the types of the stimulus.
A study of the time course of changes in MAPKs during the
pathogenesis of heart failure, such as is done in this study, is
important to sketch a comprehensive picture. Our data show
that ERK1/2 was activated early but transiently and downregulated during the heart failure stage. The early upregulation may
have been an adaptive and protective response. In contrast,
phosphorylation of p38 and JNKs kinases increased early and
persisted until the heart failure stage, suggesting that these
kinases may play a dominant role in the progression of AIC
and heart failure.
MAPKs and ADR-induced oxidative stress. It has been well
documented that oxidative stress activates MAPKs. In the
perfused rat heart and cultured cardiomycytes, H2O2 activated
all three MAPKs (1, 3, 19). N-(2-mercaptopropionyl)-glycine,
DMSO, and catalase, but not SOD, markedly depressed daunomycin-mediated activation of MAPKs, suggesting that ⫺OH
and H2O2, but not O⫺
2 are mainly involved in the activation of
MAPKs (30). All three MAPKs were also reported to be
activated in ischemia-reperfusion injury, mediated by oxidative
stress (20). It is widely accepted that ADR induces cardiomyopathy by producing reactive oxygen species as well as by
decreasing the antioxidant reserve (2). In our previous report,
with the use of the same AIC model, lipid peroxidation occurred as early as 1 h and continued to increase up to 24 h, and
there was a corresponding decrease in glutathione peroxidase
and MnSOD activities (15). In the present study, antioxidant
probucol markedly modulated ADR-induced changes in the
phosphorylation of all three MAPKs, indicating that the ADRAJP-Heart Circ Physiol • VOL
induced activation of MAPKs may also be modulated through
increased oxidative stress.
MAPKs and ADR-induced apoptosis. Isolated rat cardiomyocytes exposed to ADR showed the occurrence of apoptosis in
terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling assay, DNA laddering, and electron microscopic
evaluation (2, 9, 10). Time-dependent apoptosis was also
observed in ADR-treated rats at a cumulative dose of 15
mg/kg. The data revealed a biphasic response of apoptosis,
peaking at 4 and 21 days after ADR treatment (11). It is also
reported that apoptosis occurred in both cardiomyocytes and
endothelial cells in ADR-treated rats at a cumulated dose of 24
mg/kg for over 2 wk (29). Even a single dose of ADR at 15
mg/kg also induced apoptosis in the mice (9). In the current
study, activation of caspase-3 as well as the increase in the
ratio of Bax to Bcl-xl were shown in both early AIC and heart
failure. These changes were preceded as well as accompanied
by the activation of p38 and JNK kinases. These MAPKs have
been indicated to mediate death signaling pathways in response
to ischemia-reperfusion, oxidative stress, hypoxia, and ␤-adrenergic stimulation (4). ERK1/2 and p38 kinases were reported to play an opposite role in daunomycin-induced apoptosis. Inhibition of ERK1/2 activation was demonstrated to
increase daunomycin-induced apoptosis, whereas p38 inhibitor
reduced the numbers of apoptotic cells (30). On the basis of the
results obtained from the present study as well as existing
reports, it is suggested that MAPKs play an important role in
the ADR-induced apoptosis.
It was surprising to note that p38 mRNA expression was
downregulated while its activity was increased. It is likely that
there exists a highly differentiated p38 pathways that is mediated by different isoforms of the p38 MAPKs. For example, the
p38␣ isoform was shown to play an important role in the
induction of apoptosis, whereas p38␤ was capable of inducing
a typical hypertrophic response in cardiomyocytes (3, 9, 27).
Because cardiomyocyte apoptosis is shown to be induced in the
AIC, it is possible that in the heart failure stage of AIC, p38
mRNA expression was decreased due to the downregulation of
the p38␤ isoform, whereas p38␣ may not change or may even
increase. However, when the phosphorylation of p38 was
detected, p38␣ may be the dominant isoform in the determination of the p38 activity. The possibility of posttranslational
changes and/or reduced degradation of this protein kinase
cannot be excluded.
Apart from inducing apoptosis, the activation of caspase-3 in
the heart has also been reported to result in depressed cardiac
function, myofibril disruption, and sarcomere disorganization
through its ability to cleave cardiac myofibrillar proteins, such
as ventricular myosin light chain, ␣-actin, ␣-actinin, and troponin T (6, 18). Therefore, the activation of apoptotic signaling
proteins may mediate both cardiac dysfunction and apoptosis
in the progression of AIC.
In conclusion, the present study suggests that ADR-induced
oxidative stress may stimulate MAPKs signaling pathways,
leading to activation of proapoptotic proteins, myocardial apoptosis, and progression of heart failure. Exploring the potential
for modulating MAPK pathways would likely result in novel
approaches to prevent or treat AIC and heart failure.
288 • APRIL 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 16, 2017
Fig. 4. Time course of the ratio of Bax to Bcl-xl in the AIC. UP, myocardial
Bax and Bcl-xl proteins were measured by Western blot analysis with their
specific antibodies. Two proteins were detected from the same stripped
membrane. LP, densitometry analysis of Bax and Bcl-xl. Values are the ratio
of Bax to Bcl-xl normalized by setting the densitometer of the control samples
as 100%.
H1929
H1930
MAP KINASES AND HEART FAILURE
GRANTS
The study was supported by the Canadian Institutes of Health Research and
the Heart and Stroke Foundation of Manitoba (Interdisciplinary Health Research Team on Gene Environment). H. Lou is supported by a student
fellowship from the University of Manitoba. I. Danelisen is supported by the
student fellowship from the Heart and Stroke Foundation of Canada.
REFERENCES
AJP-Heart Circ Physiol • VOL
288 • APRIL 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 16, 2017
1. Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Tanaka M,
Shiojima I, Hiroi Y, and Yazaki Y. Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac
myocytes of neonatal rats. J Clin Invest 100: 1813–1821, 1997.
2. Arola OJ, Saraste A, Pulkki K, Kallajoki M, Parvinen M, and
Voipio-Pulkki LM. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res 60: 1789 –1792, 2000.
3. Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, Fuller SJ, Ben
Levy R, Ashworth A, Marshall CJ, and Sugden PH. Stimulation of the
stress-activated mitogen-activated protein kinase subfamilies in perfused
heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal
kinases are activated by ischemia/reperfusion. Circ Res 79: 162–173,
1996.
4. Bueno OF and Molkentin JD. Involvement of extracellular signalregulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res 91:
776 –781, 2002.
5. Clerk A, Michael A, and Sugden PH. Stimulation of multiple mitogenactivated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular
myocytes. Biochem J 333: 581–589, 1998.
6. Communal C, Sumandea M, de Tombe P, Narula J, Solaro RJ, and
Hajjar RJ. Functional consequences of caspase activation in cardiac
myocytes. Proc Natl Acad Sci USA 99: 6252– 6256, 2002.
7. Cook SA, Sugden PH, and Clerk A. Activation of c-Jun N-terminal
kinases and p38-mitogen-activated protein kinases in human heart failure
secondary to ischaemic heart disease. J Mol Cell Cardiol 31: 1429 –1434,
1999.
8. Haq S, Choukroun G, Lim H, Tymitz KM, del Monte F, Gwathmey J,
Grazette L, Michael A, Hajjar R, Force T, and Molkentin JD.
Differential activation of signal transduction pathways in human hearts
with hypertrophy versus advanced heart failure. Circulation 103: 670 –
677, 2001.
9. Kang YJ, Zhou ZX, Wang GW, Buridi A, and Klein JB. Suppression
by metallothionein of doxorubicin-induced cardiomyocyte apoptosis
through inhibition of p38 mitogen-activated protein kinases. J Biol Chem
275: 13690 –13698, 2000.
10. Kumar D, Kirshenbaum L, Li T, Danelisen I, and Singal P. Apoptosis
in isolated adult cardiomyocytes exposed to adriamycin. Ann NY Acad Sci
874: 156 –168, 1999.
11. Kumar D, Kirshenbaum LA, Li T, Danelisen I, and Singal PK.
Apoptosis in adriamycin cardiomyopathy and its modulation by probucol.
Antioxid Redox Signal 3: 135–145, 2001.
12. Kyriakis JM and Avruch J. Sounding the alarm: protein kinase cascades
activated by stress and inflammation. J Biol Chem 271: 24313–24316,
1996.
13. Lefrak EA, Pitha J, Rosenheim S, and Gottlieb JA. A clinicopathologic
analysis of adriamycin cardiotoxicity. Cancer 32: 302–314, 1973.
14. Lemke LE, Bloem LJ, Fouts R, Esterman M, Sandusky G, and Vlahos
CJ. Decreased p38 MAPK activity in end-stage failing human myocardium: p38 MAPK alpha is the predominant isoform expressed in human
heart. J Mol Cell Cardiol 33: 1527–1540, 2001.
15. Li T, Danelisen I, and Singal PK. Early changes in myocardial antioxidant enzymes in rats treated with adriamycin. Mol Cell Biochem 232:
19 –26, 2002.
16. Li T and Singal PK. Adriamycin-induced early changes in myocardial
antioxidant enzymes and their modulation by probucol. Circulation 102:
2105–2110, 2000.
17. Lou H, Danelisen I, and Singal PK. Cytokines are not upregulated in
adriamycin-induced cardiomyopathy and heart failure. J Mol Cell Cardiol
36: 683– 690, 2004.
18. Moretti A, Weig HJ, Ott T, Seyfarth M, Holthoff HP, Grewe D,
Gillitzer A, Bott-Flugel L, Schomig A, Ungerer M, and Laugwitz KL.
Essential myosin light chain as a target for caspase-3 in failing myocardium. Proc Natl Acad Sci USA 99: 11860 –11865, 2002.
19. Sabri A, Byron KL, Samarel AM, Bell J, and Lucchesi PA. Hydrogen
peroxide activates mitogen-activated protein kinases and Na⫹-H⫹ exchange in neonatal rat cardiac myocytes. Circ Res 82: 1053–1062, 1998.
20. Sawyer DB, Siwik DA, Xiao L, Pimentel DR, Singh K, and Colucci
WS. Role of oxidative stress in myocardial hypertrophy and failure. J Mol
Cell Cardiol 34: 379 –388, 2002.
21. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, and Chien
KR. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a
mitogen-activated protein kinase-dependent pathway. Divergence from
downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem
272: 5783–5791, 1997.
22. Singal PK and Iliskovic N. Doxorubicin-induced cardiomyopathy.
N Engl J Med 339: 900 –905, 1998.
23. Siveski-Iliskovic N, Kaul N, and Singal PK. Probucol promotes endogenous antioxidants and provides protection against adriamycin-induced
cardiomyopathy in rats. Circulation 89: 2829 –2835, 1994.
24. Sugden PH and Bogoyevitch MA. Intracellular signalling through protein kinases in the heart. Cardiovasc Res 30: 478 – 492, 1995.
25. Sugden PH and Clerk A. “Stress-responsive” mitogen-activated protein
kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein
kinases) in the myocardium. Circ Res 83: 345–352, 1998.
26. Wang X, Martindale JL, Liu Y, and Holbrook NJ. The cellular
response to oxidative stress: influences of mitogen-activated protein kinase
signalling pathways on cell survival. Biochem J 333: 291–300, 1998.
27. Wang Y, Huang S, Sah VP, Ross J Jr, Brown JH, Han J, and Chien
KR. Cardiac muscle cell hypertrophy and apoptosis induced by distinct
members of the p38 mitogen-activated protein kinase family. J Biol Chem
273: 2161–2168, 1998.
28. Wang Y, Su B, Sah VP, Brown JH, Han J, and Chien KR. Cardiac
hypertrophy induced by mitogen-activated protein kinase kinase 7, a
specific activator for c-Jun NH2-terminal kinase in ventricular muscle
cells. J Biol Chem 273: 5423–5426, 1998.
29. Wu S, Ko YS, Teng MS, Ko YL, Hsu LA, Hsueh C, Chou YY, Liew
CC, and Lee YS. Adriamycin-induced cardiomyocyte and endothelial cell
apoptosis: in vitro and in vivo studies. J Mol Cell Cardiol 34: 1595–1607,
2002.
30. Zhu W, Zou Y, Aikawa R, Harada K, Kudoh S, Uozumi H, Hayashi
D, Gu Y, Yamazaki T, Nagai R, Yazaki Y, and Komuro I. MAPK
superfamily plays an important role in daunomycin-induced apoptosis of
cardiac myocytes. Circulation 100: 2100 –2107, 1999.