1 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 2 Department of Biochemistry, University of Alberta at Edmonton, Edmonton, Alberta, Canada T6G 2H7
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ABSTRACT |
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Bursts in reactive oxygen species production are important mediators of contractile dysfunction during ischemia-reperfusion injury. Cellular mechanisms that mediate reactive oxygen species-induced changes in cardiac myocyte function have not been fully characterized. In the present study, H2O2 (50 µM) decreased contractility of adult rat ventricular myocytes. H2O2 caused a concentration- and time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein (MAP) kinases in adult rat ventricular myocytes. H2O2 (50 µM) caused transient activation of ERK1/2 and p38 MAP kinase that was detected as early as 5 min, was maximal at 20 min (9.6 ± 1.2- and 9.0 ± 1.6-fold, respectively, vs. control), and returned to baseline at 60 min. JNK activation occurred more slowly (1.6 ± 0.2-fold vs. control at 60 min) but was sustained at 3.5 h. The protein kinase C inhibitor chelerythrine completely blocked JNK activation and reduced ERK1/2 and p38 activation. The tyrosine kinase inhibitors genistein and PP-2 blocked JNK, but not ERK1/2 and p38, activation. H2O2-induced Na+/H+ exchanger phosphorylation was blocked by the MAP kinase kinase inhibitor U-0126 (5 µM). These results demonstrate that H2O2-induced activation of MAP kinases may contribute to cardiac myocyte dysfunction during ischemia-reperfusion.
reactive oxygen species; extracellular signal-regulated kinase; c-Jun NH2-terminal kinase; p38 mitogen-activated protein kinase
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INTRODUCTION |
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REPERFUSION OF AN ISCHEMIC myocardium leads to a burst in the production of reactive oxygen species (ROS) that is associated with contractile dysfunction, Ca2+ overload, and myocardial stunning (15). Ischemia-reperfusion injury also initiates activation of several signaling cascades, including mitogen-activated protein (MAP) kinases and tyrosine kinases such as Src (1, 7, 31, 38). However, the exact contribution of ROS to the activation of these signaling events remains unknown.
MAP kinases regulate a number of cellular events that are altered during the reperfusion of an ischemic myocardium, including phosphorylation of heat shock proteins 20 and 27 (13), regulation of apoptotic signaling cascades (see Ref. 37 for a recent review), enhanced gene expression (8), and phosphorylation of the Na+/H+ exchanger (NHE) to increase its activity (39). Activation of NHE is responsible for the postischemic restoration of intracellular pH (pHi). The increased Na+ influx that arises from NHE activation leads to a secondary increase in intracellular Ca2+ levels via an indirect inhibition of Na+/Ca2+ exchange (12, 19, 40).
Three subfamilies of MAP kinases have been clearly identified in the
heart: the extracellular signal-regulated kinases (ERK1/2), the c-Jun
NH2-terminal kinases (JNKs), and the p38 MAP kinases. We
and others have investigated the ability of ROS to regulate MAP kinase
signaling in primary cultured neonatal cardiac myocytes (3,
31). Physiological concentrations of
H2O2 (5-100 µM) activated NHE1 in an
ERK1/2-dependent manner in cultured neonatal cardiac myocytes
(31). However, extrapolation of these results to adult
cells is difficult, because there are marked phenotypic differences
between neonatal and adult cardiac myocytes, including age-dependent
changes in protein kinase C (PKC) expression (30), tyrosine kinase-dependent inhibition of L-type Ca2+
channels (20), and -adrenergic receptor signaling
(23). In addition, alterations in ERK signaling between
neonatal and adult cardiac myocytes in response to angiotensin II have
been reported (11, 32). Therefore, identification of the
cellular signaling events involved in
H2O2-induced MAP kinase activation in primary
cultured adult rat ventricular myocytes (ARVM) is important for an
understanding of in vivo changes in cardiac function during the
ischemia-reperfusion injury.
Although several studies have demonstrated the effects of high doses of H2O2 on adult cardiac myocyte function (17, 18), little is known about the effect of low, physiological concentrations of H2O2 on intracellular signal transduction. The present study demonstrates a significant decrease in ARVM contractility by low doses of H2O2. Exposure of primary culture of ARVM to these low doses of H2O2 activates ERK1/2, p38 MAP kinase, and JNK members of the MAP kinase superfamily. Of these three MAP kinases, the ERK1/2 pathway mediated H2O2-induced phosphorylation of NHE1.
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METHODS |
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Isolation of ARVM. ARVM were isolated from male Sprague-Dawley rats (250-350 g) by cardiac retrograde aortic perfusion, as previously described (35), with minor modifications. Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The hearts were rapidly dissected and perfused with Krebs buffer (mM: 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.8 CaCl2, 1.2 Mg2SO4, 1.2 KH2PO4, and 11 glucose), Ca2+-free Krebs buffer, and Ca2+-free Krebs buffer containing 100 U/ml type II collagenase (Worthington). Ventricular tissue was minced and further digested in collagenase-BSA Krebs buffer containing 25 µM CaCl2. The resulting cells were resuspended in perfusion buffer with 3% BSA, in which the concentration of CaCl2 was increased in a stepwise manner to 1 mM. Cells were resuspended in medium 199 (M199) supplemented with 5 mM creatine, 2 mM L-carnitine, and 5 mM taurine and then plated on laminin-coated dishes at a density of 4 × 105/100 mm. After 1 h, the medium was changed to remove loosely attached cells. Rod-shaped myocytes (>90%) were cultured for an additional 1 h before the start of the experiments.
Preparation of cell lysates for MAP kinase
experiments.
ARVM were incubated at 37°C in M199 containing
H2O2, inhibitors, or vehicle for various times,
lysed in ice-cold buffer containing 50 mM NaCl, 50 mM NaF, 50 mM sodium
pyrophosphate, 5 mM EDTA, 5 mM EGTA, 2 mM
Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM HEPES, pH 7.4, 0.1% Triton X-100, and 10 µg/ml leupeptin, and
immediately frozen on ethanol-dry ice. Cell lysates were then thawed on
ice, scraped, sonicated, and centrifuged at 10,000 g at
4°C for 30 min. Supernatants were used immediately or stored at
80°C. Protein concentrations were determined using a bicinchoninic acid protein assay (Pierce) according to the manufacturer's protocol.
Western blot analysis. Western blot analysis was performed as described elsewhere (31). Cell lysates (35 µg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The primary antibodies were anti-total ERK1 and ERK2 antibodies (1:5,000; Santa Cruz), anti-active ERK1/2 (1:3,000; Promega), anti-total p38 (1:1,000; New England Biolabs), and anti-phospho-p38 MAP kinase (1:2,000; New England Biolabs). Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham). Autoradiograms exposed in the linear range of film density were scanned, and densitometric analysis was performed with NIH Image software.
JNK immune complex kinase assays.
ARVM were lysed by the addition of RIPA buffer (150 mM NaCl, 1.5 mM
MgCl2, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM EGTA, 0.1 mM Na3VO4, 24 mM sodium deoxycholate, and
25 mM HEPES, pH 7.4, 1% Triton X-100, 0.1% SDS, and 10%
glycerol). Cell lysates containing 600 µg of protein were
incubated with 4 µg of anti-JNK1/SAPK antibody (UBI) overnight at
4°C. Lysates were then incubated with 40 µl of protein A-agarose
beads for 90 min at 4°C, and the beads were collected by
centrifugation. The beads were washed once with 500 µl of RIPA
buffer, once with 500 µl of wash buffer A (0.5 mM LiCl and 20 mM
Tris), once with wash buffer B (1 mM EDTA and 20 mM Tris), and once
with kinase buffer (10 mM MnCl2, 1 mM dithiothreitol, and
20 mM Tris). Immunoprecipitates were resuspended in 10 µl of kinase
buffer supplemented with 5 µM ATP, 5 mM MgCl2, 10 µCi of [-32P]ATP, and 4 µg of glutathione
S-transferase (GST)-c-Jun-(1-79) (Calbiochem). After incubation for 20 min at 20°C, the reaction was
terminated by adding Laemmli sample buffer and heating at 100°C for
10 min. c-Jun was resolved by SDS-PAGE. Incorporation of
32P into GST-c-Jun-(1-79) was determined
by densitometric analysis of the c-Jun band identified by autoradiography.
In-gel kinase assays.
NHE1 kinase activity and MAP kinase activity were analyzed by the
in-gel kinase assay as described previously (31). Cell lysates (35 µg) were fractionated by SDS-PAGE in a gel in which 0.15 mg/ml of NHE1 fusion protein (amino acids 501-815 of NHE1 coupled
to GST) or 0.1 mg/ml of myelin basic protein (MBP) had been
copolymerized. The phosphorylation assay was performed by placing the
gel in 10 ml of buffer containing 50 µM ATP with 100 µCi of
[-32P]ATP and incubating it for 1 h at 30°C.
The reaction was terminated by immersing the gel in fixative solution
(5% trichloroacetic acid and 10 mM sodium pyrophosphate). The
radioactivity was quantified by densitometric analysis of scanned images.
Measurement of single-cell contractility. Myocytes were cultured for 1-2 h in M199 on laminin-coated glass coverslips. The coverslip was placed in a perfusion chamber with internal stimulating platinum electrodes (Warner) and imaged using an inverted microscope (model IX50, Olympus) and charge-coupled device camera (Philips). Rod-shaped cells with no blebbing or spontaneous contractions were chosen for imaging. The chamber was perfused with Tyrode's basic salt solution for 2 min, and cells were paced at 2 Hz (3-ms duration). This equilibration period was followed by a 10-min perfusion with 50 µM H2O2 in Tyrode's basic salt solution and a 10-min washout period with continued stimulation throughout the experiment. Cell contractility was recorded with a video edge detection system (Crescent Electronics) and analyzed using the Digi-Med System Integrator (model 2001) and the Cell Length Analysis program (MicroMed). The average resting cell length was 123 ± 6 µm (n = 11). Percent contraction was defined as the change in cell length divided by the resting length and averaged 9.7 ± 0.4% in control cells at 2 Hz. Changes in contractile amplitude were expressed relative to the percent contraction of control cells (set at 100%).
Data analysis. Data were analyzed using InStat statistical software (Graphpad). Values are means ± SE. One-way analysis of variance with a Dunnett's post test was used to compare control with treated groups. Differences between groups were considered statistically significant at P < 0.05.
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RESULTS |
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Low concentration of H2O2
elicited a significant decrease in ARVM contractility.
A video edge detection system was used to monitor the effects of
H2O2 on single ARVM contractility (Fig.
1). ARVM were paced at 2 Hz for a 2-min
equilibration period. Exposure to 50 µM H2O2 for 10 min induced a significant, 35% decrease in the contraction amplitude of ARVM compared with control. This contractile impairment was sustained throughout the 10-min washout period.
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Activation of ERK1/2, p38 MAP kinase,
and JNK by low concentrations of
H2O2.
ARVM were exposed to H2O2 (5-200 µM) for
20 min. Activation of ERK1/2 was detected by Western blot analysis with
antibodies that recognize phosphorylated, active ERK1/2.
H2O2 caused a dose-response-dependent phosphorylation of ERK2 that was detected at 5 µM
H2O2 and was maximal at 50 µM
H2O2 (9.6 ± 1.2-fold vs. control; Fig.
2A, top blot).
H2O2 at 200 µM resulted in a decreased
ERK1/2 activation. There was no difference in ERK1/2 expression as
detected by Western blot analysis with anti-total ERK1/2 antibodies
(Fig. 2A, middle blot). ERK1/2 activation was confirmed
using in-gel kinase assays with MBP as a substrate. Compared with
control, treatment with H2O2 (5-200 µM)
resulted in a concentration-dependent increase in MBP phosphorylation
by 42- and 44-kDa kinases, the molecular weights of ERK2 and ERK1,
respectively (Fig. 2A, bottom blot). Thus both measures of
ERK1/2 MAP kinase activation yielded similar results.
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Time course for MAP kinase activation by
H2O2.
Exposure of ARVM to 50 µM H2O2 resulted in a
rapid but transient phosphorylation of ERK1/2 and p38 (Fig.
3, A and B).
Activation of both MAP kinases was detected at 5 min and was maximal at
20 min (6.2 ± 1.2-fold vs. control for ERK1/2 and 6.3 ± 0.9-fold vs. control for p38) but returned to values close to baseline at 60 min. In contrast, significant JNK activation, as assessed by
immune complex kinase assays, occurred much more slowly but was
prolonged (Fig. 3C). Significant JNK activation by 50 µM
H2O2 did not occur until 60 min (1.7 ± 0.2-fold vs. control) and was sustained at 3.5 h (1.8 ± 0.2-fold vs. control).
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Role of tyrosine kinases and PKC in
H2O2-induced MAP
kinase activation.
To determine whether one or more upstream tyrosine kinases were
involved in the H2O2-induced MAP kinase
activation in ARVM, cells were pretreated with the tyrosine kinase
inhibitor genistein (100 µM) or the selective Src family tyrosine
kinase inhibitor PP-2 (10 µM) for 45 min. Neither genistein nor PP-2
had an effect on the phosphorylation of ERK1/2 and p38 MAP kinase by 50 µM H2O2 (Fig.
6, A and B). In
contrast, genistein and PP-2 caused a 40 and 70% inhibition of
H2O2-induced JNK activation, respectively (Fig.
6C). We next examined whether PKC was involved in the
H2O2-induced MAP kinase activation in ARVM.
Pretreatment with the PKC inhibitor chelerythrine (5 µM, 45 min)
caused a partial inhibition of H2O2-induced activation of ERK1/2 (50%) and p38 MAP kinase (45%) and a complete inhibition of JNK activation (Fig. 6).
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ERK1/2-dependent NHE1 phosphorylation
by H2O2.
We previously showed that low doses of H2O2
cause a rapid, ERK1/2-dependent activation of NHE1 (31).
The primary sequence of NHE1 contains several sites that are suitable
phosphorylation sites for proline-directed MAP kinases
(12). Because NHE1 activity is regulated by
phosphorylation, we determined whether
H2O2-induced MAP kinase activation mediates
NHE1 phosphorylation in ARVM. In-gel kinase assays using a recombinant
NHE1-GST fusion protein as a substrate were performed.
H2O2 induced a concentration-dependent increase
in NHE1 phosphorylation by two proteins that corresponded to ERK1 (44 kDa) and ERK2 (42 kDa; Fig.
7A). The time course of H2O2-induced MAP kinase-dependent NHE
activation was nearly identical to that of
H2O2-induced ERK1/2 phosphorylation (compare
Fig. 7B with Fig. 3A). To confirm that the ERK
pathway is a critical step in H2O2-mediated
stimulation of NHE1, the effects of the specific MAP kinase kinase
(MEK) inhibitor U-0126 and the p38 MAP kinase inhibitor SB-203580 were
studied. Pretreatment with U-0126 (5 µM, 45 min) completely abolished
the ERK1/2-dependent phosphorylation of NHE1 induced by 50 µM
H2O2, whereas pretreatment with SB-203580 (10 µM, 45 min) had no effect (Fig. 7C). U-0126
completely inhibited H2O2-induced ERK1/2
activation (data not shown).
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DISCUSSION |
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The most important findings of the present study are that low
physiological concentrations of H2O2 result in
activation of multiple MAP kinases in adult ventricular myocytes that
are associated with altered contractile function and NHE
phosphorylation. The H2O2 concentrations
(5-100 µM) are similar to those observed during a burst in free
radical production that occurs on reperfusion of an ischemic
myocardium (15). However, 200 µM
H2O2 actually decreased MAP kinase activity,
suggesting that these higher concentrations may exert cytotoxic
effects. H2O2 induced a rapid but transient phosphorylation in ERK1/2 and p38 MAP kinase in ARVM but a slower, more
sustained JNK activation. These MAP kinase activations required only a
brief exposure to H2O2.
The physiological roles of MAP kinase activation during ischemia-reperfusion injury in the intact myocardium are somewhat controversial. For example, ischemia-reperfusion elicited an increase in ERK1/2 activation in conscious rabbits (27), isolated guinea pig hearts (39), and isolated rat hearts (21). In contrast, Bogoyevitch et al. (6) reported that 10-20 min of ischemia with or without reperfusion failed to activate ERK1/2. However, Ping et al. (27) examined MAP kinase activity in the nuclear and cytosolic fractions of heart tissue and found a significant elevation of ERK1/2 only in the nuclear compartment. On the other hand, other groups determined MAP kinase activity only on the cytosolic fraction (6, 7, 21, 39), and in the study by Bogoyevitch et al., ERK activity was measured in the cytosolic fraction after fast protein liquid chromatography on Mono Q. Thus the conflicting results may be attributed to species-related differences or variation in the preparation of myocardium samples. In contrast, our results provide direct evidence that ROS directly activate ERK1/2 MAP kinases in cardiac myocytes.
Our results also demonstrate that low concentrations of H2O2 induced a rapid but transient activation of p38 MAP kinase and a slower, sustained activation of JNK in ARVM. These data are in agreement with previous studies by Ping et al. (28), who reported a sustained activation of JNK, but only a transient activation of p38 MAP kinase, triggered by ischemic preconditioning in the hearts of conscious rabbits. Schneider et al. (34) also reported a transient activation of p38 MAP kinase, whereas Bogoyevitch et al. (6) reported a sustained activation of p38 MAP kinase in isolated rat hearts. Besides the differences in sample preparation for MAP kinase activity assays, these differences may also reflect discrepancies between animal models or ischemic protocols. Our study is the first to demonstrate, on the cellular level, that H2O2 induces a rapid but transient activation of p38 in ARVM.
The activation of MAP kinase by ROS, which contribute to cardiac myocyte dysfunction, appears at first glance to be in contradiction to the well-documented MAP kinase activation by growth factors and neurohormones. However, recent studies suggested the importance of the ERK1/2 pathway for myocyte survival in ischemic myocardium on the basis of the results that pretreatment with the MEK1 inhibitor PD-98059 reduced the number of apoptotic cells on isolated rat hearts subjected to global ischemia (44) and H2O2-treated rat neonatal myocytes (2, 3). Our results do not exclude the possibility of protective roles of MAP kinase activation in ischemic myocardium. For example, it is possible that MAP kinases could regulate the expression and activation of heat shock proteins or the expression of genes that would prevent further myocardial damage (8, 13).
It is also possible that differences in the magnitude and duration of
MAP kinase activation can result in different physiological responses.
For example, transient activation of p38 and JNK by tumor necrosis
factor- is a survival signal, whereas persistent activation induces
apoptosis (16, 29). Transient ERK1/2 activation leads to proliferation, whereas sustained activation mediates growth
arrest (4, 5). In addition, the simultaneous activations of multiple MAP kinases may result in different responses due to cross
talk. Kusuhara et al. (22) showed that activation of p38
MAP kinase leads to decreased ERK1/2 signaling due to inhibition of MEK
activity. Therefore, it is tempting to speculate that different stimuli, such as H2O2 and phenylephrine, may
elicit distinct functional responses by eliciting a specific temporal
or spatial pattern of activation of a single MAP kinase pathway or by
stimulating cross talk between multiple signaling pathways.
The activation of all three MAP kinases by H2O2
in ARVM was PKC dependent (Fig. 6). This is consistent with the finding
that PKC activates MAP kinases in neonatal myocytes (24, 31,
41), isolated rat hearts (6, 14), and in vivo
rabbit hearts (28). Activation of ERK1/2 and p38 by
H2O2 in ARVM was not tyrosine kinase dependent
(Fig. 6). In contrast, the tyrosine kinase inhibitor genistein and the
Src inhibitor PP-2 partially inhibited the activation of JNK by
H2O2. This is consistent with a study in adult
rat myocytes that -adrenoceptor stimulation of ERK1/2 was PKC
dependent, but not genistein sensitive (33). Yoshizumi et
al. (43) reported an Src- and Cas-mediated activation of
JNK, but not ERK1/2 and p38 MAP, kinase by H2O2
in smooth muscle cells and also showed that JNK activation by
H2O2 was completely blocked in cells derived from src-deficient transgenic mice. Our data suggest a
possible mechanism whereby tyrosine kinases are located upstream of JNK activation in response to H2O2, but not
upstream of ERK1/2 and p38 MAP kinase activation. We previously showed
that the activation of ERK1/2 by H2O2 is partly
modulated by the activity of upstream tyrosine kinases and PKC
(31) in neonatal rat ventricular myocytes. Thus a role for
tyrosine kinases in H2O2-induced MAP kinase
activation appears to exhibit age-related differences in cardiac
myocytes. Interestingly, Katsube et al. (20) reported that
inhibition of basal L-type Ca2+ channel currents by
genistein in rat ventricular myocytes was greater in neonatal than in
adult cells, suggesting the existence of age-related changes in the
expression of tyrosine kinases or in their coupling to channel regulation.
Many studies have established a good correlation between myocardial stunning, ROS, and Ca2+ overload with ischemia-reperfusion injury. Early studies described a reversible depression of contractility during moderate ischemia-reperfusion, which was thought to be "cardioprotective," since it would serve to maintain myocardial integrity and viability during persistent ischemia (9). Changes in ATP concentration, pHi, or Ca2+ handling have been proposed as mechanisms responsible for acute, ischemic contractile dysfunction (15). The data from our study suggest that ROS may contribute to ischemia-reperfusion injury through an ERK1/2-dependent NHE1 phosphorylation (Fig. 7). The ability of the MEK inhibitor U-0126, but not the p38 inhibitor SB-203580, to abolish the H2O2-induced increase in NHE1 phosphorylation provides evidence that the ERK1/2, but not the p38, pathway is critical for H2O2-mediated stimulation of NHE1 in ARVM.
There is a large body of evidence to suggest that NHE1 activation
during cardiac ischemia-reperfusion plays a major role in restoring pHi after an acid load. Phosphorylation of the
exchanger is thought to be the primary mechanism responsible for
increased NHE activity during ischemia-reperfusion injury
(10). However, the increases in intracellular
Na+ concentration resulting from enhanced exchanger
activity can result in Ca2+ overload via the
Na+/Ca2+ exchanger and actually exacerbate the
damage to the myocardium (19). Moreover, Myers et al.
(26) reported that the NHE inhibitor cariporide prevented
H2O2-induced impairment of postischemic
ventricular function. ERK1/2 (and the downstream kinase
p90rsk) regulate NHE activity by phosphorylation in the rat
myocardium and in endothelin-stimulated rat neonatal ventricular
myocytes (25). Snabaitis et al. (36)
demonstrated a similar requirement for ERK1/2, but not p38, in an
1-adrenoreceptor-mediated NHE activation in ARVM. On the
other hand, Kusuhara et al. (22) reported that ERK1/2 and
p38 phosphorylate NHE1 in response to angiotensin II in vascular smooth
muscle, although p38-mediated phosphorylation was associated with
decreased NHE activity. The underlying cellular basis for these
discrepancies remains unclear but may be in part due to the differences
in agonists used to stimulate NHE1 phosphorylation or in cell
type-specific regulatory mechanisms.
In conclusion, the present study is the first to characterize, at the cellular level, the activation of multiple MAP kinases in adult cardiac myocytes by ROS at physiologically relevant concentrations. It is possible that the ROS- and ERK1/2-dependent NHE1 activation may contribute to the contractile dysfunction observed during reperfusion injury. Future studies with antisense or dominant-negative strategies are needed to elucidate the exact pathophysiological roles of each kinase using models of oxidant-induced myocardial injury.
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ACKNOWLEDGEMENTS |
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S. Wei is a fellow of the American Heart Association Southeast Affiliate (Grant 00202399B). This work was supported by National Heart, Lung, and Blood Institute Grants HL-56046 and HL-63318 (P. A. Lucchesi) and grants from the Canadian Institute of Health Research and the Heart and Stroke Foundation of Canada (L. Fliegel).
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FOOTNOTES |
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A preliminary report of these findings has been published in abstract form (42).
Address for reprint requests and other correspondence: P. A. Lucchesi, Dept. of Physiology and Biophysics, University of Alabama at Birmingham, 986 MCLM, 1530 3rd Ave. S, Birmingham, AL 35294-0005 (E-mail: lucchesi{at}physiology.uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 February 2001; accepted in final form 5 July 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abe, J,
and
Berk BC.
Reactive oxygen species as mediators of signal transduction in cardiovascular disease.
Trends Cardiovasc Med
8:
59-64,
1998[ISI].
2.
Adderley, SR,
and
Fitzgerald DJ.
Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2.
J Biol Chem
274:
5038-5046,
1999
3.
Aikawa, A,
Komuro I,
Yamazaki Y,
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
4.
Albs, J,
Slager-Davidov R,
Steenbergh PH,
Sussenbach JS,
and
van der Burg B.
The role of MAP kinase in ATP-mediated cell cycle arrest of human breast cancer cells.
Oncogene
16:
131-139,
1998[ISI][Medline].
5.
Bennett, AM,
and
Tonks NK.
Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases.
Science
278:
1288-1291,
1997
6.
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 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
7.
Clerk, A,
Fuller S,
Micheal A,
and
Sugden PH.
Stimulation of "stress-regulated" mitogen-activated protein kinase (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses.
J Biol Chem
237:
7228-7234,
1998.
8.
Clerk, A,
and
Sugden PH.
Cell stress-induced phosphorylation of ATF2 and c-jun transcription factors in rat ventricular myocytes.
Biochem J
325:
801-810,
1997[ISI][Medline].
9.
Diamond, GA,
Forrester JS,
deLuz PL,
Wyatt HL,
and
Swan HJ.
Post-extrasystolic potentiation of ischemic myocardium by atrial stimulation.
Am Heart J
95:
204-209,
1978[ISI][Medline].
10.
Dyck, JRB,
Maddaford TG,
Pierce GN,
and
Fliegel L.
Induction of expression of the sodium-hydrogen exchanger in rat myocardium.
Cardiovasc Res
29:
203-208,
1995[ISI][Medline].
11.
Fischer, TA,
Singh K,
O'Hara DS,
Kaye DM,
and
Kelly RA.
Role of AT1 and AT2 receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes.
Am J Physiol Heart Circ Physiol
275:
H906-H916,
1998
12.
Fliegel, L,
and
Frohlich O.
The Na+/H+ exchanger: an update on structure regulation and cardiac physiology.
Biochem J
296:
273-285,
1993[ISI][Medline].
13.
Freshney, NW,
Rawlison L,
Guesdon F,
Jones E,
Cowley S,
Hsuan J,
and
Saklatvala J.
Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.
Cell
78:
1039-1049,
1994[ISI][Medline].
14.
Fryer, RM,
Schults JEJ,
Hsu AK,
and
Gross GJ.
Importance of PKC and tyrosine kinase in single or multiple cycles of preconditioning in rat hearts.
Am J Physiol Heart Circ Physiol
276:
H1229-H1235,
1999
15.
Goldhaber, JI,
and
Weiss JN.
Oxygen free radicals and cardiac reperfusion abnormalities.
Hypertension
20:
118-127,
1992[Abstract].
16.
Guo, YL,
Baysal K,
Kang B,
Yang LJ,
and
Williamson JR.
Correlation between sustained c-Jun N-terminal protein kinase activation and apoptosis induced by tumor necrosis factor-.
J Biol Chem
273:
4027-4034,
1998
17.
Guyton, KZ,
Liu Y,
Gorospe M,
Xu Q,
and
Holbrook NJ.
Activation of mitogen-activated protein kinase by H2O2.
J Biol Chem
271:
4138-4142,
1996
18.
Josephson, R,
Silverman H,
Lakatta E,
Stern M,
and
Zweier J.
Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.
J Biol Chem
266:
2354-2361,
1991
19.
Karmazyn, M,
Xiaohong T,
Humphreys R,
Yoshida Y,
and
Kusumoto K.
The myocardial Na+-H+ exchanger: structure, regulation, and its role in heart disease.
Circ Res
85:
777-786,
1999
20.
Katsube, Y,
Yokoshiki H,
Nguyen L,
Yamamoto M,
and
Sperelakis N.
Inhibition of Ca2+ current in neonatal and adult rat ventricular myocytes by the tyrosine kinase inhibitor, genistein.
Eur J Pharmacol
345:
309-314,
1998[ISI][Medline].
21.
Knight, RJ,
and
Buxton DB.
Stimulation of c-Jun kinase and mitogen-activated protein kinase by ischemia and reperfusion in the perfused rat heart.
Biochem Biophys Res Commun
218:
83-88,
1996[ISI][Medline].
22.
Kusuhara, M,
Takahashi E,
Peterson TE,
Abe J,
Ishida M,
Han J,
Ulevitch R,
and
Berk BC.
p38 Kinase is a negative regulator of angiotensin II signal transduction in vascular smooth muscle cells: effects on Na+/H+ exchange and ERK1/2.
Circ Res
83:
824-831,
1998
23.
Kuznetsov, V,
Pak E,
Robinson RB,
and
Steinberg SF.
2-Adrenergic receptor actions in neonatal and adult rat ventricular myocytes.
Circ Res
76:
40-52,
1995
24.
Laderoute, KR,
and
Webster KA.
Hypoxia/reoxygenation stimulates Jun kinase activity through redox signaling in cardiac myocytes.
Circ Res
80:
336-344,
1997
25.
Moor, AN,
and
Fliegel L.
Protein kinase-mediated regulation of the Na+/H+ exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways.
J Biol Chem
274:
22985-22992,
1999
26.
Myers, ML,
Farhangkhoee P,
and
Karmazyn M.
Hydrogen peroxide-induced impairment of post-ischemic ventricular function is prevented by sodium-hydrogen exchange inhibitor HOE 642 (cariporide).
Cardiovasc Res
40:
290-296,
1998[ISI][Medline].
27.
Ping, PP,
Zhang J,
Cao X,
Li RCX,
Kong D,
Tang XL,
Qiu Y,
Manchikalapudi S,
Auchampach JA,
Black RG,
and
Bolli R.
PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits.
Am J Physiol Heart Circ Physiol
276:
H1468-H1481,
1999
28.
Ping, P,
Zhang J,
Huang S,
Cao X,
Tang XL,
Li RC,
Zheng YT,
Qiu Y,
Clerk A,
Sugden P,
Han J,
and
Bolli R.
PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits.
Am J Physiol Heart Circ Physiol
277:
H1771-H1785,
1999
29.
Roulston, A,
Reinhard C,
Amiri P,
and
Williams LT.
Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor-.
J Biol Chem
273:
10232-10239,
1998
30.
Rybin, VO,
and
Steinberg SF.
Protein kinase C isoform expression and regulation in the developing rat heart.
Circ Res
74:
299-309,
1994[Abstract].
31.
Sabri, A,
Byron KL,
Samarel AM,
Bell JM,
and
Lucchesi PA.
Hydrogen peroxide activates mitogen-activated protein kinases and Na+/H+ exchange in neonatal rat ventricular myocytes.
Circ Res
82:
1053-1062,
1998
32.
Sadoshima, J,
Qiu Z,
Morgan JP,
and
Izumo S.
Angiotensin II and other hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes.
Circ Res
76:
1-15,
1995
33.
Schluter, KD,
Simm A,
Schafer M,
Taimor G,
and
Piper HM.
Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy.
Am J Physiol Heart Circ Physiol
276:
H1655-H1663,
1999
34.
Schneider, S,
Chen W,
Hou J,
Steenbergen C,
and
Murphy E.
Inhibition of p38 MAPK-/
reduces ischemic injury and does not block protective effects of preconditioning.
Am J Physiol Heart Circ Physiol
280:
H499-H508,
2001
35.
Schwarzfeld, TA,
and
Jacobson SL.
Isolation and development in cell culture of myocardial cells of the adult rat.
J Mol Cell Cardiol
13:
563-575,
1981[ISI][Medline].
36.
Snabaitis, AK,
Hiroyuki Y,
and
Avkiran M.
Roles of mitogen-activated protein kinases and protein kinase C in 1A-adrenoceptor-mediated stimulation of the sarcolemmal Na+-H+ exchanger.
Circ Res
86:
214-220,
2000
37.
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
38.
Takahashi, E,
Abe JI,
Gallis B,
Aebersold R,
Spring DJ,
Krebs EG,
and
Berk BC.
p90 RSK is a serum-stimulated NHE1 kinase: regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1.
J Biol Chem
274:
20206-20214,
1999
39.
Takeishi, Y,
Abe JI,
Lee JD,
Kawakatsu H,
Walsh RA,
and
Berk BC.
Differential regulation of p90 ribosomal S6 kinase and big mitogen-activated protein kinase 1 by ischemia/reperfusion and oxidative stress in perfused guinea pig hearts.
Circ Res
85:
1164-1172,
1999
40.
Wakabayashi, S,
Shigekawa M,
and
Pouyssegur J.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol Rev
77:
51-74,
1997
41.
Webster, KA,
and
Discher DJ.
Induction and nuclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes.
J Biol Chem
268:
16852-16859,
1993
42.
Wei, S,
Rothstein EC,
Dell'Italia LJ,
Fliegel L,
and
Lucchesi PA.
Activation of multiple MAP kinases by H2O2 is associated with Na+/H+ exchanger phosphorylation in adult rat ventricular cardiomyocytes (Abstract).
Circulation
102:
II-84,
2000.
43.
Yoshizumi, M,
Abe J,
Haendeler J,
Huang Q,
and
Berk BC.
Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species.
J Biol Chem
275:
11706-11712,
2000
44.
Yue, TL,
Wang C,
Gu JLM,
Ma XL,
Kumar S,
Lee JC,
Feuerstein GZ,
Thomas H,
Maleeff B,
and
Ohlstein EH.
Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused hearts.
Circ Res
86:
692-699,
2000