Differential MAP kinase activation and Na+/H+ exchanger phosphorylation by H2O2 in rat cardiac myocytes

Shan Wei1, Emily C. Rothstein1, Larry Fliegel2, Louis J. Dell'Italia1, and Pamela A. Lucchesi1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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 [gamma -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Effect of H2O2 on adult rat ventricular myocyte contractility. Amplitude of contraction was measured using video edge detection. Cells stimulated at 2 Hz were perfused for 2 min with Tyrode's basic salt solution, exposed to 50 µM H2O2 for 10 min, and subjected to a 10-min washout period. Values are means ± SE from 5 experiments. Data are expressed relative to control values measured at the start of every experiment. *P < 0.05; Dagger P < 0.01 vs. control.

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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Fig. 2.   Concentration dependence of mitogen-activated protein (MAP) kinase (MAPK) activation by H2O2. Adult rat ventricular myocytes (ARVM) were treated with various concentrations of H2O2 for 20 or 60 min [c-Jun NH2-terminal kinase (JNK)]. Cell lysates were size-fractionated by SDS-PAGE and subjected to Western blot analysis. Left, A: extracellular signal-regulated kinase (ERK) 1 and 2 activation was assessed with anti-active ERK1/2 antibodies (top blot) and by in-gel kinase assays with myelin basic protein (MBP) as a substrate (bottom blot). ERK1/2 expression was measured with anti-total ERK1/2 antibodies (middle blot). Left, B: p38 activation was assessed with anti-phospho-p38 (top blot) and p38 expression by blotting with anti-total p38 antibodies (bottom blot). Left, C: JNK activity was assessed by immune complex kinase assays with glutathione S-transferase (GST)-c-Jun as a substrate (top blot). JNK expression was detected using anti-JNK antibodies (bottom blot). Right: cumulative data from 3-5 experiments. *P < 0.05 vs. control.

A similar concentration-dependent activation of p38 MAP kinases and JNK by H2O2 was observed (Fig. 2, B and C). For p38 MAP kinase, phosphorylation was detected at 5 µM H2O2 and was maximal at 50 µM H2O2 (9.0 ± 1.6-fold vs. control). JNK activation was measured by immune complex kinase assays with GST-c-Jun as a substrate. Significant JNK activation was detected at 50 µM H2O2 (2.0 ± 0.3-fold vs. control), increased at 100 µM H2O2, and declined at 200 µM H2O2.

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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Fig. 3.   Time course of MAP kinase activation by H2O2. ARVM were exposed to 50 µM H2O2 for indicated times. Left: activation was determined by Western blot analysis with anti-active ERK1/2 (A) and anti-active p38 MAP kinase (B) antibodies or by immune complex kinase assays for JNK (C). MAP kinase expression was detected by Western blot analysis with anti-total MAP kinase antibodies. Right: cumulative data from 3-5 experiments. *P < 0.05 vs. control.

To determine that H2O2 was the oxidant responsible for activation of MAP kinases, the ability of the H2O2 scavenger catalase to block the effect of H2O2 was studied. Pretreatment with catalase (400 U/ml) for 10 min completely abolished the activation of all three MAP kinases by 50 µM H2O2 (Fig. 4). In contrast, pretreatment with the superoxide scavenger superoxide dismutase (1,000 U/ml) for 30 min failed to inhibit MAP kinase activation by H2O2 (Fig. 4).


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Fig. 4.   Catalase, but not superoxide disumtase (SOD), blocks H2O2-induced MAP kinase activation. ARVM were pretreated with 400 U/ml of catalase for 10 min or 1,000 U/ml of SOD for 30 min and then exposed to 50 µM H2O2 for 20 min. Activated MAP kinases were detected by Western blot analysis with anti-active ERK1/2 (top) and anti-active p38 MAP kinase (middle) antibodies or by immune complex kinase assays for JNK (bottom). Blots are representative of 3 experiments.

To mimic the free radical burst that occurs during ischemia-reperfusion injury, we tested the effect of a brief exposure of ARVM to H2O2. ARVM were treated with 50 µM H2O2 for 5 min. The medium was then replaced with M199, and ARVM were incubated for an additional 5, 15, and 55 min (205 min for JNK activation) in the absence of H2O2. Brief exposure to H2O2 was sufficient to cause a transient activation of ERK1/2 and p38 MAP kinase and a sustained activation of JNK (Fig. 5) that persisted after the removal of H2O2.


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Fig. 5.   Brief exposure to H2O2 results in prolonged MAP kinase activation. For experiments assessing the acute effects of H2O2, ARVM were treated with 50 µM H2O2 for 5 min. Medium was replaced with fresh medium 199, cells were incubated for the indicated times, and MAP kinase activation was detected by Western blot analysis with anti-active ERK1/2 (left) and anti-active p38 MAP kinase (middle) antibodies or by immune complex kinase assays for JNK (right). Cumulative data from 4 experiments are presented. *P < 0.05 vs. control.

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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Fig. 6.   Effects of tyrosine kinase and protein kinase C (PKC) inhibitors on H2O2-induced MAP kinase activation. ARVM were pretreated with 100 µM genistein (Gen), 10 µM PP-2, or 5 µM chelerythrine (Chel) for 45 min before the addition of 50 µM H2O2 for 20 min (60 min in JNK activation). Left: activation was determined by Western blot analysis with anti-active ERK1/2 (A) and anti-active p38 MAP kinase (B) antibodies or by immune complex kinase assays for JNK (C). MAP kinase expression was detected by Western blot analysis with anti-total MAP kinase antibodies. Right: cumulative data from 3-5 experiments. *P < 0.05 vs. control; +P < 0.05 vs. H2O2.

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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Fig. 7.   ERK1/2-dependent phosphorylation of Na+/H+ exchanger isoform 1 (NHE1) by H2O2. ARVM were treated with H2O2, and NHE1 phosphorylation was determined by in-gel kinase assays using a carboxy tail of NHE1 coupled to GST as a substrate. A: concentration dependence for H2O2 was determined at 20 min. B: time course with 50 µM H2O2. C: effect of pretreatment with the MAPK kinase inhibitor U-0126 (5 µM, 45 min) or the p38 inhibitor SB-203580 (10 µM, 45 min) on H2O2-induced NHE1 phosphorylation.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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 alpha -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 alpha 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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