Hydrogen Peroxide Activates Mitogen-Activated Protein Kinases and Na+-H+ Exchange in Neonatal Rat Cardiac Myocytes
Abstract—Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na+-H+ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 μmol/L hydrogen peroxide (H2O2) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H2O2 (100 μmol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H2O2. Pretreatment with chelerythrine (10 μmol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 μmol/L, 45 minutes) or herbimycin A (3 μmol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H2O2-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H2O2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 μmol/L, 60 minutes). These results suggest that low doses of H2O2 induce MAP kinase–dependent pathways that regulate NHE activity during reperfusion injury.
Reperfusion of cardiac tissue after a reversible ischemic episode results in further cardiac injury, including reduced tension development, arrhythmias, mitochondrial swelling, and Ca2+ overload. The resulting contractile dysfunction is referred to as myocardial stunning.1 There is substantial evidence that oxidant injury plays a critical role in the degenerative alterations that occur in myocardial reperfusion injury. First, reperfusion is associated with a rapid burst in OFR production.2 Second, the highly reactive hydroxyl radical (·OH) has been demonstrated to cause lipid peroxidation and myocardial injury and is thought to trigger the contractile dysfunction observed during reperfusion.3 Third, scavengers of free radicals such as catalase and SOD can reduce myocardial stunning and reperfusion arrhythmias.4 Finally, exposure of nonischemic myocardium or cultured myocytes to OFRs can produce cell injury similar to that seen in ischemia/reperfusion.1
A number of mechanisms are capable of generating OFRs within the ischemic heart. Within the myocardium, both endothelial cells and myocytes cause the oxidation of xanthine and hypoxanthine by xanthine oxidase to generate O2−, which can be further reduced to H2O2 and ·OH. In addition, during ischemia, there is infiltration of the myocardium by neutrophils that produce several types of OFRs, including superoxide (O2−) and hydrogen peroxide (H2O2).1 These free radicals are stored in granules that are released as part of the inflammatory response.
OFRs have numerous intracellular targets, including second-messenger pathways, L-type Ca2+ channels, K+ channels, ion transporters, and contractile proteins.1 In cardiac myocytes, ·O2− and H2O2 have been shown to inhibit Na+ and Ca2+ pumps, accelerate rundown of L-type Ca2+ currents, activate Na+-Ca2+ exchange,5 and deplete internal caffeine-sensitive Ca2+ stores by inhibiting the sarcoplasmic reticulum Ca2+-ATPase.1 6 These effects are independent of metabolic inhibition, since the mitochondrial uncoupler, carbonylcyanide-p-trifluoro-methoxyphenylhydrazone, or the metabolic inhibitor, 2-deoxyglucose, do not mimic the effects of OFRs.6 OFR-generating systems have also been shown to reduce myofibrillar ATPase activity, which could attenuate myofilament responsiveness to Ca2+ and force development.7
The NHE plays an important role in cardiac injury after reperfusion. Activation of NHE on reperfusion represents the major mechanism for restoration of pHi after ischemia-induced acidosis. However, activation of the exchanger produces undesirable secondary effects leading to the exacerbation of tissue injury, a phenomenon that is termed the “pH paradox.”8 As NHE is activated, the rising influx of Na+ leads to an increase in [Ca2+]i via Na+-Ca2+ exchange. This further intensifies intracellular Ca2+ overload and results in mitochondrial swelling, arrhythmias, activation of proteases, and further cell damage.8 9 10 Therefore, activation of NHE is thought to be a primary mechanism that accounts for the [Ca2+]i overload associated with reperfusion injury, and inhibitors of NHE have been shown to protect the myocardium from reperfusion injury.8
The mechanisms that modulate NHE activity during reperfusion are unknown. Exchanger activity is regulated by changes in protein expression and by phosphorylation of existing exchangers or a closely associated modulatory protein. Both types of regulation increase maximum transport capacity, whereas phosphorylation also increases the affinity of the “pHi sensor” of the exchanger for intracellular H+.11 In cardiac myocytes, the type of regulation may be dependent on the length of the ischemic period. Protein expression appears to play a major role in exchanger activity during chronic ischemia, whereas phosphorylation may be more important in response to acute ischemia/reperfusion.12 Phosphorylation of the exchanger on its carboxy-terminal tail is associated with a shift in pHi dependence toward more alkaline pH values and an increase in maximum activity (Vmax) at acidic pH values.13 14
The kinases that regulate NHE activity in cardiac cells have not yet been identified. In in vitro experiments, PKC,15 MAP kinase,16 calmodulin-dependent protein kinase,10 and a novel 97-kDa NHE-1 kinase17 all phosphorylate NHE-1. MAP kinases are likely to play a major role in regulating NHE activity for several reasons. First, it was recently demonstrated that cells overexpressing a dominant-negative mutant of MAP kinase showed decreased serum activation of NHE activity but had no effect on hypertonic activation of the protein (which does not require phosphorylation).16 Second, in nonmyocytes, MAP kinase mutants cause a significant inhibition of growth factor–induced NHE-1 activation.18 Finally, we16 and others18 have shown that MAP kinase can phosphorylate NHE-1 in vitro.
Little is known about the cellular signaling systems that are activated in cardiac myocytes by OFRs. At high doses, OFRs cause irreversible cell injury and cell death.1 9 Lower concentrations of OFRs, especially H2O2 and ·OH, have been shown to cause reversible cell injury by activating a variety of intracellular signaling processes. In other cell types, H2O2 stimulates arachidonic acid release, PKC activation, tyrosine kinases, cytoplasmic phospholipase A2 activation, and an increase cytosolic [Ca2+].19 20 21 In addition, H2O2 is necessary for growth factor mitogenic signaling in vascular smooth muscle.22 In vascular and airway smooth muscle, H2O2 and O2− activate the MAP kinase pathway in a partially PKC-dependent manner.20 23
In the present study, we demonstrate that acute exposure to low doses of H2O2 caused a prolonged decrease in contractility and a rapid activation of both 42-kDa (ERK2) and 44-kDa (ERK1) MAP kinases in cardiac myocytes. This MAP kinase activation was partially blocked by inhibitors of tyrosine kinases and PKC. H2O2 also induced a rapid activation of NHE that was blocked by a MEK inhibitor. A preliminary report has appeared.24
Materials and Methods
All experiments used primary cultures of rat ventricular myocytes obtained from 1- to 2-day-old Sprague-Dawley rats. Myocytes were isolated from ventricular tissue by enzymatic dissociation.25 Animal procedures conformed to the Guide for Care and Use of Laboratory Animals, issued by the US Institute for Laboratory Resources (publication No. [NIH] 93-23, revised 1985). Cells were plated at a density of 160 000/cm2 in serum-free PC-1 medium (BioWhittaker) supplemented with 1 mmol/L l-glutamine and antibiotic/antimycotic solution (GIBCO/BRL). Under these high-density conditions, the myocytes display spontaneous contractile activity within 24 hours of plating and form cell-cell contacts. The cells were maintained for 24 to 48 hours in a 2:1 mixture of DME/F-12 Ham:PC-1 medium. Maintenance medium was replaced with HEPES-buffered DME/F-12 Ham at least 60 minutes before the start of the experiments.
Preparation of Cell Lysates for MAP Kinase Experiments
Cells were incubated at 37°C in DME/F-12 Ham–containing H2O2, plus or minus inhibitors, or vehicle for various times. After H2O2 stimulation, cells were harvested by aspirating the medium and washing with ice-cold PBS. Cells were lysed by the addition of ice-cold lysis buffer (mmol/L: NaCl 50, NaF 50, sodium pyrophosphate 50, EDTA 5, EGTA 5, Na3VO4 2, phenylmethylsulfonyl fluoride 0.5, and HEPES 10 at pH 7.4, along with 0.1% Triton X-100 and 10 μg/mL leupeptin), followed by immediate freezing on ethanol/dry ice. The cell lysates were then thawed on ice, scraped, sonicated, and centrifuged at 14 000 rpm at 4°C for 30 minutes. Supernatants were used immediately or stored at −80°C. Protein concentrations were determined using a bicinchoninic acid protein assay kit from Pierce, according to the manufacturer’s protocol.
Western Blot Analysis
Cell lysates (25 μg) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 2 hours in 1% casein (Hammarsten-prepared), 0.05% Tween, and 0.05% azide in PBS. Western blot analysis was performed using anti–ERK1- and anti–ERK2-specific primary antibodies (Santa Cruz) and a horseradish peroxidase–conjugated goat anti-rabbit IgG (Bio-Rad). Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Amersham). Autoradiograms exposed in the linear range of film density were scanned using an LKB laser densitometer, and densitometric analysis was performed with Gelscan software. Because of insufficient resolution of the ERK1 bands, only ERK2 phosphorylation was quantified. Percent ERK2 activation was defined as the autoradiographic density (measured in arbitrary units) of phosphorylated MAP kinase (p42) divided by the total autoradiographic density of both the unphosphorylated and phosphorylated MAP kinases (p42+42)×100%.
In-Gel Kinase Assay
MAP kinase activity was analyzed by the in-gel kinase assay previously described by Lucchesi et al.26 After treatment with agonists, cells were rinsed with ice-cold PBS, harvested with lysis buffer, sonicated, and centrifuged. Cell lysates (10 μg) were then solubilized in SDS sample buffer and fractionated by SDS-PAGE in a gel in which 0.1 mg/mL of MAP kinase substrate (myelin basic protein) was copolymerized. After electrophoresis, the gel was washed twice in buffer A (50 mmol/L HEPES [pH 7.4] and 5 mmol/L β-mercaptoethanol) containing 20% isopropanol to remove SDS. Gels were then reequilibrated in buffer A alone and denatured by 2 washes (45 minutes each) in buffer A containing 6 mol/L guanidine HCl. Guanidine was removed with buffer A containing 0.04% Tween 20 (16-hour wash at 4°C). The gel was then equilibrated in buffer B (25 mmol/L HEPES [pH 7.4], 100 μmol/L sodium orthovanadate, 10 mmol/L MgCl2, and 5 mmol/L β-mercaptoethanol) for 30 minutes at 30°C. The phosphorylation assay was performed by placing the gel in 10 mL buffer B containing 50 μmol/L ATP with 100 μCi [γ-32P]ATP and incubating for 1 hour at 30°C. The reaction was terminated by immersing the gel in fixative (10 mmol/L sodium pyrophosphate and 5% trichloroacetic acid). The gel was washed with fixative until the radioactivity in the wash equaled background counts. The gel was dried, and radioactivity was quantified using an InstantImager electronic autoradiography system (Packard).
Measurement of Single-Cell Contractility
Myocytes were cultured for 24 to 48 hours in standard growth medium in Plexiglas superfusion chambers, in which the bottom was formed by a collagen-coated glass coverslip. The chamber was then placed on the stage of an inverted microscope (Nikon Diaphot), and cells were superfused with modified Krebs solution for 5 minutes at 37°C. This control period was followed by a 5-minute perfusion with 50 μmol/L H2O2 in Krebs solution and a 10-minute washout period. Cell shortening was measured using a video edge detection system (Crescent Electronics). The signal was acquired using a DataQ DI-200 board interfaced to a personal computer and stored using Windaq Software (DataQ Instruments, Inc). The cell image was also recorded on videotape for additional off-line analysis. Amplitude and frequency of contraction in the presence of H2O2 were expressed relative to control values. Contractile frequency in the absence of H2O2 averaged 133±8 bpm (n=11). Contractile amplitude in control cells was set to 100%.
Measurement of pHi
pHi was measured by monitoring the fluorescence of the pH-sensitive dye BCECF. Cells grown on collagen-coated coverslips were loaded with BCECF by incubating for 15 minutes at room temperature with the acetoxymethyl ester form (BCECF-AM, 0.5 μmol/L) in modified Krebs solution (mmol/L: NaCl 135, KCl 5.9, CaCl2 1.5, MgCl2 1.2, HEPES 11.6, and d-glucose 11.5) supplemented with 0.1% BSA and 0.02% Pluronic F127. The cells were then washed and incubated for an additional 45 minutes in fresh Krebs solution in the presence or absence of the MEK inhibitor PD 98059 (30 μmol/L). BCECF fluorescence was measured using a Perkin-Elmer LS50B fluorescence spectrophotometer. The coverslip was inserted into a 4.5-mL optical methacrylate cuvette on a 30o angle to the light beam. The solution bathing the cells was changed by perfusing fresh solution into the bottom of the cuvette while aspirating continuously from just above the coverslip. The perfusion rate (5 mL/min) during acid load and recovery from acid load was kept constant using a Harvard syringe. At the perfusion rate used, the half-time for mixing in the cuvette was ≈20 seconds. The cells were excited alternately with 490- and 440-nm light every 0.02 seconds using a rotating filter wheel in the path of the excitation light, and average fluorescence intensity ratios (490/440 nm) were recorded at 0.5-second intervals. At the end of each experiment, the fluorescence ratio values were converted to pHi using the nigericin high-K+ protocol of Thomas et al.27 Cells were perfused for 5 minutes with K+-HEPES-PSS (5 mmol/L NaCl, 130 mmol/L KCl, and 50 mmol/L HEPES/KOH) at varying pH levels (6.4 to 7.85) in the presence of nigericin (4 mg/L). There was a linear relationship between fluorescence intensity ratios and pH over this range (data not shown).
Measurement of NHE Activity
NHE activity was measured in cells after acidification using the NH4Cl prepulse technique.28 After determination of basal pHi, cells were exposed to Krebs solution containing 25 mmol/L NH4Cl for 5 minutes, followed by perfusion with Na+-free Krebs solution (Na+ isosmotically replaced with N-methylglucamine). There was no recovery from this acid load in the absence of Na+, and the pH stabilized at 6.79±0.06 (n=6). pHi recovered when the perfusate was switched to Na+-containing Krebs solution. This Na+-dependent recovery was inhibited by the NHE-specific blocker DMA (25 μmol/L) and was operationally defined as NHE activity. To quantify the rate of pHi recovery, a straight line was fitted to the initial 60 seconds after the onset of recovery, and the respective slopes were compared.
All experiments were performed at least 3 times, and results are expressed as mean±SE. Statistical analysis was performed by a Student t test (2-tailed) with significant differences determined at P<0.05.
Exposure to Low Concentrations of H2O2 Affects Contractility of Neonatal Cardiac Myocytes
A video edge detection system was used to monitor the effects of physiological doses of H2O2 on single-cell contractility (Figure 1⇓). Spontaneously beating myocytes were exposed to 50 μmol/L H2O2 for 5 minutes. Contractility was measured during the exposure to H2O2 and during a subsequent 10-minute washout period. The addition of H2O2 caused a significant decrease in contraction amplitude of spontaneously beating myocytes after 5 minutes of exposure (86.6% of control, P<0.005, n=11) that persisted throughout the 10-minute washout period (84.8%, P<0.05, n=11). There was a transient increase in contraction frequency that returned to baseline on the removal of H2O2. H2O2 also produced a significant decrease in contractile amplitude when cells were paced at a constant frequency of 1.5 to 2 Hz (data not shown), suggesting that the effects of H2O2 on amplitude were not due solely to changed contraction frequency. These results clearly demonstrate that low concentrations of H2O2 elicit significant changes in cardiac contractile function.
Hydrogen Peroxide Activates MAP Kinases in Cardiac Myocytes
Cardiac myocytes were exposed to H2O2 (1 to 200 μmol/L) for 20 minutes. Cell lysates were size-fractionated by SDS-PAGE, and activation of MAP kinases was determined by Western blot analysis with antibodies that recognize both the ERK2 (42-kDa) and ERK1 (44-kDa) MAP kinases (Figure 2⇓). Activation of MAP kinase results in a “bandshift” of both ERK1 and ERK2, because phosphorylation of MAP kinase reduces its mobility on SDS-polyacrylamide gels. As shown in Figure 2⇓, H2O2 caused a concentration-dependent phosphorylation of ERK2, with a maximal increase occurring at 50 μmol/L H2O2 (42±9.7%, P<0.01, n=7). Concentrations of H2O2 of >200 μmol/L decreased MAP kinase activation, possibly because of the cytotoxic effects of these high concentrations of free radicals.
The time course for activation of MAP kinases by H2O2 was determined by 3 different methods (Figure 3⇓). Analysis of bandshifts of ERK1 and ERK2 on Western blots showed that exposure of cardiac myocytes to 100 μmol/L H2O2 caused a rapid and sustained phosphorylation of ERK2. ERK2 was activated within 5 minutes of exposure to H2O2 (23±6%, n=7). Maximal activation occurred at 20 minutes (48±10%, n=14) and was maintained for 60 minutes (Figure 3A⇓). Because of insufficient resolution of the ERK1 bands by SDS-PAGE, we were unable to determine whether ERK1 was also phosphorylated in response to H2O2. Therefore, we used an anti–active MAP kinase antibody that recognized only the phosphorylated forms of ERK1 and ERK2 (Figure 3B⇓). These results indicated that ERK1 and ERK2 are indeed phosphorylated in response to 100 μmol/L H2O2.
To confirm that phosphorylated ERK1 and ERK2 represented activated kinases, lysates from control cells and cells treated with 100 μmol/L H2O2 were subjected to in-gel kinase assays using myelin basic protein as a substrate. Compared with untreated control myocytes, myocytes exposed to 100 μmol/L H2O2 demonstrated an increase in both ERK1 and ERK2 phosphotransferase activity (Figure 3C⇑).
To demonstrate that H2O2 was responsible for activation of MAP kinase, cardiac myocytes were treated with the H2O2 scavenger catalase (400 U/mL) for 10 minutes before exposure to H2O2. Catalase caused a complete inhibition of MAP kinase activation at all time points measured, demonstrating that either H2O2 or the ·OH radical is responsible for MAP kinase activation (Figure 3A⇑ and 3C⇑).
To mimic the free radical burst that occurs during ischemia/reperfusion injury, we tested the effect of a brief exposure of cultured myocytes to 100 μmol/L H2O2. Cells were treated with 100 μmol/L H2O2 for 5 minutes. The medium was then replaced with DME/F-12 Ham, and myocytes were incubated for an additional 15, 25, or 55 minutes in the absence of H2O2. MAP kinase activity was measured by Western blot analysis and by in-gel kinase assays using myelin basic protein as the substrate (Figure 4⇓). Brief exposure to H2O2 resulted in a sustained activation of ERK1 and ERK2 that was observed for at least 60 minutes after the removal of free radicals. These results suggest that a brief exposure of cells to H2O2 is sufficient to generate intracellular signals that persist after the stimulus is removed. In some experiments, acute exposure actually increased MAP kinase activation compared with continuous or prolonged H2O2 exposure. It may be that continuous exposure has more cytotoxic effects that would mask full MAP kinase activation. However, analysis of data pooled from 5 experiments indicated that there was no significant difference in ERK2 activation by brief versus prolonged H2O2 exposure (Figure 4C⇓).
Effects of SOD and ·O2−-Generating Systems on MAP Kinase Activation in Cardiac Myocytes
Superoxide (·O2−) is a potent activator of MAP kinase in vascular smooth muscle.29 To test the effects of superoxide on MAP kinase activation in cardiac myocytes, we pretreated myocytes with the ·O2− scavenger SOD for 30 minutes before the addition of 50 μmol/L H2O2 (Figure 5A⇓ and 5B⇓). SOD failed to inhibit MAP kinase activation induced by H2O2, suggesting that H2O2 does not exert its effects by altering the levels of ·O2−. To further rule out the role of ·O2−, we examined the ability of ·O2−-generating systems to activate MAP kinases in cardiac myocytes. Cells were treated with 100 μmol/L xanthine/5 mU/mL xanthine oxidase for 20 or 30 minutes; MAP kinase activation was assessed by in-gel kinase assays (Figure 5C⇓). Cells were also treated with 1 μmol/L LY83683, a membrane-permeant compound that generates ·O2− via metabolism by cytosolic and membrane-bound NAD(P)H oxidases30 (data not shown). Neither ·O2−-generating system resulted in significant MAP kinase activation in cardiac myocytes.
Role of Tyrosine Kinases and PKC in the Activation of MAP Kinase by H2O2
To evaluate the role of tyrosine kinases in the stimulation of MAP kinases by H2O2, myocytes were pretreated with the tyrosine kinase inhibitor genistein. Genistein caused a dose-dependent inhibition of ERK2 phosphorylation by H2O2 at all time points examined, with maximal inhibition occurring at 75 μmol/L (Figure 6A⇓). Inhibition was 75±8% at 5 minutes, 82±12% at 10 minutes, and 78±16% at 20 minutes (Figure 6B⇓). Daidzein, an inactive structural analogue of genistein, was used to determine whether the effects of genistein were due to its ability to act as a free radical scavenger. Pretreatment with concentrations of daidzein as high as 75 μmol/L had no significant effect on the ability of H2O2 to activate MAP kinases (Figure 6A⇓).
Pretreatment with 3 μmol/L herbimycin A, a potent inhibitor of nonreceptor tyrosine kinases, also caused significant inhibition of H2O2-induced MAP kinase phosphorylation (45±7% versus control at 20 minutes, n=3). These results indicate that activation of MAP kinase by H2O2 may be mediated in part by activation of tyrosine kinases.
Many free radical–stimulated signaling events are dependent on PKC activity. To assess the role of PKC in H2O2-stimulated MAP kinase activation, the effect of the specific PKC inhibitor, chelerythrine, was studied. Pretreatment (45 minutes) with 10 μmol/L chelerythrine caused a significant inhibition of ERK2 phosphorylation induced by 100 μmol/L H2O2 at all time points examined (Figure 7A⇓). The inhibition was 63±12% at 5 minutes, maximal at 10 minutes (74±5%), and still pronounced at 20 minutes (51±9%) (Figure 7B⇓). Chelerythrine also completely inhibited phorbol 12-myristate 13-acetate–stimulated MAP kinase phosphorylation (data not shown).
MAP Kinase–Dependent Activation of NHE Activity by H2O2
The mean resting pH of cardiac myocytes in bicarbonate-free Krebs solution at room temperature was 7.5±0.3 (n=6). The addition of 25 mmol/L NH4Cl caused a rapid alkalinization (Figure 8A⇓) as NH3 diffused into the cells and titrated intracellular H+. Removal of NH4+ from the external medium caused a rapid decrease in pHi (Figure 8A⇓). The cells were unable to recover from this acid load in Na+-free medium. Reintroduction of Na+ (perfusion with Krebs solution) led to a rapid recovery of pHi that approached resting values. This Na+-dependent recovery was completely abolished in the presence of the NHE inhibitor DMA (25 μmol/L, data not shown). Exposure to 50 μmol/L H2O2 caused a 1.6-fold increase in Na+-dependent recovery of pHi from an acid load (6.4±0.7×10−3 ΔpH/s [n=4] versus 3.9±0.4×10−3 ΔpH/min in control [n=5], P<0.05) that was completely blocked by DMA (data not shown). Pretreatment with the MEK inhibitor PD 98059 (30 μmol/L, 1 hour) completely blocked H2O2-induced NHE activity to control levels (Figure 8B⇓ and 8C⇓). The MEK inhibitor had no effect in the absence of H2O2. Under identical experimental conditions, PD 98059 abolished H2O2-induced MAP kinase activation (Figure 9⇓).
The results of the present study demonstrate that physiological concentrations of H2O2, similar to the levels seen during the burst in free radical production after reperfusion of an ischemic myocardium,1 activate MAP kinases and lead to alterations in cardiac myocyte contractility (Figure 1⇑) and NHE activity (Figure 8⇑). Acute exposure of cardiac myocytes to 50 to 100 μmol/L of H2O2 causes a rapid and sustained activation of MAP kinases. This effect was specific for H2O2, since it was blocked by catalase. On the other hand, superoxide-generating systems had no effect on MAP kinase activity. The time course for MAP kinase activation was much more rapid than that observed in vascular smooth muscle,20 29 required only a brief exposure to H2O2 (Figure 4⇑), and resulted in a sustained activation that persisted for at least 60 minutes. In contrast, Aikawa et al31 recently reported that high concentrations of H2O2 (1 mmol/L) caused a transient activation of MAP kinase that was decreased at 30 minutes and abolished by 60 minutes. The difference in time course for MAP kinase activation by high and low concentrations of H2O2 remains unclear but may be due to cytotoxic effects that are observed more readily at the higher H2O2 levels.
These results are in agreement with reports of MAP kinase activation by free radicals in many cell types, including airway smooth muscle, PC12 cells, and fibroblasts.20 22 23 29 32 In vascular and airway smooth muscle, H2O2 and O2− activate the MAP kinase pathway in a partially PKC-dependent manner.20 23 In cardiac myocytes, hypoxia increases MAP kinase and Jun kinase activities and causes the translocation of PKC isozymes.33 34
The mechanism by which H2O2 activates MAP kinase signaling has not been fully elucidated. In the present study, the tyrosine kinase inhibitor genistein caused a 75% inhibition of H2O2-induced MAP kinase activation, whereas the inactive analogue of genistein (daidzein) was without effect. This concentration of genistein (75 μmol/L) is within the range reported for the inhibition of nonreceptor tyrosine kinases. Moreover, the more selective nonreceptor tyrosine kinase inhibitor, herbimycin A, also blocked H2O2-induced MAP kinase activation (Figure 6C⇑). A possible interpretation of these findings is that H2O2 exerts its effects on MAP kinase by modulating the activity of upstream tyrosine kinases.
There are several candidate tyrosine kinases upstream from MAP kinase activation. A proline-rich tyrosine kinase (PYK2) has recently been identified and shown to link G protein–coupled receptors to Shc-Grb2 complex formation and MAP kinase activation.35 36 However, H2O2 failed to activate PYK2 phosphorylation in cardiac myocytes (A. Sabri, P.A. Lucchesi, unpublished data, 1997). Therefore, it is likely that other nonreceptor tyrosine kinases may be involved. For example, the src family of tyrosine kinases, including fyn and src, have also been implicated in angiotensin II37 and H2O231 signaling to MAP kinases in cardiac myocytes.
Several laboratories have reported that OFRs may also increase MAP kinase activity by inhibiting one or more tyrosine phosphatases38 rather than by stimulating tyrosine kinase activity. However, we have found that H2O2 failed to affect the expression of the transcriptionally regulated MAP kinase phosphatase, termed MKP-1 (data not shown). Additional studies will be needed to identify the kinases and signaling molecules involved in the activation of MAP kinases by H2O2.
Several PKC isoforms have also been implicated in regulation of the MAP kinase pathway, either by direct phosphorylation and activation of Raf or indirectly by stimulation of PYK2. Our results show that inhibition of PKC with 10 μmol/L chelerythrine produced an ≈50% inhibition of H2O2-induced MAP kinase activation (Figure 7⇑). These data are in agreement with recent studies demonstrating that PKC plays a critical role in angiotensin II–induced MAP kinase activation in cardiac myocytes.39 Therefore, our results are consistent with the notion that H2O2 stimulation of MAP kinases in cardiac myocytes involves both PKC-dependent and tyrosine kinase–dependent processes.
Convincing evidence for NHE involvement in myocardial reperfusion injury has come from studies demonstrating the various beneficial effects of NHE inhibitors.40 The first such study was reported by Karmazyn,41 who demonstrated a protective effect of amiloride in isolated rat hearts subjected to low-flow ischemia followed by reperfusion. Protection was associated with enhanced ventricular recovery and decreased creatine kinase efflux during perfusion. These results have been confirmed in studies using more specific blockers of NHE.42
In chronic ischemia, there is an increase in steady-state levels of NHE-1 mRNA (the only NHE-1 isoform present within the myocardium), suggesting that increased activity is due to an increase in protein expression.12 In the present study, we have shown that acute exposure to H2O2 causes a rapid activation of NHE-1 activity. The rapid time course for activation suggests that posttranslational modification rather than gene expression is the most likely explanation for the observed increase in NHE activity. More interestingly, exchanger activation by H2O2 was abolished by pretreatment with the MEK inhibitor PD 98059, suggesting that MAP kinase or a MAP kinase–regulated pathway mediates H2O2-induced NHE activation.
This is consistent with the findings that phosphorylation of the exchanger or a closely related accessory protein increases NHE activity.11 12 13 43 Furthermore, expression of a dominant-negative mutant MAP kinase in fibroblasts inhibited NHE activity by >50% in response to growth factors.18 We have recently demonstrated that MAP kinases in purified skeletal muscle extracts activate NHE activity and that MAP kinases can phosphorylate the carboxy-terminal tail of NHE-1 in gel renaturation assays.16 Future studies will be necessary to determine whether MAP kinases directly phosphorylate the NHE-1 protein in cardiac myocytes in response to H2O2 stimulation.
In addition to enhancing NHE activity, micromolar concentrations of H2O2 produced a deleterious effect on contractile function in ventricular myocytes (Figure 1⇑). These results confirm previous studies by other groups, although both Goldhaber and Liu6 and Kaneka et al44 used at least 10-fold higher concentrations of OFRs. The relationship between MAP kinase–dependent increases in NHE activity and H2O2-induced contractile dysfunction remain to be established. However, it is unlikely that changes in NHE activity alone are sufficient to explain all of the cellular mechanisms responsible for the contractile dysfunction. A detailed study of the correlation between MAP kinase activation with alterations in sarcomere assembly, myosin and troponin phosphorylation, and Ca2+ sensitivity of the myofilaments is needed to clearly define the cellular mechanisms involved in the contractile dysfunction caused by OFRs.
In summary, activation of MAP kinase signaling by H2O2 may play an important role in the alteration of NHE activity that is associated with ischemia/reperfusion injury. Future studies will elucidate the cellular signaling pathways involved in H2O2-mediated MAP kinase regulation.
Selected Abbreviations and Acronyms
|ERK||=||extracellular-regulated signal transduction kinase|
|MEK||=||MAP kinase kinase|
|MKP-1||=||MAP kinase phosphatase 1|
|NHE-1||=||NHE isoform 1|
|OFR||=||oxygen-derived free radical|
|PKC||=||protein kinase C|
This study was supported in part by grants HL-03282 (Dr Lucchesi) and HL-34328 (Dr Samarel) from the National Institutes of Health and by grants from the American Heart Association (Dr Lucchesi), from the Eugene J. and Elsie E. Weyler Endowment for Clinical Cardiology Research (Dr Byron), and from the John and Marian Falk Trust for Medical Research. We are grateful to Tina Griffin, Justin Brezina, Andrew Hwang, and Meg Farnsworth for superb technical assistance.
Previously presented as a preliminary report in abstract form (J Mol Cell Cardiol. 1996;28:A198).
- Received July 10, 1997.
- Accepted March 16, 1998.
- © 1998 American Heart Association, Inc.
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