The transmission of extracellular signals to their intracellular targets is mediated by a network of interacting proteins that include cytoplasmic protein kinases known as mitogen-activated protein kinases (MAPKs) (1-3). The MAPKs represent a superfamily of Pro-directed Ser/Thr protein kinases that are involved in signal transduction from the plasma membrane to nuclear and cytoplasmic targets (4,5). The MAPK cascades consist of at least three protein kinase families: the ERKs (extracellular signal-regulated protein kinases), SAPKs/JNKs (stress-activated protein kinases/c-Jun-NH2-terminal protein kinases), and p38-MAPkinases (5,6). These three cascades represent parallel pathways, which have different substrate specificities and are regulated by distinct stimuli. The MAPK pathways can be influenced by conditioning stresses (ionizing or ultraviolet radiation, heat shock, hyperosmotic stress, ischemia/reperfusion, etc.) as well as by pharmacologic agents, which specifically influence the activities of distinct members of the MAPKs. For example, in the heart, ERKs are activated by peptide growth factors and Gq protein-coupled receptor agonists (7-9). In contrast to the ERKs, the SAPKs/JNKs and p38-MAPK were shown to be activated mainly by cellular stresses such as UV radiation, ischemia, and ischemia/reperfusion (10-12). Specific and selective inhibitors have been used to differentiate between the three main MAP kinase pathways and to understand the role of various MAP kinases in cellular regulation by extracellular stimuli (13,14).
Short transient periods of ischemia render the myocardium more resistant to a subsequent prolonged coronary occlusion, resulting in a reduction of the infarct size (IS). This cardioprotective mechanism is known as ischemic preconditioning (IP) (15-17). Our previous studies with protein kinase stimulators and inhibitors have indicated different roles of the various protein kinases in IP (18) in pig myocardium. We have also observed a differential regulation of MAPK cascades during ischemia and reperfusion. The activity of the ERKs increased moderately during brief ischemia and markedly during reperfusion. SAPK/JNKs (46 and 55 kDa) were activated only during reperfusion. p38-MAPK was activated during ischemia, but its activity was downregulated during the following reperfusion phase and in the second period of ischemia (10,19). Activation of the SAPK/JNK pathway by treatment with okadaic acid and anisomycin was shown to augment the protective effect of IP in porcine myocardium (10,20). Conversely, the results with a specific inhibitor of the p38-MAPK pathway, SB203580, have suggested a negative role of p38-MAPK during ischemia in the same in vivo model (21). In cultured neonatal rat cardiac myocytes, sustained activation of p38-MAPK was observed during ischemia. This sustained activation of p38-MAPK was deleterious to cells, at least partly mediated by apoptosis. SB203580 protected cardiac myocytes from ischemic injury in a dose-related manner. SB induced protection only when it was present during the second sustained phase of p38-MAPK activation, not when it was exclusively present during the first, transient phase of p38 activation (22). Previously, we found that intramyocardial infusion of fibroblast growth factor-1 (FGF-1) and FGF-2 mimicked the effect of IP in the porcine myocardium (23,24). Peptide growth factors, FGFs, and Gq protein-coupled receptor agonists stimulate activation of ERKs in ischemic myocardium (3,4,8). This fact and the observed cardioprotection by FGF-1 and FGF-2 suggest a positive role of the ERK cascade in IP.
PD98059 and UO126 represent specific inhibitors of the ERK cascade. PD acts by inhibiting the activation of MEK 1/2 by Raf-kinase, while UO acts directly on both MEK 1/2 and ERK 1/2 (25,26). UO thereby acts on the distal ERK signaling pathway just before it results in activation of transcription factors. A common feature of all MAPKs is their ability to phosphorylate the transactivation domain of numerous transcription factors and thereby to modulate their transcriptional activities. The transcription factor Elk-1 is a substrate for three distinct classes of MAPKs. Different activation stimuli and targeting domains of Elk-1 exist. The ERKs and JNKs use overlapping yet distinct determinants in the D-domain for targeting Elk-1. In contrast, members of the p38 subfamily of MAPKs are not targeted to Elk-1 through this domain. The kinases responsible for phosphorylation and stimulation of Elk-1 transcriptional activity in response to mitogenic signals are believed to be ERK 1/2 (27). JNKs are responsible for Elk-1 activation in response to UV irradiation or MEKK1 expression. JNKs were first identified by their ability to phosphorylate c-Jun and ATF2, thereby stimulating their transcriptional activities (28). Stimulation of Elk-1 phosphorylation and activity in vivo by the p38-MAPK pathways may not be as pronounced as activation by either the JNK or ERK pathways. p38-MAPK appears to phosphorylate Elk-1 in a more constitutive manner that does not require rapid and efficient kinase targeting to the substrate (29,30). To test the hypothesis that ERKs are involved in IP, we studied the influence of the ERK inhibitors PD98059 and UO126 in IP. This was tested in our in vivo model using intramyocardial microinfusions of both substances (18) and systemic infusion of UO as well. The influence of ERK cascade inhibitors on infarct size was measured after a period of index ischemia and reperfusion with or without infusion of these agents before and during IP. Furthermore, the effects of PD and UO infusion on ERK activities/phosphorylations were determined by in gel phosphorylation assays and Western blot analysis.
MATERIALS AND METHODS
The experimental protocol of this study was approved by the Bioethical Committee of the District of Darmstadt, Germany. Furthermore, all animals in this study were handled in accordance with the guiding principles for care and use of animals as approved by the American Physiology Society, and the investigation conformed to the Guide for Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health.
Piritramide was purchased from Janssen Pharmaceutica (Meckenheim, Germany). Ketamine was received from Medistar GmbH (Holzwickede, Germany). Liquemin N 25000 was obtained from Roche (Grenzach-Wyhlen, Germany). UO126 (MEK 1/2 and ERK-1/2 inhibitor) was ordered from ALEXIS Biochemicals (Grünberg, Germany). PD98059 (MEK 1/2 inhibitor), α-chloralose, triphenyl-tetrazolium-chloride (TTC), and other biochemicals were from Sigma (Deisenhofen, Germany). The fluorescent zinc-cadmium sulfide microspheres (diameter, 2-15 μm) were purchased from Duke Scientific Corp. (AC Leusden, Netherlands). The monoclonal antibody against phospho-ERKs, the polyclonal antibodies against ERK, phospho-p38-MAPK, phospho-SAPK/JNK, and phospho-Elk-1 were from New England Biolabs (Schwalbach/Taunus, Germany). Nitrocellulose membranes, rainbow molecular mass markers, the horseradish peroxidase-linked goat anti-rabbit and anti-mouse immunoglobulins, the enhanced chemiluminescence (ECL) reagents, autoradiography films, and [γ-32P]-ATP were from Amersham (Pharmacia Biotech., Europe GmbH, Freiburg, Germany).
Male castrated German landrace-type domestic pigs (35.4 ± 5.6 kg) were premedicated with ketamine [20 mg/kg of body weight (BW) by intramuscular injection] and piritramide (2 mg/kg BW, subcutaneously) 10 min before the initiation of anesthesia with a bolus of α-chloralose (25 mg/kg BW). After tracheal intubation anesthesia was maintained by a continuous intravenous infusion of α-chloralose (25 mg/kg/h). The animals were ventilated with a pressure-controlled respirator (Stephan Respirator ABV; F. Stephan GmbH Gackenbach, Germany) with room air enriched with 2 L/min of oxygen. Arterial blood gases were analyzed frequently (Chiron, Diagnostics, St. Leon-Rot, Germany) to guide adjustment of the respirator settings. Additional doses of piritramide (10 mg) were given i.v. every 60 min. Both internal jugular veins were cannulated with polyethylene tubes for administration of saline, piritramide, and α-chloralose. Arterial sheath catheters (7F) (Vasurix; Guerbert GmbH, Sulzbach/Ts., Germany) were inserted into both carotid arteries. To measure arterial blood pressure, the left sheath was advanced into the aortic arch and connected with a Statham transducer (P23XL; Statham, San Juan, Puerto Rico). After midsternal thoracotomy, the heart was suspended in a pericardial cradle. Arterial pressure, heart rate, and ECG were continuously monitored and recorded on a MacLab computer. A loose reversible ligature was placed halfway around the left anterior descending artery (LAD) and was subsequently tightened to occlude the vessels. In pigs subjected to intramyocardial microinfusion, eight 26-gauge needles connected by tubing to a peristaltic pump (Minipuls 3; ABIMED Gilson; Villiersle-Bel, France) were placed in pairs along the LAD into the myocardium perpendicular to the epicardial surface. After a stabilization period of 30 min, the experimental protocols were started. The MEK 1/2 inhibitors PD98059 and UO126 were dissolved in DMSO (dimethylsulfoxide) and finally diluted in Krebs-Henseleit buffer (KHB; the maximal final concentration of DMSO was 0.1%). Infusion of KHB with DMSO served as a negative control.
The experimental design for this study is presented in Fig. 1.
Determination of infarct size
At minute 45 into the last reperfusion period, 1 g of fluorescein dissolved in 10 ml Ringer's solution was injected into the right ventricle. This stained the entire myocardium, unless there were areas of nonreperfused tissue. Hearts with traces of nonreperfused myocardium were excluded from analysis. At the end of the experimental protocol, the LAD was reoccluded, and the distal ascending aorta was clamped. Then 500 mg of zinc cadmium fluorescent microspheres (diameter, 2-15 μm) in 10 ml of Ringer's solution was injected into the proximal ascending aorta. Shortly thereafter, the animals were killed with an intravenous bolus of 20% potassium chloride to arrest the heart. After excision of both atria, the right ventricle was removed. The left ventricle was cut perpendicular to the LAD into transverse slices, each one containing a microinfusion needle pair. After systemic drug administration, the left ventricle was cut into six slices at an equal distance. Heart slices were weighed and incubated at 37°C in 1% triphenyltetrazolium-chloride (TTC) in PBS, pH 7.0, for 20 min. Myocardium at risk of infarction was identified as the nonfluorescent (by microspheres) area under UV light (366 nm). The infarcted area was demarcated by the absence of the characteristic red TTC stain. The slices were photographed by double exposure with UV and artificial daylight (Figs. 2 and 3), and the pictures were used for further planimetric evaluation (NIH image 1.62). Planimetry of the infarct areas was performed on the basal aspect of the apex, the apical and basal sides of the following four consecutive myocardial slices, and on the apical aspect of the basal section of the left ventricle. We expressed IS as the infarct area (IA) relative to the risk area (RA). ISs were then averaged per group and depicted graphically (Fig. 4A and B).
Preparation of soluble, particulate, and nuclear fractions
The biopsy tissues for the kinase assays were suspended in ice-cold buffer containing (in mM): 20 Tris-HCl, 250 sucrose, 1.0 EDTA, 1.0 EGTA, 1.0 dithiothreitol (DTT), 0.1 sodium orthovanadate, 10 NaF, and 0.5 PMSF, pH 7.4, (buffer A) and were homogenized with a Teflon-glass homogenizer. The homogenates were centrifuged for 10 min at 3,000 g, and the supernatants were centrifuged again at 14,000 g for 30 min at 4°C. The supernatants after this second centrifugation represented the soluble (cytosolic) fractions, and the pellets represented the particulate fractions. The pellets from the first (3,000 g) centrifugation were resuspended in buffer B containing (in mM): 20 Tris-HCl, 1,000 sucrose, 1.0 EDTA, 1.0 EGTA, 1.0 DTT, 0.1 sodium orthovanadate, 10 NaF, 10 KCl, 0.1 PMSF (pH 7.4), and were centrifuged for 30 min at 10,000 g (4°C). The resulting pellets were resuspended in buffer C containing 10% glycerol, 20 mM Tris-HCl, 400 mM KCl, 1.0 mM EGTA, 1.0 mM DTT, 0.1 mM sodium orthovanadate, 10 mM NaF, 0.5 mM PMSF, 0.1% Triton X-100, sonicated, and used for the detection of transcription factor Elk-1. For the preparation of electrophoretic probes, Laemmli sample buffer was added, and the proteins were denatured by heating. The denatured probes were applied to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and used for MAPK assays by "in gel" kinase assays and for Western blot analysis.
Measurement of p44/42-MAPK activities by in gel phosphorylation
Equivalent amounts of proteins were separated on 10% SDS-polyacrylamide gels containing 0.25 mg/ml myelin basic protein (MBP). After electrophoresis, the gels were washed for 1 h with 20% (vol/vol) 2-propanol in 50 mM Tris-HCl (pH 8.0), followed by 1 h with 5 mM 2-mercaptoethanol in 50 mM Tris-HCl, pH 8.0. The in gel proteins were denatured by incubation for 2 h with 50 mM Tris-HCl, pH 8.0, containing 6 M guanidine HCl. Renaturation was achieved by incubation with 50 mM Tris-HCl, pH 8.0, containing 0.1% (vol/vol) Nonidet P-40 and 5 mM 2-mercaptoethanol for 16 h. After preincubation of gels in 40 mM HEPES (pH 8.0) containing 2 mM DTT and 10 mM magnesium chloride, the "in gel" phosphorylation of substrates was performed in 40 mM HEPES (pH 8.0), 0.5 mM EGTA, 10 mM magnesium chloride, 1.0 μM protein kinase A inhibitory peptide, and 25 μM [γ-32P]-adenosine triphosphate (ATP; 5 μCi/ml) at 25°C for 4 h. After extensive washing in 5% (wt/vol) trichloracetic acid containing 2% (wt/vol) sodium pyrophosphate, the gels were dried, and quantitative analysis was performed using a Phosphorimager SF (Molecular Dynamics, Krefeld, Germany).
In some experiments, the phosphorylated ERKs from soluble fractions (200 μg of proteins) were immunoprecipitated with an antibody against phospho-ERKs. After incubation for 4 h at 4°C (continuous mixing), the immune complexes were incubated for a further 4 h with protein A agarose beads. The resulting complexes were collected by centrifugation, resuspended in Laemmli sample buffer, boiled, and immunoblot assay using the phospho-ERKs antibody was performed.
Soluble (cytosolic), particulate, and nuclear fractions of heart tissue were subjected to SDS-PAGE in 10% polyacrylamide gels, and proteins were transferred onto nitrocellulose membranes. Antibodies against ERKs, phospho-ERKs, phospho-SAPK/JNKs, phospho-p38-MAPK, and phospho-Elk-1 were used for primary immunodetection. Peroxidase labeled anti-mouse or anti-rabbit immunoglobulins were used as secondary antibodies. Bound antibodies were detected by the ECL Western blot method.
The one "factorial analysis of variance" with subsequent multiple comparisons by Bonferroni were performed to compare the infarct size of the different groups; p < 0.05 was accepted as significant. For in gel phosphorylation and Western blot assays, the PD- and UO-treated tissue biopsies were compared with controls (KHB-treated tissue (negative control). The differences were evaluated by the Student's t test. The accepted level of significance was p < 0.05.
The hemodynamic parameters remained unchanged during the local microinfusion of PD98059, UO126, or solvent (KHB/DMSO) alone and also during the systemic application of UO126. In addition, no ventricular premature beats were detected during any of the infusion regimens.
Three of 17 PD98059 (100 μM)-treated animals and two of 11 animals treated with UO126 systemically (7.5 mg/animal) died of electromechanical dissociation consistent with drug toxicity. For this reason, the PD concentration was subsequently reduced to 50 μM. PD98059 could not be given systemically because of its poor solubility and tendency to precipitate in aqueous solutions. Therefore, we administered it by local intramyocardial microinfusions. Altogether, 41 animals successfully completed the protocol and were included in the data analysis.
Effect of intramyocardial PD98059 and UO126 microinfusion before and during IP on infarct size in pig myocardium
In pilot experiments, we had not observed any evidence of cell necrosis by TTC staining as a consequence of the local microinfusion of either PD or UO into nonischemic myocardium (data not shown).
Ischemic preconditioning in our model reduced the mean infarct size from 54.0 ± 1.5% (control A, index ischemia, group I), to 2.5 ± 0.1% (control B, ischemic preconditioning, group II; Fig. 2A and B). Local microinfusions of PD98059 and UO126 15 min before IP and during both reperfusion phases of the IP protocol significantly reduced the IP-induced cardioprotection, as measured by IS (i.e. the IA relative to the RA). In areas of 50 μM PD and 50 mM UO126 microinfusions, significant wedge-shaped infarcts were observed. With decreasing concentrations of PD and UO, the IS decreased significantly, thereby demonstrating a clear dose-response effect (Figs. 3A-E and 4A, C, and D). The infusion of DMSO in KHB (negative control) had no effect on the degree of IP-induced cardioprotection (Figs. 2B and 3B).
Effect of systemic application of UO126 before and during IP on infarct size in pig myocardium
The systemic infusion of UO126 30 min before IP and during both reperfusion phases of the IP protocol reduced and even cancelled the IP-induced cardioprotection in a concentration-dependent fashion (Figs. 2C and 4B). The infusion of DMSO in KHB (negative control) had no influence on the IP-induced cardioprotection (Figs. 2B and 4B).
Effect of intramyocardial PD98059 and UO126 microinfusions on ERK activities/phosphorylation
The ERK 1/2 activities were determined by means of in gel phosphorylation assays of myelin basic protein (MBP). After IP, the cytosolic activities of both ERK-1 (44 kDa) and ERK-2 (42 kDa) were significantly increased compared with baseline in nonpreconditioned tissue.
Microinfusion of KHB induced an additional increase of ERKs activities. This suggests some nonspecific stimulation of the ERKs pathway by the microinfusion procedure, possibly related to mechanical stress. The intramyocardial microinfusion of UO126 and PD98059 for 15 min before IP and during both reperfusion cycles of IP significantly inhibited ERKs activities (as compared with the KHB/DMSO microinfusion). The in gel phosphorylation of MBP revealed an inhibition of ERK-1 and ERK-2 activities after treatment with 50 μM PD98059 (Fig. 5A and B). Similar results were obtained by treatment with 50 μM UO126 (Fig. 6A and B).
Western blot analysis with a phosphospecific ERK (Thr 202/Tyr 204) antibody showed a decreased content of cytosolic phospho-ERK-1 and phospho-ERK-2 at the end of IP after PD treatment compared with KHB/DMSO alone (Fig. 5C, lanes 1-7; left). Similar but more pronounced results were obtained by prior immunopre-cipitation of the cytosolic fractions with a phosphospecific ERK antibody (Fig. 5C, lanes 1-4; right).
A decrease of phosphorylated ERK-1 and ERK-2 was also demonstrated in the cytosolic fractions of UO-treated myocardium (Fig. 6C and D). There were no significant changes of ERKs phosphorylation in the particulate and nuclear fractions of PD-, DMSO/KHB-treated tissue (data not shown). By Western blot analysis, we observed no changes of levels or cellular distribution of ERKs after IP with or without PD or UO treatment (data not shown).
Effect of intramyocardial PD98059 infusion on phosphorylation of Elk-1
Further to characterize the in vivo effect of PD98059 on ERK activities, we also determined the phosphorylation of the transcription factor Elk-1. This transcription factor serves as an endogenous substrate for ERKs, and we investigated its phosphorylation after microinfusion of PD98059 (or KHB as negative control) before and at the end of the IP protocol. We found PD98059 to significantly inhibit the phosphorylation of Elk-1 (Fig. 7A and B).
Effect of systemic infusion of UO126 on ERK phosphorylation after IP
After systemic UO126 infusion (7.5 mg/animal), we observed a decreased content of cytosolic phospho-ERK-1 and phospho-ERK-2 (Fig. 8A and B) when compared with tissue from preconditioned myocardium (systemic treatment with KHB/DMSO) of control animals. We found no changes in levels or in cellular distribution of total ERKs during the experimental procedure (data not shown).
Effect of intramyocardial infusion of PD98059 and UO126 on p38-MAPK and SAPK/JNK phosphorylation
We also investigated the degree of p38-MAPK and SAPK/JNKs phosphorylation after completion of the IP protocol. For this purpose, we used a specific anti-phospho-p38-MAPK antibody (Thr180/Tyr182) and specific anti-phospho-SAPK/JNK antibody (Thr183/Tyr185). We found no changes in levels or in cellular distribution of phosphorylated p38-MAPK and SAPK/JNKs on completion of the IP protocol. At the tenth minute of index ischemia, we observed an increased phosphorylation of p38-MAPK in PD treated (P), KHB/DMSO (D), and untreated (K) ischemic myocardium versus nonischemic myocardium (Fig. 9A and B).
We demonstrate for the first time in vivo that specific inhibition of the ERK cascade counteracts the IP-related cardiac protection. We also show that both agents used, PD98059 and UO126, inhibit the phosphorylation pathway of ERKs (p44/42 MAPKs) in a specific way without influence on the p38-MAPK and SAPK/JNK cascades. As we have previously shown, ischemia alone activates p38-MAPK and ERKs (10), whereas reperfusion specifically activates the SAPK/JNKs and further potentiates the ischemia-activated ERKs. In this study we observed that the local infusion of both PD98059 and UO126 as well as systemic application of UO126 before and during the preconditioning (IP) protocol reversed the IP-induced cardioprotection and specifically inhibited the activities of ERKs. These results suggest that the activation of ERKs protects cardiomyocytes (cardiac cells) during ischemia/reperfusion.
PD98059 [2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] is the specific inhibitor of MEK 1/2 (MAPKK 1/2), and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenyl-thio]butadiene) represents a novel specific inhibitor of MEK 1/2 and ERK 1/2 (26). UO126 is a selective and potent (100-fold higher affinity for MEK than PD98059) inhibitor of the dual-specificity kinases MEK1 and MEK2 both in the in vitro enzymatic assays and intracellularly where UO126 blocked phosphorylation and activation of ERKs (31). Blockade of ERK activation would prevent downstream phosphorylation of a number of factors, including p62TCF (Elk-1) (31). The compound UO126 appears to inhibit MEK1 directly but with higher specificity for the constitutive mutant wild-type enzyme. In contrast, PD98059 inhibits MEK 1/2 activation by binding to the inactive enzyme, preventing its activation by Raf-kinase, indirectly inhibiting activation of ERK 1/2. Therefore, the compound UO126 acts one step further downstream of PD98059 and blocks ERK 1/2 activity (31). UO126 and PD98059 in general seem to accomplish the same results, although in a mechanistically distinct fashion.
MEK inhibition affects many different signaling events through a variety of cell-surface receptors. Growth factor-mediated proliferation, chemotactic responses, and the production of IL-2, TNF-α, GMCSF, interferon-τ, and IL-6 are blocked by PD98059 (32). PD98059 also inhibits ERKs phosphorylation in myocytes exposed to phenylephrine but fails to block atrial natriuretic factor expression (33).
MAPKs play an important role in signal transduction in eukaryotic cells, where they modulate many cellular events such as growth, differentiation, and stress response. The MAPK superfamily includes ERKs, JNKs, and p38-MAPKs that are found in three interwoven signal-transduction cascades (5,34). These kinases phosphorylate and thus activate transcription factors in response to mitogens, growth factors, or various forms of stress. ERK-1 and -2 are activated on phosphorylation by MAPKKs, also known as MEK1 and MEK2, MAPK of ERK kinase (35). This phosphorylation occurs at an activation domain on the threonine and tyrosine residues in the sequence pTEpY. Previous studies have described signal-transduction systems that operate during myocardial ischemia or ischemia/reperfusion (2). In our study we focused on the extracellular signal-regulated protein kinase (ERK) cascade that belongs to the group of MAPKs. Previously we have found that the activities of the ERKs increased moderately during brief ischemia and even markedly during the following reperfusion period (10). Our previous studies have also indicated a rapid activation of ERKs, with subsequent inactivation of these enzymes during prolonged exposure to ischemic stress. The activities of ERKs in our experimental model peaked after 10-15 min of ischemia and thereafter declined. In a recent study, Shimizu et al. (36) ascertained that ERKs, JNKs, and p38-MAPK were activated by ischemia after experimentally induced in vivo myocardial infarction in rats. Myocardial ERKs were activated and reached a peak within 5 min after coronary ligation in vivo. After 30 min, the activities of these kinases returned to baseline. Also in isolated rat cardiac myocytes under hypoxia and reoxygenation, activation of 42-kDa and 44-kDa MAPKs (ERKs) was observed by in gel kinase phosphorylation assays. The kinases activities reached a maximum peak at 5-10 min by hypoxia and at 15 min by reoxygenation after 60 min of hypoxia (37-39). The role of ERKs by hypoxic or ischemic stress has been confirmed by an in vitro tissue culture model of ischemia (depletion of ATP after exposure of myocytes to potassium cyanide and deoxyglucose) with upregulation of the activities of protein kinases with molecular masses of 42 and 44 kDa (40). These kinases appeared to correspond to the ERKs. Contrary to these results obtained in rat cardiomyocytes, in canine kidney epithelial cells, the ERKs were not activated after ATP depletion with cyanide and deoxyglucose (41). This indicates possible differences in stimulation and regulation of this protein kinase cascade in different cellular systems, and species differences cannot be excluded.
We have previously shown that brief coronary occlusions lead to upregulation of the protooncogenes c-jun and c-fos(42,43). Because exogenous application of TRK (tyrosine kinase) ligands like IGF-2, aFGF, and bFGF exerted (23) trophic effects with a survival value of their known effects on MAPKs, especially ERKs, it became obvious that ischemic preconditioning and TRK ligands share a common pathway. In our present study, we showed that at the end of our ischemic preconditioning protocol, the ERKs are activated and that inhibition of ERK 1/2 by specific inhibitors PD98059 and UO126 has a negative effect on the cardioprotection by ischemic preconditioning. Recently, we have also seen an augmentation of IS after ischemia without IP by PD and UO treatment (unpublished data). Thus, the ERK activation appears to have a protective role in response of the myocardium to ischemia. This hypothesis is supported by results of some other studies. Aikawa et al. (44) demonstrated that oxidative stress activates ERKs in cultured neonatal cardiomyocytes. When H2O2-induced activation of ERKs was selectively inhibited by PD98059, the number of cardiac cells that showed apoptotic cell death was increased. Adderley et al. (45) found that death of cardiomyocytes due to exposure to H2O2 and doxorubicin (oxidative stresses) was limited by an increase of prostacyclin formation. This increase reflected the induction of cyclooxygenase-2 (COX-2) expression mediated by ERK 1/2 activation, which could be abolished by PD98059. The positive role of ERKs in cell protection is supported by a study of Sheng et al. (46). The authors found that cardiotrophin 1 (CT-1), a cardiac cytokine, prevented cell death by inhibiting apoptosis of serum-deprived neonatal rat myocytes. This effect was associated with an activation of both ERK-1 and ERK-2. Ten millimolar PD98059 was able to inhibit completely the survival effects of CT-1. All these findings suggest that activation of ERKs is important for protecting cardiac myocytes against cell death injury.
In contrast to most other studies, including our own, Bogoyevitch et al. (47) showed that global ischemia and ischemia followed by reperfusion did not activate p42- and p44-ERK. Also in partial contrast (regarding the role of the SAPK/JNKs in ischemia) is a recent study by Shimizu et al. (36) showing that ERK, JNK and p38-MAPK were activated by ischemia experimentally in the in vivo myocardial infarction. Part of the differences may be because the patterns of activation of both ERKs differ depending on whether the experimental conditions were in vivo or in vitro.
In conclusion, we have shown that inhibition of ERK 1/2 during ischemic preconditioning has a detrimental effect on cardioprotection and infarct development. Thus, the ERKs play a protective role in the myocardium during ischemia/reperfusion. The effector site for the protective action of ERK is not known. Transcriptional and translational processes appear somewhat unlikely because of the fast onset of signal transduction in IP. However, the proximity of the UO-blocked step in the signal cascade to the first transcriptional event does not rule out a transcriptional component. Experimental approaches for both hypotheses are imaginable.
Acknowledgment: We thank Armin Helisch for critical reading of the manuscript and suggestions about figures.
1. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem
2. Downey J, Cohen M. Signal transduction in ischemic preconditioning
. Adv Exp Med Biol
3. Seger R, Krebs EG. The MAPK signalling cascade. FASEB J
4. Pelech SL, Charest DL. MAP kinase-dependent pathway in cell cycle control. Prog Cell Cycle Cont
5. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol
6. Kyriakis JM, Banerjee P, Nikolalkaki E, et al. The stress-activated protein kinase subfamily of c-Jun kinases. Nature
7. Aharonovitz O, Aboulafia-Etzion S, Leor J, Battler A, Granot Y. Stimulation of 42/44 kDa mitogen-activated protein kinases by arginine vasopressin in rat cardiomyocytes. Biochim Biophys Acta
8. Bogoyevitch MA, Glennon PE, Andersson MB, et al. Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes: the potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem
9. Fischer TA, Singh K, O'Hara DS, Kaye DM, Kelly RA. Role of AT1
receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes. Am J Physiol
10. Htun P, Barancik M, Schaper W. Brief ischemia/reperfusion
activates transcellular signaling cascades and leads to proto-oncogene expression and growth factor production. In: Abiko Y, Karmazyn M, eds. Protection against ischemia/reperfusion damage of the heart.
Tokyo: Springer, 1998:179-92.
11. Knight RJ, Buxton DB. Stimulation of c-jun kinase and mitogen-activated protein kinase by ischemia and reperfusion in the perfused rat hearts. Biochem Biophys Res Commun
12. Sudgen PH, Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res
13. Nagarkatti DS, Sha'afi RI. Role of p38 MAP kinase in myocardial stress. J Mol Cell Cardiol
14. Ma XL, Kumar S, Gao F, et al. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation
15. Murry C, Jennings R, Reimer K. Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation
16. Yellon DM, Baxter GF, Garcia-Dorado D, Heusch G, Sumeray MS. Ischaemic preconditioning: present position and future directions. Cardiovasc Res
17. Schott RJ, Rohmann S, Braun ER, Schaper W. Ischemic preconditioning
reduces infarct size in swine myocardium. Circ Res
18. Vogt AM, Htun P, Arras M, Podzuweit T, Schaper W. Intramyocardial infusion of tool drugs for the study of molecular mechanisms in ischemic preconditioning
. Basic Res Cardiol
19. Cano E, Hazzalin CA, Mahadevan LC. Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol Cell Biol
20. Barancik M, Htun P, Schaper W. Okadaic acid and anisomycin are protective and stimulate the SAPK/JNK pathway. J Cardiovasc Pharmacol
21. Barancik M, Htun P, Strohm C, Schaper W. Inhibition of the cardiac p-38-MAPK pathway by SB203580 delays ischemic cell death. J Cardiovasc Pharmacol
22. Mackay K, Mochly-Rosen D. An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia. J Biol Chem
23. Htun P, Ito WD, Hoefer IE, Schaper J, Schaper W. Intramyocardial infusion of FGF-1 mimics ischemic preconditioning
myocardium. J Mol Cell Cardiol
24. Htun P, Barancik M, Ito W, Schaper W. Cardioprotection by aFGF and bFGF is induced by activation of the MAP kinases p42 and p44 [Abstract]. Circulation
25. Dudley T, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A
26. Duncia JV, Santella JBr, Higley CA, et al. MEK inhibitors: the chemistry and biological activity of UO126
, its analogs, and cyclization products. Bioorg Med Chem Lett
27. Cavigelli M, Dolfi F, Claret FX, Karin M. Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation. EMBO J
28. Gupta S, Campbell D, Derijard B, Davis RJ. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science
29. Yang SH, Whitmarsh AJ, Davis RJ, Sharrocks AD. Differential targeting of MAP kinases to the ETS-domain transcription factor Elk-1. EMBO J
30. Yang SH, Yates PR, Whitmarsh AJ, Davis RJ, Sharrocks AD. The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif. Mol Cell Biol
31. Favata MF, Horiuchi KY, Manos EJ, et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem
32. Dumont FJ, Staruch MJ, Fischer P, DaSilva C, Camacho R. Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK MAP kinase or calcineurin signaling pathways results in differential modulation of cytokine production. J Immunol
33. Post G, Goldstein D, Thuerauf D, Glembotski C, Brown J. Dissociation of p44 and p42 mitogen-activated protein kinase activation from receptor-induced hypertrophy in neonatal rat ventricular myocytes. J Biol Chem
34. Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascade. Adv Cancer Res
35. Cano E, Mahadevan L. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci
36. Shimizu N, Yoshiyama M, Omura T, et al. Activation of mitogen-activated protein kinases and activator protein-1 in myocardial infarction in rats. Cardiovasc Res
37. Seko Y, Tobe K, Takahashi N, Kaburagi Y, Kadowaki T, Yazaki Y. Hypoxia and hypoxia/reoxygenation activate Src family tyrosine kinases and p21 ras in cultured rat cardiac myocytes. Biochem Biophys Res Commun
38. Seko Y, Tobe K, Ueki K, Kadowaki T, Yazaki Y. Hypoxia and hypoxia/reoxygenation activate Raf-1, mitogen-activated protein kinase kinase, mitogen-activated protein kinases, and S6 kinase in cultured rat cardiac myocytes. Circ Res
39. Seko Y, Takahashi N, Tobe K, Kadowaki T, Yazaki Y. Hypoxia and hypoxia/reoxygenation activate p65PAK, p38 mitogen-activated protein kinase (MAPK), and stress-activated protein kinase (SAPK) in cultured rat cardiac myocytes. Biochem Biophys Res Commun
40. Bogoyevitch MA, Ketterman AJ, Sugden PH. Cellular stresses differentially activate c-jun N-terminal protein kinases and extra-cellular signal-regulated protein kinases in cultured ventricular myocytes. J Biol Chem
41. Pombo CM, Bonventre JV, Avruch J, Woodgett JR, Kyriakis JM, Force T. The stress-activated protein kinases are major c-Jun amino-terminal kinases activated by ischemia and reperfusion. J Biol Chem
42. Brand T, Sharma H, Fleischmann K, et al. Proto-oncogene expression in porcine myocardium subjected to ischemia and reperfusion. Circ Res
43. Knöll R, Arras M, Zimmermann R, Schaper J, Schaper W. Changes in gene expression following short coronary occlusions studied in porcine hearts with run-on assays. Cardiovasc Res
44. Aikawa R, Komuro I, Yamazaki T, et al. Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest
45. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem
46. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, Chien KR. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway: divergence from downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem
47. Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, et al. Stimulation of the stress-activated mitogen-activated protein kinases and c-jun N-terminal kinases are activated by ischemia/reperfusion
. Circ Res