Stimulation of the δ1-opioid receptor has been shown to trigger ischemic preconditioning (IPC). Additionally, myocardial ischemia/reperfusion induces the activation of extracellular signal-regulated kinase (ERK). Therefore, we examined the role of ERK in acute cardioprotection induced by δ1-opioid receptor stimulation or IPC. Infarct size (IS) was expressed as a percentage of the area at risk (AAR). Control animals had an IS/AAR of 60.6 ± 1.8. IPC and δ1-opioid receptor stimulation with TAN-67 reduced IS/AAR (8.2 ± 1.3 and 30.2 ± 2.4). Inhibition of ERK with the selective MEK-1 antagonist, PD 098059 during IPC or TAN-67 administration significantly reduced cardioprotection (41.5 ± 6.4 and 63.0 ± 4.8). Western Blot analysis and subsequent densitometry corroborated these observations. Control, TAN-67-, or IPC-treated hearts were harvested after 0, 5, 15, and 30 min of ischemia or 5, 30, and 60 min of reperfusion and separated into cytosolic and nuclear fractions. Both isoforms of ERK (p44 and p42) rapidly increased to greater levels throughout reperfusion in the nuclear fraction of IPC- and opioid-treated versus control rats, however, this increase was not attenuated by PD 098059. Conversely, the rapid activation of the 44-kDa isoform of ERK after 5 min of reperfusion in the cytosolic fraction was significantly increased in IPC- and opioid-treated hearts versus control, and this increase was abolished by pretreatment with PD 098059. Additionally, p42 was activated in the cytosolic fraction of IPC-treated animals. These results suggest a key role for the 44-kDa isoform of ERK in the cytoplasm during cardioprotection induced by either IPC or stimulation of the δ1-opioid receptor.
We have previously demonstrated cardioprotection against sustained ischemia in the rat heart via both ischemic preconditioning (IPC) (Schultz et al., 1997; Fryer et al., 2000a) and pharmacological preconditioning with opioid receptor agonists (Schultz et al., 1995,1996). Schultz et al. (1998a) first demonstrated that opioid-induced cardioprotection was mediated by the δ1, but not the μ- or κ-opioid receptor and subsequently demonstrated that this effect was also mediated by the activation of a Gi/o protein (Schultz et al., 1998b). Additionally, opiates are thought to induce cardioprotection via activation of protein kinase C (PKC) (Miki et al., 1998) and activation of the mitochondrial ATP-sensitive potassium (KATP) channel (Schultz et al., 1996, 1998b;Fryer et al., 2000a,b).
IPC may induce cardioprotection via a similar signal transduction pathway within the cardiac myocyte, and this phenomenon has been intensely examined. It is thought that IPC induces the activation of specific families of tyrosine kinases and the activation and translocation of specific PKC isoforms (Liu et al., 1994; Speechly-Dick et al., 1994; Ytrehus et al., 1994; Miyawaki et al., 1996; Albert and Ford, 1999; Fryer et al., 1999b). These kinases may act as a link to the mitogen-activated protein (MAP) kinase cascade, where, upon activation, MAP kinases directly modulate cellular function or alternatively may translocate to the nucleus of the cell and subsequently influence gene transcription and translation.
It has been suggested that inhibition of the stress-activated MAP kinases, c-jun N-terminal kinase (JNK) and p38, may be cardioprotective via a reduction in apoptosis (Mackay and Mochly-Rosen, 1999), a delay in ischemic cell death (Barancik et al., 2000), and an improvement in cardiac function after ischemia (Ma et al., 1999). However, Weinbrenner et al. (1997) have suggested that p38 MAP kinase phosphorylation of tyrosine 182 correlates with protection afforded by IPC and that anisomycin, an activator of JNK and p38, can induce cardioprotection equal to that of IPC. Despite evidence both pro and con as to the importance of p38 and JNK in IPC, little data exists concerning a role for ERK in cardioprotection following IPC or opioid receptor stimulation.
ERK is activated upon phosphorylation by the upstream kinase MEK-1 on a threonine and tyrosine residue. This activation may be PKC-dependent. Indeed, opioid- and IPC-induced cardioprotection has previously been shown to be regulated by a PKC-sensitive mechanism (Ytrehus et al., 1994; Miki et al., 1998; Fryer et al., 1999b). Indeed, Ping et al. (1999) have demonstrated that the activation of PKC-ε during IPC correlates with p44/p42 MAP kinase activation when PKC-ε was selectively overexpressed in rabbit cardiomyocytes. Additionally,Schonwasser et al. (1998) have demonstrated that transfection of PKC-α, βI, δ, ε, η, or ζ into Cos-7 cells can activate p42 MAP kinase. Similarly, evidence from our laboratory suggests a role for PKC in δ1-opioid receptor-mediated cardioprotection and that PKC-δ plays an important role in infarct size reduction, since the selective δ-isoform inhibitor, rottlerin, could abolish TAN-67-induced reduction in infarct size. Additionally, evidence from our laboratory suggests that opioid receptor stimulation induces the selective translocation of PKC-α, βI, δ, and ε to distinct cellular loci, which may be responsible for cardioprotection, possibly via activation of a MAP kinase signaling cascade (R. M. Fryer, P. F. Pratt, A. K. Hsu, and G. J. Gross, unpublished observation).
The role of ERK in cardioprotection from δ1-opioid receptor stimulation may be important, since opioids can stimulate members of the MAP kinase family. Gutstein et al. (1997) have demonstrated in COS cells that μ- and δ-opioid receptor stimulation can potently activate ERK but only weakly activate the stress-activated MAP kinases. Therefore, ERK activation is a likely signaling pathway by which opioid agonists induce cardioprotection. Therefore, we examined the role of ERK in acute cardioprotection against ischemia via δ1-opioid receptor stimulation and IPC and hypothesize that ERK activation may be an important component of IPC- or opioid-mediated signal transduction during ischemia/reperfusion injury.
Materials and Methods
This study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.
General Surgical Preparation.
Male Wistar rats, 350 to 450 g, were used for all phases of this study. The rats were anesthetized via i.p. administration of inactin (100 mg/kg), a long-acting barbiturate. A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (model CIV-101, Columbus Instruments, Columbus, OH, or model 683, Harvard Apparatus, South Natick, MA). The rats were ventilated with room air supplemented with O2 at 60 to 65 breaths per minute. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5 to 10 mm H20. Arterial pH, PCO2, and PO2were monitored at control, 15 min of occlusion, and 60 and 120 min of reperfusion by a blood gas system (AVL 995 pH/blood gas analyzer, Roswell, GA) and maintained within a normal physiological range (pH 7.35–7.45; PCO2 25–40 mm Hg; and PO2 80–110 mm Hg) by adjusting the respiratory rate and/or tidal volume. Body temperature was maintained at 38°C by the use of a heating pad and bicarbonate was administered i.v. as needed to maintain arterial blood pH within normal physiological levels.
The right carotid artery was cannulated to measure blood pressure and heart rate via a PE50 or PE23 pressure transducer (Gould, Cleveland, OH) connected to a polygraph (model 7, Grass, Quincy, MA). The right jugular vein was cannulated for saline, bicarbonate, and drug infusion. A left thoracotomy was performed at the fifth intercostal space followed by a pericardiotomy and adjustment of the left atrial appendage to reveal the location of the left coronary artery. A ligature (6-0 prolene) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle. The ends of the suture were threaded through a propylene tube to form a snare. Pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat elicited occlusion of the coronary artery and resulted in regional left ventricular ischemia. Epicardial cyanosis and a subsequent decrease in blood pressure verified coronary artery occlusion. Reperfusion of the heart was initiated via unclamping the hemostat and loosening the snare and was confirmed by visualizing an epicardial hyperemic response. Heart rate and blood pressure were allowed to stabilize before the following protocols were initiated.
Inactin (thiobutabarbital sodium) was purchased from Research Biochemicals International (Natick, MA). 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical Co. (St. Louis, MO). TAN-67 was synthesized and kindly furnished by Dr. Hiroshi Nagase of Toray Industries (Kanagawa, Japan) and dissolved in saline. Inactin was dissolved in distilled water. PD 098059 was purchased from Research Biochemicals International and dissolved in ethanol and saline. All drugs were dissolved in approximately 0.9 ml of vehicle for administration at all concentrations.
Study Groups and Experimental Protocols.
The protocols used to determine a role for ERK in cardioprotection are shown in Fig.1. All animals were subjected to 30 min of ischemia and 2 h of reperfusion (Control). TAN-67 (2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino[2,3,3-g]isoquinoline), a δ1-opioid agonist, was infused 15 min before ischemia and reperfusion (TAN-67). IPC was induced via one cycle of a 5-min coronary artery occlusion and 5 min of reperfusion. The effect of the MEK-1 inhibitor, PD 098059, in the absence of opioid receptor stimulation or IPC was investigated by administering this compound 20 min before the control protocol. The effect of ERK inhibition during IPC or opioid treatment was investigated via administration of a bolus of PD 098059 10-min before IPC or 5-min before TAN-67 administration (PD 098059 + IPC and PD 098059 + TAN-67, respectively).
Determination of Infarct Size.
Upon completion of the above protocols, the coronary artery was reoccluded and the area at risk (AAR) was determined by negative staining. Patent blue dye was administered via the jugular vein to effectively stain the nonoccluded area of the left ventricle. The rat was euthanized with a 15% KCl solution. The heart was excised, and the left ventricle was removed from the remaining tissue and subsequently cut into six thin cross-sectional pieces. This allowed for the delineation of the normal area, stained blue, versus the AAR that subsequently remained pink. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 min with a 1% TTC stain in 100 mM phosphate buffer (pH 7.4) at 37°C. TTC is an indicator of viable and nonviable tissue and is reduced by dehydrogenase enzymes present in the myocardium, resulting in a formazan precipitate and inducing a deep red color in the viable tissue while the infarcted area stains gray (Klein et al., 1981). Tissues were stored in vials of 10% formaldehyde overnight, and the infarcted myocardium was dissected from the AAR under the illumination of a dissecting microscope (Cambridge Instruments, Monsey, NY). Infarct size (IS) and AAR were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR).
Tissue Sample Preparation.
Tissues samples were processed from the area at risk in the left ventricle of control animals, animals treated with TAN-67, and animals subjected to IPC, at 0, 5, 15, or 30 min of ischemia or 5, 30, or 60 min of reperfusion for the determination of protein expression and activity of either p44 or p42 MAP kinase as previously described (Ping et al., 1999). Myocardial tissue samples, frozen at −80°C until use, were powdered with a prechilled mortar and pestle. Total cellular proteins were isolated via glass-glass homogenization of the powdered tissue in lysis buffer A (0.3% β-mercaptoethanol, 50 mM Tris-HCl, 5 mM EDTA, 10 mM EGTA, 50 μg/ml phenylmethylsulfonyl fluoride, 200 μM sodium orthovanadate, 1 ml/20 g of tissue Sigma Protease Inhibitor Cocktail P8340 containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A,trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane (E-64), bestatin, leupeptin, and aprotinin in dimethyl sulfoxide).
Preparation of the Nuclear and Cytosolic Fractions.
The homogenate was loaded onto a sucrose cushion, containing 2 ml of 1 M sucrose in lysis buffer A, and was centrifuged at 1600g for 10 min to allow for pelleting of the nuclear fraction. The pellet was washed with dH2O and resuspended in lysis buffer B (lysis buffer A containing 0.5% Igepal, 0.1% deoxycholate, and 0.1% Brij-35) for 60 min on ice and subsequently recentrifuged at 7850g for 5 min. The supernatant became the nuclear fraction. The supernatant from the initial 1,600gcentrifugation was loaded onto a second 1 M sucrose cushion and was centrifuged at 150,000g for 60 min. The supernatant became the cytosolic fraction. Total protein concentrations in the respective fractions were determined via the Bradford (Bio-Rad, Hercules, CA) protein assay. Preliminary experiments were carried out to ensure that storing the tissues at −80°C until use, and powdering of the frozen tissue did not fractionate the myocardial nuclei. The purity of the fractions was assessed with specific antibody markers for the cytosolic and nuclear compartments, β-actin and histone deacetylase-1 (HDAC-1), respectively, and pure separation was verified by Western blot analysis.
Western Blot Analysis of Subcellular ERK1/2 Distribution.
Thirty micrograms of total protein from the nuclear fraction or 60 μg of total protein from the cytosolic fraction of tissue homogenate was electrophoresed on a 10% SDS-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride membrane. Molecular weight markers and controls were also electrophoresed to confirm that the molecular mass of the bands were 44 and 42 kDa and for comparison between samples during densitometric analysis, respectively. The controls used for densitometric comparison between groups in the nuclear fraction came from the nuclear fraction of a rat subjected to 30 min of ischemia and 60 min of reperfusion. The positive control for the cytosolic fraction came from another rat subjected to the same protocol. Gel transfer efficiency was verified via transfer of the molecular weight markers to the membrane. Nonspecific background staining was blocked in nonfat dry milk, and the membrane was incubated with the appropriate primary antibody at 1:5000 dilution. The membrane was washed and incubated with the appropriate horseradish peroxidase-linked secondary antibody in blocking buffer. The membrane was washed again and stained with a chemiluminescence system (ECL kit, Amersham Pharmacia Biotech, Arlington Heights, IL). Densitometry was performed on each sample and analyzed via NIH IMAGE software. Phospho-specific polyclonal antibodies against p44/p42 MAP kinase were purchased from New England BioLabs (Beverly, MA). The polyclonal antibody to HDAC-1 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal β-actin antibody was a gift from Dr. Nancy Rusch, originally purchased from Sigma Chemical Co.
A total of 40 rats successfully completed the above protocols for infarct size analysis. An additional 72 rats completed the above protocols for Western blot analysis. Rats were excluded from data analysis if they exhibited severe hypotension (<30 systolic blood pressure) or if we were unable to maintain adequate blood gas values within a normal physiological range due to metabolic acidosis. Exclusion of animals from the present study was evenly distributed among the protocol groups.
Statistical Analysis of Data.
All values are expressed as mean ± S.E.M. Analysis of variance (ANOVA) with Newman-Keuls post test was used to determine whether any significant differences existed among groups for hemodynamics, IS, and AAR. Significant differences for infarct size and hemodynamic analysis were determined atp < 0.05. The same post test was used to determine significant differences in relative density of p44/p42 MAP kinase at the various times of heart excision in each group for the nuclear fraction. Significant differences were determined at p< 0.05. Additionally, significant differences in the relative density of the cytosolic fraction at 5 min of reperfusion were determined by an unpaired t test at p < 0.05.
Table 1 summarizes the hemodynamic data obtained for the following experiments. There were no consistent differences in any group versus control for heart rate, mean blood pressure, or rate pressure product. Heart rate was less than control animals in the IPC animals at 120 min of reperfusion and in animals treated with PD 098059 in the presence of TAN-67 or IPC at 15 min of ischemia and 120 min of reperfusion, respectively. Mean blood pressure was increased in preconditioned rats versus control rats at baseline and 120 min of reperfusion and in IPC rats treated with PD 098059 at baseline. No differences existed for the rest of the groups for heart rate or mean blood pressure, and no significant differences were found between groups for the rate pressure product.
Gravimetric analysis of body weight, left ventricular (LV) weight, AAR/LV, and IS/AAR was determined. The body weight was slightly less in TAN-67 animals treated with PD 098059, however, the LV weight was only significantly increased versus control in animals treated with PD 098059 alone. AAR, expressed as a percentage of the LV, was not significantly different in any of the groups. IS, expressed as a percentage of the AAR, is listed in Table2 and represented graphically in Fig.2. In control animals IS/AAR averaged 60.6 ± 1.8. IPC and TAN-67 significantly reduced IS/AAR (8.2 ± 1.3 and 30.2 ± 2.4, respectively). The MEK-1 inhibitor, PD 098059, did not affect IS/AAR in animals when administered 20 min (67.0 ± 2.3) before the control protocol. However, when PD 098059 was administered before IPC or TAN-67, cardioprotection was markedly attenuated or abolished (41.5 ± 6.4 and 63.0 ± 4.8, respectively). Separation of the nuclear and cytosolic fractions was confirmed by Western blot analysis with histone deacetylase-1 and β-actin, respectively (Fig. 3).
The relative density in the nuclear fraction of p44 and p42 MAP kinase in control, TAN-67, and IPC-treated animals at 0, 5, 15, and 30 min of ischemia and at 5, 30, and 60 min of reperfusion are represented graphically in Figs. 4, 5, and 6. In the nuclear fraction of control animals, p44 and p42 levels, respectively, were increased at 5 min of coronary artery reperfusion (1.73 ± 0.17 and 1.42 ± 0.19). This increase gradually fell by 30 (0.85 ± 0.10 and 1.08 ± 0.10) and 60 (1.06 ± 0.11 and 1.07 ± 0.12) min of reperfusion but was maintained at higher levels than those found before (0.09 ± 0.04 and 0.10 ± 0.02), or during, ischemia. TAN-67-treated hearts followed a similar pattern of p44/p42 activation, respectively, as in control hearts. However, the p44/p42 MAP kinase was increased to significantly higher levels than control before ischemia (0.37 ± 0.06 and 0.48 ± 0.09). This increase in MAP kinase activation fell by 5 min of ischemia but reappeared by 30 (0.42 ± 0.07 and 0.38 ± 0.08) min of ischemia. Additionally, this activation was sustained at a higher level than control throughout reperfusion after 5 (2.01 ± 0.09 and 2.27 ± 0.18), 30 (1.78 ± 0.13 and 2.39 ± 0.17), or 60 (1.79 ± 0.16 and 2.40 ± 0.23) min. A similar pattern was seen for IPC-treated hearts, however, p44/p42 levels, respectively, were significantly increased versus both control and TAN-67-treated rats at 0 (2.10 ± 0.32 and 1.73 ± 0.19), 5 (0.66 ± 0.05 and 0.84 ± 0.11), and 15 (0.49 ± 0.09 and 0.53 ± 0.10) min of ischemia. Additionally, ERK was maintained at higher levels versus control for both isoforms at 30 (0.42 ± 0.04 and 0.41 ± 0.11) min of ischemia and at 5 (2.28 ± 0.15 and 2.60 ± 0.20), 30 (1.79 ± 0.02 and 2.44 ± 0.14), and 60 (1.46 ± 0.07 and 1.87 ± 0.26) min of reperfusion. However, these increases in ERK at 0 min of ischemia in IPC animals and 5 and 60 min of reperfusion in the nuclear fraction of control, opioid-treated, and IPC animals were not abolished by PD 098059 (Fig.7).
Activation of ERK in the cytosolic fraction did not follow the same pattern of activation as the nuclear fraction. We were not able to detect ERK activation during ischemia in any group, however, after 5 min of reperfusion p44 MAP kinase was significantly increased versus control in IPC- and opioid-treated hearts (0.07 ± 0.04, 0.69 ± 0.33, and 0.55 ± 0.22, respectively; Fig.8). However, the levels were not increased versus control at 30 or 60 min of reperfusion (data not shown). Additionally, p44 activation could be abolished at all time points by PD 098059. Finally, p42 activation was observed in the cytosolic fraction of IPC hearts, but not opioid or control animals. This effect was also abolished by PD 098059 (Fig.9).
We demonstrate that both IPC and δ1-opioid receptor stimulation induce cardioprotection via activation of an isoform- and cellular loci-specific ERK signaling pathway. These observations are supported by the finding that the ERK antagonist, PD 098059, could attenuate or abolish cardioprotection induced by either IPC or the δ1-opioid receptor agonist TAN-67. We have previously demonstrated that this dose of TAN-67 is selective for the δ1-opiate receptor and could be abolished by the δ1-opioid receptor antagonist 7-benzylidenenaltrexone (Schultz et al., 1998b). Additionally, we corroborate our in vivo results with biochemical evidence and demonstrate that, upon myocardial reperfusion, activation of the 44-kDa isoform of ERK in the cytosolic fraction is more pronounced after IPC or opioid receptor stimulation versus control animals. Because the increase in cytosolic phosphorylated ERK, but not nuclear phosphorylated ERK, could be abolished by PD 098059, these data suggest that the activation of cytosolic p44 MAP kinase is an important component of acute cardioprotection. Additionally, cytosolic p42 activation in IPC animals may suggest that this isoform is associated with the increased cardioprotection observed in IPC versus opioid-treated animals.
These results are in agreement with Strohm et al. (2000). They demonstrated in the porcine myocardium that intramyocardial infusion of PD 098059 dose dependently abolished IPC-induced cardioprotection and ERK activation as determined by infarct size analysis and in gel phosphorylation of myelin basic protein, respectively. Although in their investigation they did not demonstrate cellular redistribution of ERK during IPC, they suggested that cytosolic activation of ERK may be important to induce cardioprotection. Interestingly, they reported that PD 098059 decreased phosphorylation of both isoforms at the end of IPC in the cytosolic fraction; however, PD 098059 did not induce significant changes in ERK phosphorylation or cellular redistribution in the particulate or nuclear fractions. We report that opiates and IPC elevated cytosolic ERK to higher levels than nontreated rats only at 5 min of reperfusion, additionally, p44 MAP kinase was also increased at 30 and 60 min of reperfusion (data not shown). However, this increase in p44 MAP kinase at 30 and 60 min of reperfusion was not greater than that observed in control animals but was still abolished via pretreatment with PD 098059. Thus, it is likely that the increase in cytosolic ERK activation following 30 or 60 min of reperfusion is unimportant in acute cardioprotection, since most tissue damage is thought to occur during prolonged ischemia or during the initial few minutes of reperfusion following ischemia.
ERK has been shown to induce cardioprotection via the inhibition of an apoptotic signaling cascade. Yue et al. (2000) have demonstrated that inhibition of ERK enhances ischemia/reoxygenation-induced apoptosis and exaggerates reperfusion injury. Similarly, Zhu et al. (1999) have demonstrated that the chemotherapeutic agent, daunomycin, can induce apoptosis of cardiac myocytes that can be exaggerated via inhibition of ERK but reduced via p38 inhibition. Apoptosis may occur via stimulation of the ERK substrate, p90 ribosomal S6 kinase (p90RSK), which may function to regulate gene expression via the phosphorylation of the proapoptotic protein, Bad, leading to suppression of Bad-mediated apoptosis (Shimamura et al., 2000). Similarly, p90RSK can induce the phosphorylation of the cAMP-response element-binding protein, which may be important in cell survival (Bonni et al., 1999).
We also demonstrate in this investigation that both IPC and opioids can induce a more potent ERK activation in the nuclear fraction both before prolonged ischemia and during reperfusion versus control animals. However, because this activation could not be attenuated by PD 098059, these data suggest that nuclear ERK activation is not important for acute cardioprotection, but may serve a more important role for delayed cardioprotection. Indeed, MAP kinase involvement during delayed cardioprotection has been previously reported. Carroll and Yellon (2000) recently demonstrated in a human cardiomyocyte-derived cell line that IPC- or adenosine-induced delayed cardioprotection, as measured by lactate dehydrogenase release and propidium iodide exclusion, could be abolished with SB 203580, implicating a p38-mediated signal transduction mechanism. Additionally, we have recently demonstrated that opioids induce delayed cardioprotection (Fryer et al., 1999a) that is dependent on a p38 and ERK signaling cascade (Fryer et al., 2001).
Evidence prior to the current studies suggested that IPC or opiates induce activation of a MAP kinase signaling cascade. Maulik et al. (1996) suggested that IPC triggers an increase in total MAP kinase activity and MAP kinase activated protein kinase 2 in rat hearts, which is dependent on a tyrosine kinase-sensitive mechanism. However, their studies do not differentiate between the activation of ERK, JNK, and p38. Burt et al. (1996) have shown that the δ-opioid peptide agonist, [d-Ala2,d-Leu5]-enkephalin, can induce activation of both isoforms of ERK following expression of the mouse δ-opioid receptor in rat-1 fibroblasts. Similar activation of the MAP kinase cascade has been demonstrated by Gutstein et al. (1997) who showed that opioid stimulation of the δ- or μ-, but not κ-, opioid receptor induces the potent activation of ERK but only weak or no activation of JNK or p38. Additionally, it is thought that μ- and δ-opioid receptor agonists can induce the activation of MAP kinases in the absence of receptor internalization (Kramer and Simon, 2000) and that this activation is regulated by Ras and involves the Gβγ subunit of the opioid receptor protein (Belcheva et al., 1998). Despite the above reports supporting a role for ERK in IPC or after opiate administration, it is possible in our investigation that the increased necrosis in control animals directly reduced detectable ERK activation in these tissues.
JNK and p38, thought to be activated by cellular stress (Sugden and Clerk, 1998), may also have an important role in ischemia/reperfusion or hypoxia/reoxygenation injury (Seko et al., 1997). Weinbrenner et al. (1997) demonstrated that IPC-induced cardioprotection resulted in the phosphorylation of tyrosine 182 of p38 MAP kinase in the rabbit myocardium and that the p38 and JNK activator, anisomycin, could induce cardioprotection equal to that of IPC. Maulik et al. (1998) also demonstrated a role for p38 MAP kinase in IPC utilizing the selective inhibitor, SB 203580. They have shown that myocardial adaptation to ischemia triggers a tyrosine kinase-mediated mechanism leading to the translocation and activation of p38 MAP kinase into the nucleus. Additionally, they suggested the importance of activation of MAP kinase-activated protein (MAPKAP) kinase 2, which may induce activation of heat shock protein 27. Similarly, Armstrong et al. (1999) have demonstrated that IPC enhances the dual-phosphorylation of p38 induced by ischemia and also demonstrated that ischemia induces the transient phosphorylation of the small heat shock protein 27.
However, contrasting evidence also supports inhibition of p38 in cardioprotection. Barancik et al. (2000) demonstrated that p38 inhibition with SB 203580 is cardioprotective in the ischemic in vivo porcine model and suggest that ischemia/reperfusion activates different signaling cascades with opposing effects on cellular viability, of which ERK and JNK favor survival, whereas the p38 MAP kinases accelerate cell death. Furthermore, Mackay and Mochly-Rosen (1999)demonstrated that two distinct phases of p38 activation are present during ischemia and that SB 203580 reduced the activation of caspase-3, a key event in apoptosis. Ma et al. (1999) also demonstrated that p38 inhibition decreases myocardial apoptosis and improves postischemic cardiac function. Nagarkatti and Sha'afi (1998) suggested that the duration or intensity of p38 activation might determine whether this kinase is beneficial or deleterious to the cell. Inhibition of p38 during pharmacological preconditioning with adenosine or the PKC agonist, phorbol 12-myristate 13-acetate, decreased cell viability after ischemia as indicated via an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide bioreduction assay, however, in the absence of IPC or pharmacological preconditioning, inhibition of p38 MAP kinase increased cellular viability versus nontreated cells, which may provide a possible explanation for these discrepancies.
The present investigation suggests an integral role for cytosolic p44 ERK in cardioprotection. These observations may have clinical implications. Indeed, Talmor et al. (2000) have demonstrated in the human myocardium that ERK is markedly activated during coronary artery bypass grafting surgery. They show in biopsies taken from the right atrial appendage that both isoforms of ERK are increased approximately 2- and 8-fold versus baseline during and after cross-clamping, respectively.
In conclusion, we demonstrate that at 5-, 30-, and 60-min reperfusion, nuclear p44 and p42 MAP kinase is activated to a greater extent in IPC- and opioid-treated hearts than in controls and cytosolic p44 MAP kinase is activated to a greater extent in IPC- and opioid-treated hearts versus control animals. Additionally, we demonstrate that in IPC-treated animals that p42 MAP kinase is also activated. These increases in cytosolic ERK activation and the reduction in infarct size following IPC or opioid administration were abolished by the MEK-1 inhibitor, PD 098059. Therefore, we suggest that these isoforms of ERK are differentially regulated and that the cytosolic activation of p44 MAP kinase may be an important component of the acute cardioprotection produced by IPC or opioid agonists.
We acknowledge the helpful advice of Dr. Rolf Jakobi in the successful completion of this project.
- Received August 17, 2000.
- Accepted October 12, 2000.
Send reprint requests to: Garrett J. Gross, Ph.D., Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail:
This study was funded in part by a predoctoral research grant from the American Heart Association (R.M.F.) and National Institutes of Health Grant HL08311 (G.J.G.).
- ischemic preconditioning
- area at risk
- extracellular signal-regulated kinase
- histone deacetylase-1
- heart rate
- infarct size
- c-jun N-terminal kinase
- ATP-sensitive potassium channel
- left ventricular weight
- mitogen-activated protein
- protein kinase C
- 2,3,5-triphenyltetrazolium chloride
- PD 098059
- The American Society for Pharmacology and Experimental Therapeutics