Activation of Cardiac c-Jun NH2-Terminal Kinases and p38-Mitogen–Activated Protein Kinases With Abrupt Changes in Hemodynamic Load
Abstract—The role of mitogen-activated protein kinase (MAPK) pathways as signal transduction intermediates of hemodynamic stress leading to cardiac hypertrophy in the adult heart is not fully established. In a rat model of pressure-overload hypertrophy, we examined whether activation of MAPK pathways, namely, the extracellular signal–regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), and the p38-MAPK pathways, occurs during rapid changes in hemodynamic load in vivo. A slight activation of ERK2 and marked increases in JNK1 and p38-MAPK activities were observed 30 minutes after aortic banding. The increase in p38-MAPK activity was accompanied by an increase in the phosphorylation of the p38 substrate MAPK–activated protein kinases 2 and 3. Activation of these kinases was coincident with an increase in phosphorylation of c-Jun and activating transcription factor-2 (ATF-2) and enhanced DNA binding of activator protein-1 factors. Thus, hemodynamic stress of the adult rat heart in vivo results in rapid activation of several parallel MAPK kinase cascades, particularly stress-activated MAPK and p38-MAPK and their target transcription factors c-Jun and ATF-2.
Mechanical stress is known to induce a hypertrophic response of the postnatal mammalian myocardium. The cellular mechanisms contributing to the transition from increased hemodynamic load to altered gene expression and dysfunction of the human ventricle remain incompletely defined. The genetic alteration of the myocardium is characterized by a recapitulation of an embryonic pattern of expression of a number of genes, including atrial natriuretic peptides, β-myosin heavy chain, and α-skeletal actin, among others.1 In isolated neonatal cardiac myocytes, most of these changes can be mimicked by several peptide and nonpeptide autacoids, including the angiotensins and endothelins in vitro (see review2 ). These autacoids as well as inflammatory cytokines transduce their receptor-mediated cellular responses through specific intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) superfamily of signaling proteins. In particular, the extracellular signal–regulated protein kinases (ERKs) are activated by several growth factor molecules, including angiotensin II and endothelin-1. More recently, the “stress-activated” MAPK pathway (SAPK) with its 2 subfamilies, the c-Jun NH2-terminal kinases (JNKs) and the p38-MAPKs, has also been identified as signaling molecules in cardiac cells induced by several cell stressors, such as sorbitol, by exposure to reactive oxygen species, and also by endothelins (see review3 ). Thus, the ERKs and the stress-responsive kinases may serve as signaling mediators through which many extracellular-initiated signals must be funneled before a nuclear response is initiated.
Recent evidence supporting the paradigm that multiple parallel MAPK pathways are involved in the hypertrophic response of heart muscle is derived from myocyte transfection assays in in vitro model systems of cardiac myocyte hypertrophy. Transfection of neonatal cardiac myocytes with the constitutively active kinases MKK3 and/or MKK6, both of which activate p38-MAPK, stimulate the expression of atrial natriuretic factor and α-skeletal actin and mediate changes in myofibrillar organization.4 A number of downstream targets of MAPKs have been identified, including the transactivating domains of specific transcription factors. Two of the nuclear substrates of the JNK and p38-MAPK signal transduction cascade have been described. JNK phosphorylates the transcription factors c-Jun and activating transcription factor-2 (ATF-2) in their transactivating domains.5 6 p38 also phosphorylates and activates ATF-2.7 Both c-Jun and ATF-2 belong to the family of activator protein-1 (AP-1) transcription factors, which form homodimers or heterodimers with other AP-1 factors, such as c-Fos. AP-1 binding sites are present in the promoter regions of many genes that are upregulated by a number of extracellular signals, including stimulation by growth factors and cytokines.8 In cultured neonatal cardiac myocytes, both c-Jun and ATF-2 are phosphorylated in response to stress.9
Despite many in vitro studies on this subject, the role of these pathways in the response to physiologically relevant stimuli in vivo is less clear. The activation of MAPK and SAPK pathways in the adult heart in response to a sudden hemodynamic injury, such as an acute increase in blood pressure, has not been studied in detail. In the present study, we show that multiple MAPK pathways are activated by abrupt hemodynamic stress in the pressure-overloaded rat left ventricle. This is followed by phosphorylation and activation of their target nuclear transcription factors, suggesting a significant role of the MAPK signaling pathway in heart that is due to abrupt hemodynamic stress.
The investigation conforms with the Guide for the Care and the Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Adult female Wistar rats (Charles River, Kingston, Stone Ridge, NY) with a body weight of 205±3 g and an age of 8.3±0.1 weeks were subjected to either an abrupt pressure overload by banding of the proximal aorta or to a sham operation. Baseline phasic (systolic and diastolic) and mean arterial pressures and heart rate were recorded. Pulse pressure was calculated as the difference between phasic systolic and diastolic pressures. A detailed protocol of the procedure has been described previously.10 Briefly, the right carotid artery was cannulated with a saline-filled polyethylene catheter (PE-50) and connected in series to a 5F Millar micromanometer. A midsternal thoracotomy was performed by heat cauterization to expose the ascending aorta for the banding procedure. Thymus tissue was blunt-dissected, and the proximal aorta and the aortic arch, including their major branches, were carefully freed by blunt dissection and isolated by 2.0 silk sutures. The carotid arterial catheter was secured with its tip in the proximal aorta. A 0.7-mm-diameter wire was placed along the aortic arch between the origins of the left common carotid and right brachiocephalic arteries. Previously placed sutures were tightened against the wire, which was then promptly removed to produce a defined constriction across the aortic arch. Sham-operated animals underwent identical surgical procedures, except that the ligature was not tightened against the wire.
Heart Sample Preparation
After the hemodynamic studies, hearts were removed at specific time points and cleared of traces of blood in ice-cold PBS. Separation of atria and ventricles was performed at 4°C, and traces of connective tissue, including adjacent vessels, were removed and discarded. Left ventricles were frozen in liquid nitrogen and pulverized mechanically. Equivalent amounts of pulverized tissue were resuspended in an immunoprecipitation buffer (see below), vortexed for 2 minutes with intermittent chilling, and placed on ice. Total protein was measured by the Bio-Rad Bradford assay or the Bio-Rad DC assay with the use of microtiter plates (Bio-Rad).
In-Gel MAPK Assay
Total protein from animal tissue lysates was extracted in ice-cold lysis buffer, and the assay was performed as described in detail previously.11 Briefly, 100 μg of total protein from each sample was boiled in equal volumes of 2× SDS sample buffer and subjected to SDS-PAGE in the presence of myelin basic protein (MBP, Sigma Chemical Co). After electrophoresis and denaturation in 6 mol/L guanidinium, the protein was renatured in buffer containing 0.04% Tween 40 at 4°C overnight, and after equilibration in kinase buffer, the reaction was started by adding a mixture of [γ-32P]ATP and unlabeled ATP. The reaction was terminated by washing the gel in 5% trichloroacetic acid containing 1% sodium pyrophosphate until the activity of the buffer became negligible. The gel was transferred to 3 MM Whatman paper, dried under a vacuum, and exposed to autoradiography at −70°C. The identity of specific ERKs and JNK-2 in the in-gel kinase assays has been described previously.11
Immune Complex Kinase Assays and Western Blots for ERKs, JNKs, p38, and MAPKAP Kinases 2 and 3
The cDNA for the kinase inactive mutant of the p38 substrate 3pK (3pK K>M) was cloned in a pGEX-KG vector and bacterially expressed as glutathione S-transferase (GST) fusion proteins essentially according to the Pharmacia protocol. The pGEX-KG-c-Jun(1-135) vector for expression of GST-c-Jun(1-135) was a kind gift from J. Kyriakis and L. Zon (Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charleston). GST fusion proteins were purified with glutathione agarose (Pharmacia) and either eluted from the agarose beads with free glutathione [GST-c-Jun(1-135)] or cleaved from the GST portion with thrombin (3pK K>M). Purified recombinant GST-c-Jun(1-135) and 3pK K>M were used as substrates for JNK and p38, respectively.12 Purified MBP as a substrate for ERK2 was purchased from Sigma-Aldrich. Pulverized tissue was lysed in modified ice-cold RIPA buffer as described.12 Cell debris was removed by centrifugation at 4°C. Supernatants of total protein were incubated with antibodies immunoprecipitating active kinases specific for ERK2, JNK1, p38, and an antibody recognizing MAPK–activated protein (MAPKAP) kinases 2 and 3 (Santa Cruz) for 3 hours at 4°C on a rotating platform in the presence of protein A–agarose (Boehringer-Mannheim). Immunoprecipitated kinases were washed twice in modified RIPA buffer at 4°C, centrifuged, and washed twice in kinase buffer. Immune complexes were assayed in kinase buffer containing appropriate substrates for individual kinases: MBP for ERKs, GST-c-Jun(1-135) for JNKs, MAPKAP-3 (3pK) for p38, and heat-shock protein 27 (Hsp27) for MAPKAP kinases 2 and 3 in the presence of [γ-32P]ATP. The reaction mix was incubated for 15 minutes at 30°C and was stopped by adding 5× SDS containing Laemmli buffer. After the samples were heated, an aliquot of the samples was subjected to SDS-PAGE, and gels were blotted onto 0.2-μm nitrocellulose membranes. Autoradiograms of each membrane were developed for 12 to 24 hours at −70°C. After exposure, the membranes were incubated with primary antibodies and washed, and after conjugation with appropriate secondary antibodies, total protein was visualized by chemiluminescence to ensure appropriate loading of kinases onto each gel.
Immunoblot for c-Jun and ATF-2 Phosphorylation
Lysates of left ventricles were subjected to SDS-PAGE. After transfer to polyvinylidene difluoride membranes (Millipore), samples were analyzed by immunoblot with the use of affinity-purified polyclonal c-Jun and ATF-2 antibodies recognizing phosphorylated c-Jun at serine 73 and ATF-2 at threonine 71 (New England Biolabs). After secondary antibody incubation and washes, signals were visualized by ECL reagent (Amersham). The membranes were stripped at 58°C for 1 hour in 62.5 mmol/L Tris-HCl (pH 6.8), 100 mmol/L 2-mercaptoethanol, and 2% SDS. After a reblocking in 5% nonfat dry milk, stripped filters were incubated with appropriate c-Jun and ATF-2 primary antibodies and processed as indicated above to ensure equivalent loading of protein.
Electrophoretic Mobility Shift Assays
Crude nuclear extracts of pulverized heart ventricles were prepared as described by Avots et al.13 Nuclear protein (5 μg) was preincubated on ice with 2 μg poly(dI-dC) (Boehringer-Mannheim) and 1 μg BSA in an electrophoretic mobility shift assay reaction buffer (60 mmol/L HEPES [pH 7.9], 3 mmol/L dithiothreitol, 3 mmol/L EDTA, 150 mmol/L KCl, and 12% Ficoll). After 15 minutes, 12.5 fmol of 32P-labeled oligonucleotide (equivalent to ≈50 000 cpm) was added in a total volume of 10 μL, incubated at room temperature for another 10 minutes, and loaded onto 5% nondenaturing polyacrylamide gels in 0.5% Tris borate–EDTA buffer. After electrophoresis, gels were dried and exposed to autoradiography. Gel-shift oligonucleotides for AP-1 with the consensus binding motif TGACTCA and a mutated AP-1 sequence (TGACTTG) were from Santa Cruz.
Results are expressed as mean±SEM, except as otherwise indicated. For analysis, animals were partitioned into age- and weight-matched study groups. A Student t test or an appropriate nonparametric test (Mann-Whitney rank sum test) was used for final data calculations. For the hemodynamic data, a 2-way repeated-measures ANOVA was applied. A value of P<0.05 was considered statistically significant.
Hemodynamic Stress Results in Activation of Multiple Kinase Pathways in Ventricular Muscle
Baseline hemodynamics of sham-operated and aortic-banded animals are summarized in Table 1⇓. The banding procedure resulted in a rapid and profound increase in systolic and mean aortic pressures. In the banded animals, mean arterial blood pressure increased from 101±5 to 141±6 and 131±5 mm Hg at 15 and 30 minutes, respectively, after the initiation of banding. Pulse pressure was increased from 54±3 to 121±7 and 98±16 mm Hg, corresponding to a 2.1- and 2.6-fold increase after 15 and 30 minutes, respectively.
The presence of ERK1/ERK2 in adult rat ventricular muscle was verified by Western blotting with the use of ERK1- and ERK2-specific antibodies (data not shown). An increase in ERK2 phosphorylation could be detected as early as 15 minutes after banding by the in-gel MBP kinase assays (Figure 1⇓). In addition to the ERKs, a prominent band at a higher molecular weight, which we have identified previously as JNK-2, was also noted .11 The response was maximal within the first 30 minutes after the initiation of hemodynamic load to the heart, thus indicating a very early and transient response to hemodynamic stress.
We next assessed the extent of activation of selected kinase pathways by using specific antibodies to precipitate activated kinases in protein extracts of sham-operated and banded ventricles, followed by incubation with substrates specific for each kinase. In soluble fractions of the left ventricle, ERK-2 phosphorylation increased 2.6±0.9-fold at 15 minutes and 2.1±0.5-fold at 30 minutes, whereas activation of JNK-1 showed respective 5. 2±2.1- and 5.0±2.1-fold increases for both time points (Figure 2⇓). These results indicate that although activation of the ERK pathway does occur in this animal model, the effect of an abrupt increase in blood pressure is followed by a more pronounced activation of the JNK pathway.
We also determined whether activation of p38-MAPK occurred in response to aortic banding. p38 phosphorylation increased 10.7±2.5-fold at 15 minutes (P<0.05) and 41.4±8.4-fold at 30 minutes after the initiation of hemodynamic load (P<0.01 by unpaired t test). The magnitude of this increase in p38 phosphorylation was verified by use of an antibody that recognized only phosphorylated p38 (data not shown). Because MAPKAP kinases 2 and 3 are downstream kinase substrates for activated p38, stimulation of MAPKAP kinases 2 and 3 is a useful indicator of p38 activation. Activated MAPKAP kinases 2 and 3 phosphorylate Hsp27. Immune complex kinase assays with MAPKAP kinases 2 and 3 revealed an increase of 2.6±0.5 and 6.6±4.1-fold at 15 and 30 minutes, respectively, after the start of aortic banding (Figure 2⇑), providing further evidence for activation of the p38-MAPK pathway in response to hemodynamic stress.
Hemodynamic Load Increases AP-1 Binding and Phosphorylation of c-Jun and ATF-2
We next examined specific transcription factors induced by abrupt hemodynamic load. As shown in Figure 3⇓, the incubation of nuclear extracts with labeled consensus AP-1 oligonucleotides resulted in the formation of a DNA-protein complex that was enhanced at 30 minutes after aortic banding (6.3±1.9 versus 23.9±1.8, P<0.05 by unpaired t test). Incubation of the same protein with unlabeled AP-1 oligonucleotide showed the expected concentration-dependent inhibition of the AP-1 DNA complex that was reversed in the presence of mutant AP-1 oligonucleotide. Some AP-1 factors, such as c-Jun and ATF-2, are phosphorylated by activated JNKs and p38; this activation results in the potentiation of their transcriptional activity. Using phospho-specific antibodies that exclusively recognized phosphorylated c-Jun at serine 73 and phosphorylated ATF-2 at threonine 71, we could demonstrate an increase in phosphorylation of these transcription factors, which coincides with the activation of JNK and p38 (Figure 4⇓ and Table 2⇓).
Taken together, our results demonstrate that hemodynamic stress early after the initiation of load toward the heart not only results in a rapid activation of JNKs and p38-MAPKs but also leads to upregulation and phosphorylation of their target transcription factors c-Jun and ATF-2.
Cardiac hypertrophy is a fundamental adaptation to mechanical load. However, it is still not known how these hemodynamic forces are transduced into myocyte growth and how they affect myocyte survival after injury. In model systems such as primary cultures of neonatal rat cardiac myocytes, a large number of experimental studies are available; these studies demonstrate that some peptide growth factors, including angiotensin II and endothelin-1, among others, are “hypertrophic” in terms of increased protein synthesis and myocyte enlargement. This includes not only peptides that signal through G protein–coupled receptors but also receptor tyrosine kinases and cytokine receptors (see review14 ). As a common feature, identification of their downstream signaling pathways has resulted in the identification of several activated MAPK pathways as targeted signaling molecules in cardiac myocytes after exposure.
The ERKs, JNKs, and p38-MAPKs have all been implicated in the development of cardiac hypertrophy and, on a cellular level, are involved in the regulation of myocyte apoptosis. In a variety of cell systems, JNKs are activated by multiple cell stresses, such as hyperosmotic shock, hypoxia, and reactive oxygen species.15 16 The activation of multiple parallel MAPK pathways is sufficient to induce several marker genes for cardiac hypertrophy, such as brain natriuretic peptide, in cultures of neonatal cardiac myocytes.17 The application of mechanical strain to cultured neonatal cardiac myocytes on a deformable matrix has been shown to induce JNK activation.18 In the perfused adult rat heart, JNKs are activated by ischemia/reperfusion and by reactive oxygen species, and rapid phosphorylation of p38 is observed in the perfused heart by ischemia, ischemia/reperfusion, and high-pressure perfusion.19 At least in the model system of “neonatal cardiac myocyte hypertrophy,” all 3 pathways have been shown to be sufficient to induce a hypertrophic response if they are constitutively activated, but there is ongoing debate and criticism over which of the pathways is mandatory for these responses to physiologically relevant stimuli (see review20 ).
In the present study, we document the rapid activation of the 3 predominant MAPK signaling pathways in response to increased hemodynamic stress. Although the magnitude of activation of the ERK kinases was low and did not exceed more than a 2-fold increase in the phosphorylation state of ERK1, our data indicate that shortly after initiation of a rapid increase in blood pressure, SAPKs, including both JNKs and the p38-MAPK pathways, become substantially activated. Their increased activation was clearly more prominent than that observed for the ERKs and achieved activation levels comparable to that induced by environmental stress inducers in cell culture experiments. In addition, MAPKAP kinases 2 and 3, downstream effectors of activated p38-MAPK, were also activated in this model of hemodynamic overload. MAPKAP kinases 2 and 3 can directly phosphorylate Hsp27, which in turn has been shown to be implicated in actin organization and cell migration in human endothelial cells.21
Our data are supported by a recent in vivo study in which adenovirus-mediated gene transfer of a dominant-negative inhibitor of SEK-1, the immediate upstream activator of the SAPKs, into the adult heart of rats blocks SAPK activation by pressure overload, with consequent inhibition of pressure-induced increase in left ventricular wall thickness.22 Therefore, based on these in vivo findings, evidence exists that activation of the stress-regulated protein kinase pathways may contribute to the adaptive response of the left ventricle because of an abrupt increase in ventricular load. Increased activity of JNKs and p38-MAPKs in response to hemodynamic stress is suggestive of an increase of target transcription factor phosphorylation induced by these SAPKs. Thus, we also investigated whether AP-1 transcription factors show increased DNA binding activity and, furthermore, whether the AP-1 factors c-Jun and ATF-2 are phosphorylated in response to hemodynamic stress. Our data indicate that downstream from activated SAPKs, the nuclear targets of JNK and p38 not only are upregulated but also show increased phosphorylation, which may account for the transcriptional activation of AP-1 target genes.
Nevertheless, the precise cellular mechanism by which this increase in stress-regulated kinase activities occurs in vivo remains unknown. Data obtained in isolated Langendorff-perfused hearts suggest that local release of peptide growth regulatory factors, such as basic fibroblast growth factor and angiotensin II, among others, may be involved in this response.23 24 However, in the normoxic adult rat heart, the quantities of these secreted proteins are often very low or undetectable10 (also indicated by authors’ unpublished data, 1995). Furthermore, treatment of freshly isolated adult ventricular myocytes in such low concentrations with angiotensin II or bFGF did not induce even ERK-1 activation (authors’ unpublished data, 1995). Thus, clear in vivo evidence for a significant role of any one system is lacking. It is also unlikely that the stimulation of both SAPK pathways is simply the result of global ischemia of the heart with aortic banding, inasmuch as JNKs are not activated by ischemia alone (ie, without reperfusion).19
In our model, both mean arterial pressure (ie, steady-state perfusion pressure) and pulse pressure (ie, phasic increase in blood pressure) are substantially elevated. However, because of the experimental design of the procedure, the dampening effect of the ascending aorta below the constriction is negligible, as in the contribution of reflected waves from the arterial tree below the area of constriction. Thus, we cannot distinguish whether the observed changes in kinase activities are the result of increased mean left ventricular systolic pressure, per se, or changes in pulse pressure.
The data reported in the present study provide evidence of early changes in protein kinase signaling pathways immediately after an initiation of stress to the heart and support recent studies demonstrating significant, although substantially smaller, changes in ERK and JNK activities in hypertensive animals and in the hypertrophied hearts of patients with heart failure secondary to ischemic heart disease25 (also see review26 ). In addition to their role in regulation myocyte hypertrophy, there is increasing evidence for a role for activated stress-response kinases in the regulation of myocyte survival. Overexpression or increased activation of JNKs and p38-MAPKs promotes apoptotic myocyte death,27 28 whereas inhibition of p38-MAPKs decreases apoptosis after myocardial ischemia and reperfusion,29 suggesting that the ERKs and the SAPKs have antagonistic roles in mediating apoptotic cell death.30 Thus, the characterization of myocyte survival pathways indicates that activation of these pathways may drive the onset of myocyte and subsequent muscle remodeling in response to biomechanical stress.
In summary, our data indicate that an abrupt increase in hemodynamic stress induces activation of parallel signal transduction cascades in the heart in vivo, particularly of the JNK and p38-MAPK pathways. The concurrent induction of these signaling pathways indicates that the initiation of MAPK signaling events is an immediate consequence of an abrupt increase in systemic blood pressure. This rapid response of the overloaded myocardium enables the ventricular muscle to respond to physiological stress, which may help promote an adaptive response to stress or injury.
This work was supported in part by grants from the National Heart, Lung, and Blood Institute (HL-36141) to Dr Kelly and from the Deutsche Forschungsgemeinschaft (Lu 477/2-3 and 2-4) to Dr Ludwig. This manuscript is dedicated to Janice M. Pfeffer and to Thomas W. Smith, who died during the performance of this study. We thank Suzanne M. Lohman and Ulrich Walter, Institute of Clinical Biochemistry, University Medical Center, Wurzburg, Germany for generous support of this work. We thank Maximilian and Jacqueline Fischer for their comments while preparing this manuscript.
- Received September 21, 2000.
- Revision received October 10, 2000.
- Accepted October 26, 2000.
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