Cardiovascular Research

Cardiovascular Research 1998 38(1):116-124; doi:10.1016/S0008-6363(97)00327-1
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Activation of mitogen-activated protein kinases and activator protein-1 in myocardial infarction in rats

Naruhito Shimizu^a, Minoru Yoshiyama^a,*, Takashi Omura^a, Akihisa Hanatani^a, Shokei Kim^b, Kazuhide Takeuchi^a, Hiroshi Iwao^b and Junichi Yoshikawa^a

^aFirst Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahimachi, Abeno-ku, Osaka 545-0051, Japan
^bDepartment of Pharmacology, Osaka City University Medical School, 1-5-7 Asahimachi, Abeno-ku, Osaka 545-0051, Japan

^* Corresponding author. Tel. (+81-6) 645 2106; Fax (+81-6) 645 2107.

Received 21 July 1997; accepted 9 December 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The purpose of this study was to examine the activation of mitogen-activated protein kinases (MAPK) plus activator protein-1 (AP-1) and nuclear factor-B (NF-B) DNA binding activities, all of which seem to be important in a signal transduction cascade upstream of the increased level of mRNA expression observed after myocardial infarction. Methods: Myocardial infarction was produced in Wistar rats. The activities of MAPKs in the ischemic region were measured using an in-gel kinase method or an in vitro kinase method. AP-1 and NF-B binding was determined using an electrophoretic mobility shift assay. Levels of transforming growth factor β1(TGF-β1) and collagen I and III mRNAs were analyzed by Northern blot hybridization. Results: p42 Extracellular signal-regulated kinase (ERK), p44ERK and p38MAPK activities increased 5.2-fold, 4.3-fold and 1.9-fold (P<0.01), respectively, at 5 min after coronary artery ligation but returned to normal levels by 30 min. p55 c-Jun NH₂-terminal kinase (JNK) and p46JNK activities increased 4.0-fold and 3.2-fold (P<0.01), respectively, at 15 min and returned to normal levels by 24 h after ligation. AP-1 DNA and NF-B binding activities increased 8.7-fold and 7.1-fold (P<0.01), respectively, at 3 days but returned to normal levels by 7 days after ligation. Interestingly, analyses of the levels of TGF-β1, collagen I and III mRNAs revealed increases of 6.3-fold, 15.2-fold and 12.0-fold (P<0.01), respectively, at 1 week after myocardial infarction. Conclusions: Myocardial ischemia increased MAPK activities, which were followed by enhancement of AP-1 and NF-B DNA binding activity in areas of myocardial infarction in rats. These signal transduction mechanisms may contribute to the myocardial ischemia and injury associated with myocardial infarction by causing an increased expression of TGF-β1 mRNA, collagen I and III in the area.

KEYWORDS Myocardial infarction; Mitogen-activated protein kinase; Extracellular signal-regulated kinase; c-Jun NH₂-terminal kinase; p38MAPK; Activator protein-1; Nuclear factor-B; Transforming growth factor β1; Collagen I; Collagen III; Ternary complex factor/Elk-1; Serum response factor

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
After myocardial infarction, infarct expansion occurs as an acute dilatation and thinning of the infarcted zone, which is associated with progressive chamber dilatation and the increased incidence of sudden death and congestive heart failure [1, 2]. Therefore, an understanding of the remodeling process that occurs in the heart after acute myocardial infarction is important both for the design of appropriate therapeutic interventions and for the study of the molecular events associated with wound repair as a relevant model. The level of expression of various genes, such as those encoding transforming growth factor β1(TGF-β1), collagen I and III, which might lead to scar formation, have been reported to increase in the infarcted region [3–7]. These changes in gene expression may emerge as a key regulatory mechanism, allowing cells to respond and eventually adapt to changes in hypoxia or ischemia. Hypoxia, ischemia and ischemia/reperfusion induce mitogen activated protein kinase (MAPK) and transcriptional changes in cardiac myocytes or in a variety of noncardiac cells [8, 9]. However, the signal transduction system modulating gene expression has not been examined in in vivo myocardial infarction.

MAPKs are a ubiquitous group of protein serine/threonine kinases and are important mediators of the signal transduction pathways responsible for cell growth and proliferation. MAPKs are mediators of signal transduction from the cell surface to the nucleus and are activated in response to a wide array of extracellular stimuli. One nuclear target of these MAPK signaling pathways is the transcriptional factor activator protein-1 (AP-1). MAPKs regulate AP-1 transcriptional activity by multiple mechanisms. For instance, several MAPKs, including extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs), phosphorylate ternary complex factor (TCF)/Elk1, [10–17]and increase its ability to form ternary complexes with serum response factor (SRF) at the c-fos serum response element [11, 13, 15, 17]. The c-jun promoter elements Jun1 and Jun2 are constitutively occupied and preferentially bind heterodimers of c-Jun and activating transcription factor-2(ATF-2). Phosphorylation of c-Jun on 2 critical N-terminal serines by JNKs increases c-Jun transcriptional activity [18, 19]. Fos and Jun proteins combine to form transcriptional factor AP-1, [20]which regulates the expression of various genes implicated in the pathogenesis of myocardial injury. Other transcription factors, such as nuclear factor-B (NF-B), may mediate the inflammation process [21]and may have important roles in the wound repair process after myocardial infarction. The previous findings on the regulation and function of MAPKs have largely come from in vitro studies using cultured cells. Thus, the roles of MAPKs and transcription factors (AP-1 or NF-B) in ischemic heart disease in vivo remain unclear.

To elucidate the signal-transduction pathway involved in the process of ischemic injury, we examined enzymatic activities of ERKs, JNKs and p38MAPK, plus AP-1 and NF-kB DNA binding activities after myocardial infarction in rats.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985).

2.1 Myocardial infarction production and the collection of samples
Male Wistar rats weighing 290–310 g (Clea Japan, Osaka, Japan) were used in the experiments. Myocardial infarction was produced in rats as described previously [22, 23]. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (35 mg/kg, i.p.). After intratracheal intubation, a left thoracotomy was performed under volume-controlled mechanical ventilation. The heart was raised from the thorax and a ligature with 6–0 prolene suture was placed around the proximal left anterior descending coronary artery, then the chest was closed. The same surgical procedures were performed in sham-operated rats, except that the suture around the coronary artery was not tied. All samples were collected from the infarcted area.

2.2 Preparation of cardiac protein extracts
At 5, 15, 30 min, 1, 3 and 24 h after myocardial infarction, infarcted and sham-operated rats were decapitated, and the heart was rapidly removed and washed in precooled phosphate-buffered saline (PBS) (pH 7.4) containing 2.5 mM ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), 2 mM β-glycerophosphate, 10 mM NaF, 1 mM Na₃VO₄ and 1 mM phenylmethylsulfonyl fluoride (PMSF) (for each group; n=5). The ischemic areas of the left ventricles were homogenized on ice with a Polytron homogenizer (PCU-11, Kinematica, Littau/Luzern, Switzerland) in cell lysis buffer (20 mM HEPES (pH 7.2), 25 mM NaCl, 2 mM ethylene glycol bis N,N,N',N'-tetraacetic acid (EGTA), 0.2 mM dithiothreitol (DTT), 60 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM PMSF, 50 mM NaF, 1 mM Na₃VO₄, 25 mM β-glycerophosphate). After incubation at 4°C for 30 min, the homogenates were sonicated (SONIFIER 250, Branson Ultrasonics, Danbury) on ice for 1 min, then centrifuged at 15 000 rpm at 4°C for 30 min. After centrifugation, the supernatants were stored at –80°C until use.

2.3 Measurement of ERK activity
ERK activity was assayed using an in-gel kinase method as described previously [24–26]. ERK activity was determined by phosphorylation of myelin basic protein (MBP) (Sigma Chemical, St. Louis, MO) as the substrate. The samples of protein extracts (10 µg) were boiled for 5 min in Laemmli's sample buffer [27]containing 2 mM Na₃VO₄ and subjected to electrophoresis on SDS-polyacrylamide gels (12%) containing MBP (0.5 mg/mL). After electrophoresis, the gels were incubated in 20% isopropanol containing 50 mM Tris-HCl (pH 8.0) for 1 h, and then washed in 5 mM 2-mercaptoethanol containing 50 mM Tris-HCl (pH 8.0) for 1 h. After denaturation of the kinases with 6 M guanidine-HCl, 5 mM 2-mercaptoethanol and 50 mM Tris-HCl (pH 8.0) for 1 h, the kinases in the gels were renatured by incubation in 0.04% Tween-40, 5 mM 2-mercaptoethanol and 50 mM Tris-HCl (pH 8.0) at 4°C for 12 h, and equilibrated in kinase buffer (40 mM HEPES (pH 7.5), 0.1 mM EGTA, 20 mM MgCl₂, and 2 mM DTT) for 1 h. For the kinase reaction, the gels were incubated in kinase buffer with 25 µM ATP and 25 µCi (-³²P) ATP at 25°C for 1 h. The reaction was terminated by washing the gels in 5% trichloroacetic acid and 1% sodium pyrophosphate. The gels were then dried and subjected to autoradiography. To estimate the kinase activities, the densities of bands on autoradiograms were analyzed with a bioimaging analyzer (BAS-2000, Fuji Photo Film, Tokyo, Japan).

2.4 Measurement of JNK activity
JNK activity was assayed using an in-gel kinase method as described previously [18, 26]. JNK activity was estimated as the ability to phosphorylate glutathione-S-transferase (GST)-c-Jun(1-79) protein. Briefly, the GST-c-Jun(1-79) plasmid, provided by Dr. Hibi (Osaka University School of Medicine), [18]was used to express GST-fusion protein in E.coli BL21(DE3) (Novagen, Madison, WI) by incubation with 0.4 mM isopropylthiogalactopyranoside at 28°C for 3 h, and the expressed GST-c-Jun(1-79) protein was purified using glutathione-sepharose 4B according to the manufacturer's instructions (Pharmacia Biotech, Uppsala, Sweden).

Protein extracts (40 µg) were boiled for 5 min in Laemmli's sample buffer containing 2 mM Na₃VO₄, and subjected to electrophoresis on SDS-polyacrylamide gels (12%) containing 0.1 mg/ml of GST-c-Jun(1-79). After electrophoresis, removal of SDS from the gels, denaturation and subsequent renaturation of kinases in the gels, the kinase reaction was carried out under the same conditions as the in-gel kinase assay of ERK described above.

2.5 Measurement of p38MAPK activity
Endogenous p38MAPK activity was measured by in vitro kinase assay. Triton (final concentration 1%) was added to protein extracts (200 µg) followed by incubation with p38MAPK polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4°C. After incubation, the immune complex was precipitated using protein A agarose (Upstate Biotechnology, Lake Placid, NY), washed, resuspended in 20 µl of the kinase buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 0.2 mM DTT, 0.5 mM EGTA and 1 µCi (-³²P) ATP) and incubated with 1 µg of ATF-2 (Santa Cruz Biotechnology, Santa Cruz, CA) as a substrate at 30°C for 60 min. After incubation, the reaction was terminated by addition of Laemmli's sample buffer and boiling for 5 min. The supernatants were electrophoresed on SDS-polyacrylamide gels (12%), and the gels were dried and subjected to autoradiography. To estimate the kinase activity, the densities of bands on autoradiograms were analyzed with a bioimaging analyzer (BAS-2000, Fuji Photo Film, Tokyo, Japan).

2.6 Preparation of nuclear extracts
At 1, 3 h, 1, 2, 3 days and 1 week after myocardial infarction, infarcted and sham-operated rats were decapitated, and the heart was rapidly removed (sham-operated rats n=5; infarcted rats n=7). For the electrophoretic mobility shift assay, nuclear protein extracts were prepared according to the method of Schreiber et al. [28], with minor modifications. About 100 mg of myocardium from the infarcted region was immediately washed in precooled PBS (pH 7.4) containing 2.5 mM EDTA, 2 mM β-glycerophosphate, 10 mM NaF, 1 mM Na₃VO₄ and homogenized with a Dounce homogenizer in 1 ml of cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl₂, 10 mM NaF, 1 mM Na₃VO₄, 0.5 mM PMSF, 1 mM DTT, 20 mM β-glycerophosphate, 60 µg/ml aprotinin and 2 µg/ml leupeptin). The tissue homogenates were transferred into Eppendorf tubes and the cells were allowed to swell on ice for 15 min, after which 62.5 µl of 10% Nonidet P-40 was added and the tubes were vigorously vortexed for 10 s. The nuclei were pelleted by centrifugation at 5000 rpm for 10 min at 4°C. The nuclear pellets were resuspended in 150 µl of cold buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl₂, 20% glycerol, 10 mM NaF, 1 mM Na₃VO₄, 0.5 mM PMSF, 0.2 mM DTT, 20 mM β-glycerophosphate, 60 µg/ml aprotinin and 2 µg/ml leupeptin) and the tubes were rocked for 10 min at 4°C. Following centrifugation at 15 000 rpm for 10 min at 4°C, the supernatants containing nuclear protein were collected and stored at –80°C until use.

2.7 Oligonucleotide probes
The sequence of the double-stranded oligonucleotides used in the present study was as follows:

consensus AP-1, 5'-CGCTTGATGACTCAGCCGGAA-3'

consensus NF-B, 5'-AGTTGAGGGGACTTTCCCAGGC-3'

The oligonucleotide probes were labeled with (-³²P) ATP at the 5' end, using T4 polynucleotide kinase, and the labeled probes were purified by chromatography on a Bio-Spin column (Bio-Rad, Richmond, CA).

2.8 Electrophoretic mobility shift assays
For the binding reactions, 5 µg aliquots of nuclear extracts were incubated with labeled oligonucleotide probes and 2 µg of poly[dI-dC] (Pharmacia Biotech, Uppsala, Sweden) in 20 µl of binding buffer (20 mM HEPES (pH 7.9), 1 mM DTT, 80 mM NaCl, 0.2 mM EDTA, 0.2 mM EGTA, 0.3 mM MgCl₂, 0.1 mM PMSF, 10% glycerol) for 15 min at room temperature. The reaction mixtures were then loaded onto 4% nondenaturing polyacrylamide gels in 6.7 mM Tris-HCl(pH 7.5), 3.3 mM sodium acetate, 1 mM EDTA and 2.5% glycerol. Electrophoresis was performed at 200 V in 6.7 mM Tris-HCl(pH 7.5), 3.3 mM sodium acetate, 1 mM EDTA at 4°C. The gels were dried and subjected to autoradiography. To demonstrate the specificity of DNA-protein binding, the reactions were performed in the presence of non-labeled consensus oligonucleotide competitors. In addition, a supershift assay for AP-1 was carried out using rabbit polyclonal antibodies against Fos or Jun (Santa Cruz Biotechnology, Santa Cruz, CA) to examine the AP-1 complex containing Fos and Jun. Rabbit polyclonal antibodies against p50-NF-B or p65-NF-B (Santa Cruz Biotechnology, Santa Cruz, CA) were used for the supershift assay in NF-B. Specific antibodies were added to samples after the initial binding reaction between nuclear protein extracts and labeled consensus oligonucleotide, and the reaction was incubated at room temperature for 1 h.

2.9 Northern blot hybridization
At 4 days, 1, 2 and 3 weeks after operation, infarcted and sham-operated rats were decapitated, and the heart was rapidly removed (for each group, n=6). The infarcted region was separated from the left ventricle. The methods of RNA extraction and Northern blot hybridization have been previously described in detail [29]. The probes used were specific for rat TGF-β1, [30]rat 1 (I) collagen cDNA (1.3 kb PstI/BamHI fragment), [31]mouse 1 (III) collagen cDNA (1.8 kb EcoRI/EcoRI fragment), [32]rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(1.3 kb PstI/PstI fragment) [33]and rat 18S ribosomal RNA.

An optical scanner (EPSON GT-8000, Seiko, Tokyo, Japan) was used for digitization of autoradiograms to allow measurement of mRNA levels. The densities of autoradiographic bands in digitized images were measured using the public domain NIH Image program. For all RNA samples, the densities of individual mRNA bands were divided by that of the 18 S ribosomal RNA band to correct for differences in RNA loading and/or transfer.

2.10 Statistics
The results are expressed as means±S.E. Statistical significance was determined using unpaired Student's t-test or ANOVA and Duncan's multiple range test. Differences were considered statistically significant when P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 ERK and JNK activities
As shown in Fig. 1, at 5 min after coronary artery ligation, p42ERK and p44ERK activities increased 5.2-fold (P<0.01) and 4.3-fold (P<0.01), respectively, compared with sham-operated rats. Activities gradually decreased to control levels by 30 min after coronary artery ligation.

View larger version (40K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 In-gel kinase assay of extracellular signal-regulated kinases (ERK). Cardiac protein extracts were electrophoresed on SDS-polyacrylamide gels (12%) containing myelin basic protein as substrate. The upper panel shows a representative autoradiogram of ERK activities. Bar graphs show p42ERK and p44ERK activities. The mean value of sham-operated rats at each time point is represented as 1. Values were corrected relative to sham-operated rats and represent means±S.E. *P<0.01 compared with sham-operated controls. (sham n=5, MI n=5).

 
Fig. 2 shows that p55JNK activities increased 2.1-fold (N.S.), 4.0-fold (P<0.01), 3.4-fold (P<0.01), 1.7-fold (N.S.), 1.7-fold (N.S.) and 1.2-fold (N.S.) at 5, 15, 30 min, 1, 3 and 24 h after coronary artery ligation, respectively. p46JNK increased 1.3-fold (N.S.), 3.2-fold (P<0.01), 3.0-fold (P<0.01), 2.7-fold (P<0.01), 2.7-fold (P<0.01) and 1.4-fold (N.S.) at 5, 15, 30 min, 1, 3 and 24 h, respectively. Although p55JNK activity peaked at 15 min after ligation at a somewhat higher level than p46JNK, levels of the latter remained elevated for slightly longer periods. ERK and JNK activities did not change significantly after the sham operation.

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 In-gel kinase assay of c-jun NH2-terminal kinases (JNK). The cardiac protein extracts electrophoresed on SDS-polyacrylamide gels (12%) containing glutathione-S-transferase(GST)-c-jun(1-79) as substrate. The upper panel shows a representative autoradiogram of JNK activities. Bar graphs show p46JNK and p55JNK activities. The mean value of sham-operated rats at each time point is represented as 1. Values were corrected relative to sham-operated rats and represent means±S.E. *P<0.01 compared with sham-operated controls. (sham n=5, MI n=5).

 
3.2 p38MAPK activity
As shown in Fig. 3, at 5 min after coronary artery ligation, p38MAPK activity increased 1.9-fold (P<0.01) compared with sham-operated rats. However, by 15 and 30 min after coronary ligation, p38MAPK activities decreased to the control level. p38MAPK activities did not change significantly after the sham operation.

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 In-vitro kinase assay of p38 mitogen-activated protein kinases (MAPK). p38MAPK was immunoprecipitated from cardiac protein extracts, reacted with ATF-2 substrate and then the samples were electrophoresed on SDS-polyacrylamide gels (15%). The upper panel shows representative autoradiograms of p38MAPK activities. Bar graphs show p38MAPK activities. The mean value of sham-operated rats at each time is represented as 1. Values were corrected relative to sham operated rats and represent means±S.E. *P<0.01 compared with sham-operated controls. (sham n=5, MI n=5).

 
3.3 DNA binding activity of nuclear extract
As shown in Fig. 4a, the incubation of nuclear protein extracts with labeled consensus AP-1 oligonucleotide resulted in the formation of one broad band. This shifted band was certified to be specific for AP-1, since the additional of unlabeled AP-1 consensus oligonucleotide resulted in a decrease in the intensity of the band. The addition of anti-Fos or anti-Jun antibodies reduced the intensity of the bands, and both antibodies induced the appearance of supershifted bands.

View larger version (48K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (a) A competition assay for activator protein-1 (AP-1) was carried out in the presence of a 25 fold excess of unlabeled AP-1 oligonucleotide(competitor). Supershift analysis was performed with specific anti-c-Fos or anti-c-Jun antibodies. Lane 1 competitor(–), antibody(–), Lane 2 anti-c-Fos antibody(+), Lane 3 anti-c-Jun antibody(+), Lane 4 competitor(–), antibody(–), Lane 5 25 fold excess of unlabeled AP-1 oligonucleotide competitor(+). SS, supershifted AP-1 band, AP-1, AP-1 protein and labeled oligonucleotide binding, NS, non specific binding, F, free probe. (b) Electrophoretic mobility shift assays of AP-1 DNA binding activity of nuclear extracts. The panel shows a representative autoradiogram of AP-1 DNA binding activity. Sham control 1 day after operation (Lane 1), Myocardial infarction 1 day (Lane 2) Myocardial infarction 2 days (Lane 3) Myocardial infarction 3 days (Lane 4) Myocardial infarction 7 days (Lane 5) after operation. (c) Bar graphs show AP-1 DNA binding activities. The mean value of sham-operated rats at each time is represented as 1. Values were corrected relative to sham-operated rat and represent means±S.E. *P<0.01 compared with sham-operated controls. (sham n=5, MI n=7).

 
As shown in Fig. 4b–c, AP-1 DNA binding activity increased 5.7-fold (P<0.01), 7.3-fold (P<0.01), 8.7-fold (P<0.01) and 4.3-fold (P<0.01) at 1, 2, 3 days and 1 week after coronary ligation, respectively. For the time periods measured, the peak of DNA binding activity of AP-1 was 3 days after myocardial infarction.

As shown in Fig. 5a, the incubation of nuclear protein with labeled consensus NF-B oligonucleotide resulted in the formation of one broad band. The addition of unlabeled NF-B consensus oligonucleotide or anti-p50-NF-B antibody decreased the intensity of this broad band. The addition of anti-p50-NF-B or anti-p65-NF-B antibodies induced the appearance of supershifted bands. These data strongly suggest that the shifted band corresponds to NF-B.

View larger version (51K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 (a) A competition assay for nuclear factor-B (NF-B) was carried out in the presence of a 25 fold excess of unlabeled NFB oligonucleotide (competitor). Supershift analysis was performed with specific anti-p50-NFB or anti-p65-NFB antibodies. Lane 1 competitor(–), antibody(–), Lane 2 anti-p50-NFB antibody(+), Lane 3 anti-p65-NFB antibody(+), Lane 4 competitor(–), antibody(–), Lane 5 25 fold excess of unlabeled NFB oligonucleotide competitor(+). SS, supershifted NFB band, NFB, NFB protein and labeled oligonucleotide binding, NS, non specific binding, F, free probe. (b) Electrophoretic mobility shift assays of NFB DNA binding activity of nuclear extract. The panel shows representative autoradiograms of NFB DNA binding activity. Sham control 1 day after operation (Lane 1), Myocardial infarction 1 day (Lane 2) Myocardial infarction 2 days (Lane 3) Myocardial infarction 3 days (Lane 4) Myocardial infarction 7 days (Lane 5) after operation. (c) Bar graphs show NFB DNA binding activities. The mean value of sham-operated rats at each time is represented as 1. Values were corrected relative to sham-operated rat and represent means±S.E. *P<0.01 compared with sham-operated controls. (sham n=5, MI n=7).

 
As shown in Fig. 5b–c, NF-B DNA binding activity increased 1.6-fold (N.S.), 2.2-fold (P<0.01), 7.1-fold (P<0.01) and 3.7-fold (P<0.01) at 1, 2, 3 days and 1 week after coronary ligation, respectively.

3.4 mRNA expression
The TGF-β1 mRNA level was measured in the infarcted region using Northern blotting analysis. At 2 and 4 days, plus 1, 2 and 3 weeks after myocardial infarction, the TGF-β1 mRNA level increased 3.1-fold (P<0.01), 4.6-fold (P<0.01), 6.3-fold (P<0.01), 3.9-fold (P<0.01) and 2.6-fold (P<0.01), respectively. TGF-β1 gene expression levels were the same throughout duration of experiment for sham operated rats. Collagen I mRNA level in infarcted region increased 2.7-fold (P<0.01), 7.8-fold (P<0.01), 15.2-fold (P<0.01), 13.0-fold (P<0.01) and 12.1-fold (P<0.01), respectively. Collagen III mRNA in the infarcted region level increased 2.6-fold (P<0.01), 3.7-fold (P<0.01), 12.0-fold (P<0.01), 9.0-fold (P<0.01) and 8.4-fold (P<0.01), respectively. GAPDH mRNA in the infarcted region level was 0.8-fold (N.S.), 0.8-fold (N.S.), 0.8-fold (N.S.), 0.9-fold (N.S.) and 0.9-fold (N.S.), respectively (Fig. 6).

View larger version (40K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Northern blotting analysis of transforming growth factor β1 (TGF-β1), Collagen I, Collagen III and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. The upper panel shows representative autoradiograms of northern blotting analysis of the ischemic left ventricular mRNAs for TGF-β1, Collagen I, Collagen III and GAPDH and 18S ribosomal RNA. Bar graphs show TGF-β1, Collagen I, Collagen III and GAPDH mRNA expression. The mean value of sham-operated rats at each time point is represented as 1. Values were corrected relative to sham-operated rats and represent means±S.E. *P<0.01 compared with sham-operated controls. (sham n=6, MI n=6).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Several previous in vitro studies have described signal transduction systems which are operating during myocardial ischemia or ischemia/reperfusion. In cultured rat cardiac myocytes, it has been reported that both hypoxia and hypoxia/reoxygenation caused rapid activation of the mitogen-activated protein kinase kinase kinase enzymatic activity of Raf-1, which was followed by the sequential activation of mitogen-activated protein kinase kinase, 42-kD and 44-kD ERK, and S6-kinase [8]. In perfused rat hearts, Bogoyevitch et al. showed that global ischemia or ischemia reperfusion did not activate the 42-kD or 44-kD ERK. In contrast, the 55-kD and 46-kD JNKs were not activated by ischemia alone, but were activated by reperfusion following ischemia [34]. In the present study, we ascertained that although myocardial MAPK activities did not change significantly after sham operations, MAPKs were activated by ischemia following experimentally induced myocardial infarction. The activation of ERKs we observed in regions of myocardial infarction in rats was similar to the observations in cultured rat myocytes. However, the patterns of activation of both ERKs and JNKs were different from those in perfused hearts. We cannot explain the reason why ERK and JNK activities did not increase in perfused hearts. However, because our animal model is more of a physiological condition than the perfused hearts, we suggest that there are activated signal transduction by MAPKs in in vivo ischemic hearts.

In this study, we observed the time course of MAPK activities until they returned to control levels. JNK returned to a normal level by 24 h, while ERK and p38MAPK returned to normal levels by 30 min. Interestingly, we found that the initial period of ERK and p38MAPK activation was different from JNK. ERKs and p38MAPK activities increased more rapidly than that of JNKs. Recent studies have demonstrated that, in several cell types, JNKs are activated by proinflammatory cytokines and environmental stress, such as a tumor necrosis factor and ultraviolet irradiation, and is not mediated via a Ras-dependent pathway [35, 36]. In contrast, the ERK pathway is activated by growth factors via a Ras-dependent signal-transduction pathway [36, 37]. Interestingly, although the signal pathways of JNKs is very similar to p38MAPK in the in vitro study, [38]the timing of JNK activation is distinct from p38MAPK activation in myocardial infarcted rats. Myocardial infarction causes inflammation in the infarcted region and this process may induce JNK activation to last at least 3 h after myocardial infarction. These results suggest that each MAPK family may play a different role during myocardial ischemia.

It is well known that AP-1 DNA binding activity can be induced by a wide array of stimuli including growth factors, cytokines, neurotransmitters and cellular stress. The transcription factor AP-1 is the best studied target of the MAP kinase signal transduction cascades. ERKs and JNKs can phosphorylate TCF/Elk-1 transcription factor, which forms ternary complexes with SRF, leading to the induction of c-fos gene expression. Recently, JNKs were shown to increase c-Jun transcriptional activity by phosphorylating c-Jun on two critical N-terminal serines [19]. AP-1 proteins such as c-Jun and c-Fos become homo- or hetero-dimerized to bind to the specific sequence (AP-1 site) in the enhancers of the various genes and then to regulate their expression. Thus, the activation of ERKs and JNKs is likely to cause the activation of AP-1. However, little is known whether MAPKs activates AP-1 in myocardial infarction in rats. In the present study, AP-1 DNA binding activity was shown to be augmented in myocardial infarction. Supershift analysis with anti-Fos or anti-Jun antibody indicated that myocardial AP-1 binding complexes activated by ischemia contain Fos and/or Jun proteins.

We showed an increase of NF-B DNA binding activity in the infarcted region of myocardial infarcted rats. NF-B consists of homodimers and heterodimers of structurally related DNA-binding subunits [39, 40]. NF-B is activated by a great variety of mostly pathogenic conditions, including viral and bacterial infections, ultraviolet light and inflammatory cytokines [21, 40]. Although we measured the peak DNA binding activities for both AP-1 and NF-B at 3 days after myocardial infarction, the onset of increased NF-B activity was delayed in comparison to the appearance of increased AP-1 activity. One possibility for this observation is that AP-1 binding activity may be increased by ischemia, while NF-B may be induced by an inflammatory response process following ischemic injury.

We have shown that myocardial TGF-β1 gene expression increased in the ischemic region as reported previously [5, 41]. We determined that increased TGF-β1 expression reached the peak at 1 week and lasted for at least 3 weeks after myocardial infarction. TGF-β1 has an important role in extracellular matrix formation and contributes to scar formation during the healing process in the infarcted site. Although the mechanism of the enhanced myocardial TGF-β1 expression remains to be determined, it is worth noting that the TGF-β1 gene has an AP-1 consensus sequence in its promoter region [42–44]. These observations, taken together with the fact that AP-1 is responsible for the increase in TGF-β1 in cultured cells, [42, 43]suggest that the activation of AP-1 is, at least in part, involved in the enhanced expression of myocardial TGF-β1 by ischemia.

Our experiments also quantitated increases mRNA for collagen I and III in the infarcted region. The peak and time course of increased expression of collagen I and III was approximately the same as TGF-β1. It is well known that TGF-β1 induces collagen formation [45, 46]. However, in our study the increase in TGF-β1 mRNA did not precede the changes in mRNA levels for the collagen I and III. Therefore, we assume that the level of TGF-β1 mRNA at 5 or 6 days after infarction may have been greater than the 6.3-fold increase seen at 7 days. Our data showed that mRNA levels for GAPDH, which are routinely used as controls, decreased slightly but were not significantly changed. Thus, the increased activities observed in our study are not just some general physiological/cellular phenomenon following experimental myocardial infarction.

In conclusion, we have shown, in vivo, that myocardial ERKs, JNKs and p38MAPK are activated by ischemia, and followed by increased AP-1 and NF-B DNA binding activity. These signal transduction mechanisms may contribute to structural rearrangement in the infarcted zone after myocardial infarction. The myocardial infarct region contains myocytes, fibroblasts, smooth muscle cells, endothelial cells and inflammatory cells. Therefore, we were unable to determine what type of cells is actually concerned with these activities. It has recently been recognized that cellular stress activates MAPKs and transcription factors in myocytes and a variety of noncardiac cells [8, 9, 20, 21, 25, 35, 36]. The regulated signal transducting pathways in the hearts should be analyzed by the combination of in vitro and in vivo studies, which will make it possible to examine signal transduction system more precisely in the ischemic hearts.

Time for primary review 28 days.

	Acknowledgements

 
We thank Drs. Hiroyuki Yamagishi, Iku Toda, Masakazu Teragaki and Kaname Akioka for their valuable opinions and Hiroko Kajiwara, Ayako Kobayashi, Harumi Baba and Emi Utsunomiya for their secretarial assistance.

	References

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