gp130 Plays a Critical Role in Pressure Overload-induced Cardiac Hypertrophy*

Hiroki Uozumi, Yukio Hiroi, Yunzeng Zou, Eiki Takimoto, Haruhiro Toko, Pei Niu, Masaki ShimoyamaDagger , Yoshio Yazaki§, Ryozo Nagai, and Issei Komuro||

From the Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Dagger  1st Department of Internal Medicine, Faculty of Medicine, Tottori University, Tottori 680-8550, § International Medical Center of Japan, Tokyo 162-8655, and  Department of Medicine III, Chiba University School of Medicine, Chiba 260-8670, Japan

Received for publication, January 29, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

gp130, a common receptor for the interleukin 6 family, plays pivotal roles in growth and survival of cardiac myocytes. In the present study, we examined the role of gp130 in pressure overload-induced cardiac hypertrophy using transgenic (TG) mice, which express a dominant negative mutant of gp130 in the heart under the control of alpha  myosin heavy chain promoter. TG mice were apparently healthy and fertile. There were no differences in body weight and heart weight between TG mice and littermate wild type (WT) mice. Pressure overload-induced increases in the heart weight/body weight ratio, ventricular wall thickness, and cross-sectional areas of cardiac myocytes were significantly smaller in TG mice than in WT mice. Northern blot analysis revealed that pressure overload-induced up-regulation of brain natriuretic factor gene and down-regulation of sarcoplasmic reticulum Ca2+ ATPase 2 gene were attenuated in TG mice. Pressure overload activated ERKs and STAT3 in the heart of WT mice, whereas pressure overload-induced activation of STAT3, but not of ERKs, was suppressed in TG mice. These results suggest that gp130 plays a critical role in pressure overload-induced cardiac hypertrophy possibly through the STAT3 pathway.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because recent clinical studies have suggested that cardiac hypertrophy is an independent risk factor of cardiac morbidity and mortality (1), it has become even more important to clarify the mechanism of how cardiac hypertrophy is developed. Cardiomyocyte hypertrophy can be induced by a variety of factors such as mechanical stress (2), cathecholamines (3), angiotensin II (4), endothelin-1 (5), and cytokines (6). Among them, hemodynamic overload, namely mechanical stress, is clinically most important. We and others (7-9) have reported that mechanical stress induces cardiomyocyte hypertrophy through vasoactive peptides such as angiotensin II and endothelin-1.

Cardiotrophin-1, a member of the interleukin 6 (IL-6)1 family, was isolated and found to have a potent hypertrophic effect on cultured cardiomyocytes (10). The IL-6 family of cytokines promotes cell type-specific pleiotropic effects by engaging multimeric receptor complexes that share the common affinity converter/signal-transducing subunit gp130 (11-13). Cardiotrophin-1 has been reported to induce hypertrophy of cardiac myocytes in vitro (14). It has been reported that transgenic mice expressing both IL-6 and soluble IL-6 receptor, in which the gp130 is continuously activated, showed marked hypertrophy of the ventricular myocardium (15) and that targeted disruption of gp130 leads to severe anemia and a hypoplastic ventricular myocardium in the embryo (16). These results suggest that activation of gp130 induces cardiac hypertrophy, and it is still unknown whether gp130 mediates load-induced cardiac hypertrophy. Cardiotrophin-1 has been reported to promote survival of cardiac myocytes (17). Ventricular-restricted gp130 knockout mice showed marked ventricular wall dilatation with marked cardiomyocyte apoptosis and died in a week by pressure overload (18). These results suggest that gp130 signalings prevent cardiomyocytes from apoptotic cell death during the pressure overload.

In the present study, to determine the physiological significance of gp130 in load-induced cardiac hypertrophy, we generated transgenic (TG) mice, which express a dominant negative form of gp130 specifically in the heart and examined hypertrophic responses by pressure overload produced by constriction of the abdominal aorta.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transgene Construction and Generation of TG Mice-- A dominant negative mutant of gp130 (D.N.gp130) was constructed by converting cysteine at 702 to a stop codon as described previously (19). D.N.gp130 cDNA was inserted into the unique KpnI site of palpha MHCSA, which carries the mouse alpha  myosin heavy chain (alpha MHC) promoter (20). alpha MHC promoter-D.N.gp130-poly(A) DNA was excised by XhoI and NotI and microinjected into the pronuclei of fertilized BDF1 mouse eggs. Offsprings from eggs microinjected with the DNA were selected by Southern blot analysis and polymerase chain reaction.

Pressure Overload Model-- Male TG and littermate wild type (WT) mice of 20 weeks old were used in the present study. Mice were housed under climate-controlled conditions with a 12-h light/dark cycle and were provided with standard food and water ad libitum. All protocols were approved by local institutional guidelines. Pressure overload was produced by constriction of the abdominal aorta as described previously in our laboratory (21, 22). Briefly, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The abdominal aorta was constricted at the suprarenal level with 7-0 nylon strings by ligation of the aorta with a blunted 27-gauge needle, which was pulled out thereafter.

Echocardiographic Measurement-- Transthoracic echocardiography was performed with HP Sonos 100 (Hewlett-Packard Co.) with a 10-MHz imaging transducer as described previously (22, 23). Mice were anesthetized with ketamine (10 mg/kg intraperitoneal) and xylazine (15 mg/kg intraperitoneal). After a good quality two-dimensional image was obtained, M-mode images of the left ventricle were recorded. Left ventricular end-diastolic internal diameter (LVEDD), left ventricular end-systolic internal diameter (LVESD), intraventricular septum thickness, and left ventricular posterior wall thickness were measured. All measurements were performed by use of the leading edge-to-leading edge convention adopted by the American Society of Echocardiography (24). Fractional shortening was calculated as fractional shortening = ((LVEDD - LVESD)/LVEDD) × 100. Ejection fraction was calculated by the cubed method as follows: ejection fraction = ((LVEDD)3 - (LVESD)3)/LVEDD3.

Histological Analysis-- For histological analysis, hearts were fixed with 10% formalin by perfusion fixation. Fixed hearts embedded in paraffin were sectioned at 4-µm thickness and stained with hematoxylin-eosin. Cross-sectional areas of cardiac myocytes were measured from 10 sections. Suitable cross-sections were defined as having nearly circular capillary profiles and nuclei.

RNA Preparation and Northern Blot Analysis-- The left ventricle was excised, and total RNA (10 µg) was prepared using ZolB (Biotecx Laboratories, Inc.), fractionated in 1% formaldehyde agarose gel, and transferred to nylon membrane. The blots were hybridized with the cDNA fragments of gp130, brain natriuretic factor (BNP), and sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) genes.

Western Blot Analysis of STAT3 Phosphorylation-- Polyclonal antibody to STAT3 (C-20) was purchased from Santa Cruz Biotechnology, Inc. The left ventricle was excised and lysed in lysis buffer (25 mM Tris-HCl, 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 10 nM okadaic acid, 0.5 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride), and equal amounts (100 µg) of protein were incubated with 1 µg of anti-STAT3 for 1 h at 4 °C. The immune complexes were precipitated with protein A-Sepharose, and the immuneprecipitates were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The membranes were then blocked and incubated with anti-phosphotyrosine antibody (4G10), and the phosphotyrosine was detected by ECL (Amersham Pharmacia Biotech).

Assay of Extracellular Signal-regulated Kinases (ERKs)-- The activities of ERKs were measured using myelin basic protein-containing gel (25). In brief, lysates of the left ventricles were subjected to electrophoresis on an SDS polyacrylamide gel containing 0.5 mg/ml myelin basic protein. ERKs in the gel were denatured in guanidine HCl and renatured in Tris-HCl (pH 8.0) containing 0.04% Triton X-100 and 2-mercaptoethanol (5 mM). Phosphorylation activities of ERKs were assayed by incubating the gel with [gamma -32P]ATP.

Terminal Deoxynucleotidyl Transferase Assay (TUNEL)-- The 4-µm thickness paraffin sections were deparaffinized by immersing in xylene, rehydrated, and incubated with proteinase K (20 µg/ml). Next, the sections were incubated in methanol with 3% H2O2 to inactivate endogenous peroxidases, washed in phosphate-buffered saline, and incubated with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate-dUTP for 90 min and horseradish peroxidase-conjugated anti-fluorescein isothiocyanate for 30 min at 37 °C using an apoptosis detection kit (Takara Biochemicals). The sections were stained with diaminobenzine and hematoxylin and mounted for light microscopic observations.

Statistics-- Differences within groups were compared by the one-way analysis of variance (ANOVA) and Dunnett's t test. The accepted level of significance was p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of TG Mice-- The carboxyl-terminal region of gp130 containing box3 is considered to play a critical role in gp130-mediated biological responses (26). D.N.gp130 TG mice were generated by overexpression of a box3-deleted form of gp130 under the control of alpha MHC promoter (Fig. 1). Six founders containing the transgene were identified, and three transgenic lines, in particular number 3, were used in this study. TG mice were apparently healthy and fertile, and there was no difference in the heart weight to body weight ratio between TG mice and WT mice, at baseline (Fig. 2).


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Fig. 1.   Diagram of the transgene construct and expression in the heart. A, schematic structure of the alpha MHC promoter-D.N.gp130-poly(A) transgene construct. A 2.4-kilobase of truncated murine gp130 cDNA was subcloned between the murine alpha MHC promoter and poly(A). The truncated form of gp130 contains the membrane-proximal 63-amino acid part of the cytoplasmic region of gp130 but lacks box3. B, expression of the truncated form of gp130. 10 µg of total RNA from the hearts were hybridized with labeled gp130 cDNA. TM, transmembrane.


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Fig. 2.   Heart weight/body weight ratio. Pressure overload was produced by constriction of the abdominal aorta for 4 weeks. Mean ± S.E. of the heart weight/body weight ratio was shown (n = 3 in sham-operated mice, n = 4 in constricted mice). *p < 0.05; N.S., not significant.

Cardiac Hypertrophy Induced by Pressure Overload-- Constriction of the abdominal aorta is an established method to produce pressure overload-induced cardiac hypertrophy (21, 22). 4 weeks after the operation, all mice were healthy, and the blood pressure monitored at right carotid artery was elevated as reported before in our laboratory (22) without any differences between TG mice and WT mice (data not shown). Echocardiography at 4 weeks after the operation revealed that left ventricular wall thickness was markedly increased (intraventricular septum thickness, sham 0.50 ± 0.03 versus constriction 0.81 ± 0.03; left ventricular posterior wall thickness, sham 0.54 ± 0.04 versus constriction 0.83 ± 0.08) in WT mice; however, TG mice showed less increase in left ventricular wall thickness (intraventricular septum thickness, sham 0.48 ± 0.03 versus constriction 0.59 ± 0.00; left ventricular posterior wall thickness, sham 0.52 ± 0.00 versus constriction 0.64 ± 0.04) (Table I). Furthermore, constriction of the abdominal aorta for 4 weeks increased the heart weight/body weight ratio by 40 ± 4% in WT mice and 15 ± 3% in TG mice (Fig. 2). The left ventricle of WT mice showed more marked hypertrophy than that of TG mice (Fig. 3A). Microscopic analysis showed that cross-sectional areas of cardiac myocytes of WT mice were more enlarged by pressure overload than those of TG mice (Fig. 3B). These results suggest that cardiac hypertrophy induced by pressure overload was attenuated in D.N.gp130 TG mice.

                              
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Table I
Analysis of cardiac size and function
LVESD, left ventricular end-systolic internal diameter; IVST, intraventricular septum thickness; LVPWT, left ventricular posterior wall thickness; FS, fractional shortening; EF, ejection fraction.


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Fig. 3.   Histological analysis. A, transverse sections of the hearts at papillary muscle level at 4 weeks after constriction of the abdominal aorta. Data are representative of three or four independent experiments with nearly identical results. B, cross-sectional areas of cardiac myocytes from 10 sections. Normalized values in WT mice without constriction are arbitrarily expressed as 1.0. *p < 0.05.

Expression of Cardiac-specific Genes-- Pressure overload by the constriction of the abdominal aorta up-regulates fetal-type cardiac genes and down-regulates SERCA2 gene (27). We therefore examined the expression of BNP and SERCA2 in the hearts of WT and TG mice at the early and late phase after constriction of the abdominal aorta. BNP gene was slightly up-regulated at 2 days after pressure overload and markedly at 28 days in WT mice (Fig. 4). In TG mice, although BNP gene was also up-regulated by pressure overload, the expression levels were quite low compared with those of WT mice. In WT mice, SERCA2 gene was down-regulated from 2 days by pressure overload (Fig. 4). The down-regulation of SERCA2 gene by pressure overload was also attenuated in TG mice.


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Fig. 4.   Expression of cardiac genes. Hearts were excised from WT and TG mice at 2 and 28 days after constriction of the abdominal aorta. Expression of cardiac-specific genes were examined by Northern blot analysis. A, a representative autoradiogram was shown. Ethidium bromide staining of 18 S RNA was shown below. B, The band intensities of BNP, SERCA2, and 18 S RNA were quantified by densitometer. Each histogram represents the fold of BNP and SERCA2 compared with 18 S RNA from three independent experiments (mean ± S.E.). *p < 0.05 versus control.

Activation of STAT3 and ERKs-- Activation of gp130 evokes two distinct pathways, Janus kinase-STAT pathway and Ras-ERKs pathway (28). We examined which pathway is important in the pressure overload-induced cardiac hypertrophy (Fig. 5). Pressure overload activated both STAT3 and ERKs in the heart of WT mice. However, in TG mice, activation of STAT3 was barely detectable. In contrast, there was no difference in activation of ERKs between TG and WT mice. These results collectively suggest that pressure overload-induced activation of STAT3, but not of ERKs, is dependent on gp130 and that STAT3 may play a critical role in pressure overload-induced cardiac hypertrophy.


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Fig. 5.   Activation of ERKs and STAT3 by pressure overload. The abdominal aorta was constricted for indicated periods of time, and the activities of ERKs and STAT3 were measured as described under "Materials and Methods." Each histogram represents the fold of controls from three independent experiments (mean ± S.E.). *p < 0.05.

No Increase in a Number of TUNEL-positive Cardiomyocytes-- It has been reported that activation of gp130 promotes survival of cardiac myocytes (17) and that ventricular-restricted gp130 knockout mice showed marked cardiomyocyte apoptosis and marked ventricular wall dilatation by pressure overload (18). We therefore examined whether TG mice showed cardiomyocyte apoptosis during pressure overload. As shown in Fig. 6, there was no increase in the number of TUNEL-positive cardiomyocytes in the heart of TG mice at the basal and by pressure overload compared with those of WT mice.


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Fig. 6.   TUNEL assay of the heart. The abdominal aorta was constricted for 4 weeks, and cardiomyocyte apoptosis was examined by TUNEL as described under "Materials and Methods." 500 cardiomyocytes were counted from each side. Each histogram represents the number of TUNEL-positive cells from five independent experiments (mean ± S.E.). Pretreatment with micrococcal DNase I (1 mg/ml) to induce DNA strand breaks was carried out as positive controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It has been reported that gp130 is implicated in regulating cell growth, differentiation, and cell death in response to external stimuli in various tissues. In the present study, the TG mice were apparently healthy with no cardiac abnormalities at basal condition, suggesting that gp130 is not necessary for the development and the physiological function of the heart after birth. In contrast, gp130 plays a critical role in pressure overload-induced cardiac hypertrophy. By constriction of the abdominal aorta, TG mice showed less increase in the heart weight/body weight ratio and less changes in expression of BNP and SERCA2 genes compared with WT mice. We used number 3 line of transgenic mice, which expressed D.N.gp130 most abundantly. Mice of number 1 and number 2 lines also showed similar results with mild degree (data not shown). These results suggest that D.N.gp130 dose-dependently suppresses cardiac hypertrophy.

The intracellular signaling pathways evoked by gp130 activation include the Janus kinase-induced STAT pathway and the Ras-ERKs pathway (28). Activated STAT3 has been reported to form a homodimer that subsequently forms cis-inducing factor complexes and induces hypertrophy of cardiac myocytes (29). It has been reported that STAT3 plays a critical role in generating the hypertrophic signal (30) and that mice overexpressing STAT3 showed marked cardiac hypertrophy (31). In the present study, pressure overload activated ERKs and STAT3 in the heart of WT mice, whereas pressure overload-induced activation of STAT3, but not of ERKs, was suppressed in TG mice. These results suggest that pressure overload-induced activation of STAT3, but not of ERKs, is dependent on gp130 and that STAT3 may play a critical role in pressure overload-induced cardiac hypertrophy.

Hirota et al. (18) have reported that ventricular-restricted knockout mice of gp130 showed dilatation of left ventricular wall and died within a week by constriction of transverse aorta without progression of adaptive hypertrophy of left ventricle (18). Although cardiac hypertrophy was attenuated, there was no sign of heart failure such as dilatation of left ventricular wall and reduced cardiac function nor death by constriction of the abdominal aorta in the D.N.gp130 TG mice. There are several possibilities for this discrepancy. First, the methods applied to produce the pressure overload were different. Transverse aortic constriction, applied in their experiment, induces stronger pressure overload than constriction of the abdominal aorta. It is possible that if the pressure overload is too strong, heart failure, but not adaptive cardiac hypertrophy, would be induced. To determine the role of gp130 in pressure overload-induced cardiac hypertrophy, we used a mild pressure overload model of abdominal aortic constriction. Second, they used gp130 null mice, whereas we used D.N.gp130-overexpressing mice. Because pressure overload-induced activation of STAT3 was abolished in the heart of D.N.gp130 TG mice, endogenous gp130 activity is thought to be strongly suppressed by overexpression of D.N.gp130. However, there is a possibility that the two mice have different gp130 activity. The transgene lacks box3 but has box1 and box2 regions of the cytoplasmic domain of gp130. Although box3 is indispensable for the Janus kinase-induced activation of STAT3 (26), there is a possibility that box1 and box2 have some biological functions. There is a possibility that the two mice have different ERK activities, which they did not examine (18). It has been reported that the ERK signaling pathway is important for the gp130-dependent cell survival of cardiac myocytes (17) and that the tyrosine-containing motif, Tyr116-Xaa-Xaa-Val, of the cytoplasmic domain of gp130 is indispensable for the activation of SHP-2, a key molecule for the gp130-mediated signaling pathway leading to ERKs (26, 32). Although the transgene lacks this motif, pressure overload induced activation of ERKs also in TG mice. These results suggest that some other factors such as angiotensin II and endothelin-1, which exert their effects through G protein-coupled receptors, play a predominant role in the activation of ERKs and cardiomyocyte survival in the TG mice. Although it remains to be determined how pressure overload induces production of the IL-6 family of cytokines in the heart, the present study suggests that gp130 plays a critical role in pressure overload-induced cardiac hypertrophy possibly through the STAT3 pathway.

    ACKNOWLEDGEMENTS

D.N.gp130 cDNA was generously provided by Tetsuya Taga (Kumamoto University, Japan).

    FOOTNOTES

* This work was supported in part by a grant-in-aid for scientific research, developmental scientific research, and scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan and by grants from the Japan Cardiovascular Foundation, Japan (to I. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Medicine III, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel./Fax: 81-43-226-2553; E-mail: komuro-tky@umin.ac.jp.

Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M100814200

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; TG, transgenic; D.N., dominant negative; MHC, myosin heavy chain; WT, wild type; LVEDD, left ventricular end-diastolic internal diameter; LVESD, left ventricular end-systolic internal diameter; BNP, brain natriuretic factor; SERCA, sarcoplasmic reticulum Ca2+ ATPase; STAT, signal transducers and activators of transcription; ERK(s), extracellular signal-regulated kinase(s); TUNEL, terminal deoxynucleotidyl transferase assay.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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