gp130 Plays a Critical Role in Pressure Overload-Induced Cardiac Hypertrophy

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 a 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 upregulation of BNP gene and downregulation of SERCA2 gene were attenuated in TG mice. Pressure overload activated extracellular signal-regulated kinases (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. than those of TG mice (F IG . 3 B ). These results suggest that cardiac hypertrophy induced by pressure overload was attenuated in D.N.gp130 TG mice. pressure overload induces production of the IL6 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.


INTRODUCTION
Since 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 have reported that mechanical stress induces cardiomyocyte hypertrophy through vasoactive peptides such as angiotensin II and endothelin-1 (7-9).
Cardiotrophin-1 (CT-1), a member of the interleukin 6 (IL6) family, was isolated and found to have a potent hypertrophic effect on cultured cardiomyocytes (10). The IL6 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)(12)(13). CT-1 has been reported to induce hypertrophy of cardiac myocytes in vitro (14). It has been reported that transgenic mice expressing both IL6 and soluble IL6 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. CT-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 loadinduced cardiac hypertrophy, we generated transgenic (TG) mice which express a dominant negative form of gp130 specifically in the heart and examined hypertrophic responses by 2 by guest on March 17, 2020 http://www.jbc.org/ Downloaded from pressure overload produced by constriction of the abdominal aorta.

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 pαMHCSA which carries the mouse α myosin heavy chain (αMHC) promoter (20). αMHC promoter-D.N.gp130-polyA 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 PCR.

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-hour 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 (IP) 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 IP) and xylazine (15 mg/kg IP). After a good-quality two-dimensional image was obtained, M-mode images of the left ventricle were recorded.

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 Ca 2+ ATPase 2 (SERCA2) genes.

Assay of extracellular signal-regulated kinases (ERKs)
The activities of ERKs were measured using myelin basic protein (MBP)-containing gel (25). In brief, lysates of the left ventricles were subjected to electrophoresis on a SDSpolyacrylamide gel containing 0.5 mg/ml MBP. ERKs in the gel were denatured in guanidine HCl and renatured in Tris-HCl (pH 8.0) containing 0.04% Triton X-100 and 2mercaptoethanol (5 mM). Phosphorylation activities of ERKs were assayed by incubating the gel with [γ-32 P]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 FITC-dUTP for 90 min, and horseradish peroxidase-conjugated anti-FITC 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 ANOVA and Dunnett's t test. The accepted level of significance was P<.05.

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 αMHC promoter (FIG. 1). Six founders containing the transgene were identified, and three transgenic lines, in particular #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).

Cardiac hypertrophy induced by pressure overload
Constriction of the abdominal aorta is a established method to produce pressure overload-induced cardiac hypertrophy (21,22). Four 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)  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 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.

Expression of cardiac specific genes
Pressure overload by the constriction of the abdominal aorta upregulates fetal-type cardiac genes and downregulates 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 upregulated at 2 days after pressure overload and markedly at 28 days in WT mice (FIG. 4). In TG mice, although BNP gene was also upregulated by pressure overload, the expression levels were quite low compared with those of WT mice. In WT mice, SERCA2 gene was downregulated from 2 days by pressure overload (FIG. 4). The downregulation of SERCA2 gene by pressure overload was also attenuated in TG mice.

Activation of STAT3 and ERKs
Activation of gp130 evokes two distinct pathways, JAK-STAT pathway and Ras-ERKs pathway (28). We examined which pathway is important in the pressure overloadinduced 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 overloadinduced cardiac hypertrophy.

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.

DISCUSSION
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 #3 line of transgenic mice, which expressed D.N.gp130 most abundantly. Mice of #1 and #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 JAKinduced STAT pathway and the Ras-ERKs pathway (28). Activated STAT3 has been reported to form a homodimer which 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. 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 tyrosinecontaining motif, Y116XXV, of the cytoplasmic domain of gp130 is indispensable for the activation of SHP-2, a key molecule for 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 IL6 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.