Cardiotrophin-1 mRNA Research Articles

The Expression of Adipose Tissue-Derived Cardiotrophin-1 in Humans with Obesity.

Cardiotrophin-1 (CT-1) is a gp130 cytokine that was previously characterized for its effects on cardiomyocytes and identified as a marker of heart failure. More recent studies reported elevated circulating levels of CT-1 in humans with obesity and metabolic syndrome (MetS). However, a subsequent rodent study implicated CT-1 as a potential therapeutic target for obesity and MetS. Adipose tissue (AT) is broadly acknowledged as an endocrine organ and is a substantial source of CT-1. However, no study has examined the expression of adipose-derived CT-1 in humans. We present the first analysis of CT-1 mRNA expression in subcutaneous AT and its association with clinical variables in 22 women with obesity and 15 men who were 40% overfed for 8-weeks. We observed that CT-1 expression was higher in the subcutaneous abdominal (scABD) than the femoral (scFEM) depot. Importantly, we reveal that scFEM but not scABD, CT-1 expression was negatively associated with visceral adiposity and intrahepatic lipid, while positively correlated with insulin sensitivity in obese women. Also, men with higher CT-1 levels at baseline had less of a decline in insulin sensitivity in response to overfeeding. Our data provide new knowledge on the regulation of adipose-derived CT-1 in obesity and during weight gain in response to overfeeding in humans and suggest that CT-1 may play a protective role in obesity and related disorders.

Abstract 18362: Cardiac-targeted Therapy With Cardiotrophin-1 Rescues Defective Right Ventricular Adaptation in Severe Pulmonary Arterial Hypertension in Fischer Rats

Introduction: In patients with pulmonary arterial hypertension (PAH), right ventricular (RV) dysfunction is one of the strongest predictors of poor prognosis. Therapies targeting RV failure could lead to significant improvement in exercise tolerance and survival in patients with severe PAH. Cardiotrophin-1 (CT-1) is a member of the IL-6 cytokine family that promotes physiological cardiac hypertrophy and cardiomyocyte survival. Hypothesis: We hypothesized that RV-specific therapy using CT-1 therapy will improve RV adaptation to PAH. Methods: PAH was induced by a single subcutaneous injection of SU5416 (SU; 20 mg/kg) followed by exposure to chronic hypoxia (CH; 10% O2) for 3 weeks. On day 22 post SU, human recombinant cardiotrophin-1 (CT-1, Fate Therapeutics) (6 ug/kg/hr) or vehicle (PBS) was administered via subcutaneously implanted mini-osmotic pumps until end-study at 7 weeks. Echocardiography (Vevo 2100, VisualSonics) was performed weekly starting at baseline (day 21). Results: We observed a ~5-fold reduction in expression of endogenous CT-1 mRNA in the RV of rats treated with SU5416+hypoxia compared to healthy controls. Administration of CT-1 to Fischer rats with PAH had no significant effect on pulmonary hemodynamics (pulmonary acceleration time, RVSP), nor RV/LV+S. At 6 weeks post SU, CT-1 significantly reduced RV dilatation (end diastolic RV/LV internal diameter) (p<0.001), improved RV function (cardiac output, RV fractional area change, p<0.05) (Figure 1), which was associated with an increase in RV capillary density (p<0.05) and a 2-fold improvement in survival (31 vs 12%). Conclusions: Targeted cardiac therapy with CT-1 therapy improved RV adaptation in the SU/CH model of severe PAH without altering pulmonary hemodynamics, which in part was mediated by enhancing RV myocardial angiogenesis. These findings support the development of RV-targeted therapeutics for patients with severe PAH and RV failure.

A Potential Profibrogenic Role of Biliary Epithelium-Derived Cardiotrophin-1 in Pediatric Cholestatic Liver Disease.

As a cytokine of the interleukin-6 family, cardiotrophin-1 (CT-1) has been shown to be an important endogenous protector in liver injury. Our study aimed to investigate the role of CT-1 in liver fibrosis in pediatric cholestatic liver disease (PCLD). CT-1 mRNA and protein expression levels were upregulated in PCLD liver biopsy tissues compared with controls. Immunohistochemistry and confocal microscopy of liver sections showed that CT-1 was predominantly expressed by biliary epithelium cells. Serum CT-1 was elevated significantly in the children with PCLD compared with controls. Serum CT-1 levels exhibited a moderate positive correlation with the Scheuer stage of hepatic fibrosis and serum TB levels and a weak correlation with serum ALP levels. In vitro analysis indicated that LX-2 cells preconditioned with CT-1 exhibited significant increments in proliferation and accumulation of extracellular matrix components, while also positively regulating the STAT3 and p38MAPK pathways. In conclusion, biliary epithelium-derived CT-1 may exert a profibrogenic potential in PCLD.

Downregulation of the Cardiotrophin-1 Gene Expression by Valsartan and Spironolactone in Hypertrophied Heart Rats In Vivo and Rat Cardiomyocyte H9c2 Cell Line In Vitro

The incidence, prevalence, and hospitalization rates associated with heart failure (HF) are projected to increase substantially in the world. Among all medications used clinically to treat HF, valsartan (VAL) and spironolactone (SPL) have been shown to reduce morbidity and mortality. Recently, a novel cardiac gene cardiotrophin-1 (CT-1) has been shown to play a crucial role in the pathogenesis of HF. However, the ability of VAL and SPL to modulate the expression of CT-1 has not been investigated yet. Therefore, healthy and isoproterenol (ISO)-induced hypertrophy adult male Wistar albino rats were treated with either VAL or SPL for 14 days. Thereafter, cardiac markers of cardiotoxicity and hypertrophy, creatine kinase, heart weight/body weight ratio, and atrial natriuretic peptide mRNA levels were measured. In addition, CT-1 mRNA and protein levels were determined by real-time polymerase chain reaction and Western blot analysis. Our results showed that the increases in all HF markers, creatine kinase, heart weight/body weight ratio, and atrial natriuretic peptide mRNA levels in ISO-treated rats were significantly restored to their normal levels by VAL and SPL. In addition, induction of cardiac hypertrophy by ISO caused remarkable induction in CT-1 mRNA and protein expression levels by approximately 3.5- and 3-fold, respectively. Importantly, VAL and SPL significantly decreased the induction of CT-1 gene at the mRNA and protein levels in heart hypertrophied rats. On the other hand, treatment of cardiac-derived rat myoblast H9c2 cells with VAL and SPL significantly decreased angiotensin II-induced CT-1 mRNA levels through transcriptional mechanism, as demonstrated by the effect of transcription inhibitor, actinomycin D. In conclusion, VAL and SPL exhibited their cardioprotective effect through inhibiting the expression of CT-1 gene in cardiac hypertrophied rats.

HIF-1-mediated up-regulation of cardiotrophin-1 is involved in the survival response of cardiomyocytes to hypoxia

Cardiotrophin-1 (CT-1) is a cytokine of the interleukin-6 superfamily which is up-regulated in cardiac diseases, in part via hypoxia-dependent mechanisms. However, no evidence for a direct regulation of CT-1 gene (CTF1) promoter by hypoxia inducible factor-1 (HIF-1) has been provided. Hypoxia increased CT-1 mRNA levels in the murine adult cardiomyocyte cell line HL-1 in a time-dependent manner. Interestingly, in a murine model (C57BL/6), we show that systemic hypoxia also significantly up-regulated CT-1 in myocardial tissue. The effect of hypoxia on CT-1 expression was mediated through a transcriptional mechanism, since hypoxia increased luciferase activity of constructs containing CTF1 promoter sequences. The increase in CT-1 levels was significantly reduced by drugs that prevent calcium mobilization, such as lercanidipine, or that inhibit the activation of the PI3K/Akt pathway (wortmannin) or mammalian target of rapamycin (rapamycin). The CT-1 elevation was similarly induced by HIF-1α over-expression in co-transfection experiments and prevented by HIF-1α silencing. The direct interaction of HIF-1α with the CTF1 promoter was confirmed through site-directed mutagenesis of hypoxia response elements, electrophoreric mobility shift, and ChIP assays. Hypoxia induced HL-1 apoptosis (measured as annexin-V binding or caspase 3/7 activity) which was increased when CT-1 was silenced in knocked-down cells by lentiviral vectors. Hypoxia increased CT-1 levels in cardiac cells (in vitro and in vivo) through a direct regulation of CTF1 promoter by HIF-1α. This CT-1 activation by hypoxia may protect cells from apoptosis, thus supporting a protective role for CT-1 as a survival factor for cardiomyocytes.

Enalapril decreases cardiac mass and fetal gene expression without affecting the expression of endothelin-1, transforming growth factor β-1, or cardiotrophin-1 in the healthy normotensive rat

Angiotensin II can induce cardiac hypertrophy by stimulating the release of growth factors. ACE inhibitors reduce angiotensin II levels and cardiac hypertrophy, but their effects on the healthy heart are largely unexplored. We hypothesized that ACE inhibition decreases left ventricular mass in normotensive animals and that this is associated with altered expression of cardiac fetal genes, growth factors, and endothelial nitric oxide synthase (eNOS). Wistar rats (n = 7 per group) were orally administered with enalapril twice daily for a total daily dose of 5 mg·kg(-1)·d(-1) (ENAP5) or 15 mg·kg(-1)·d(-1) (ENAP15) or vehicle. Systolic blood pressure was measured by the tail-cuff method. Left ventricular expression of cardiac myosin heavy chain-α (MYH6) and -β (MYH7), atrial natriuretic peptide (ANP), endothelin-1 (ET-1), transforming growth factor β-1 (TGFβ-1), cardiotrophin-1 (CT-1), and renal renin were examined by real-time PCR, and eNOS using Western blot. Blood pressure was decreased only in ENAP15 animals (p < 0.05 vs. Control), whereas left ventricular mass decreased after both doses of enalapril (p < 0.05 vs. Control). MYH7 and ANP were reduced in ENAP15, while no changes in ET-1, TGFβ-1, CT-1, and MYH6 mRNA or eNOS protein were observed. Renal renin dose-dependently increased after enalapril treatment. Enalapril significantly decreased left ventricular mass even after 1 week treatment in the normotensive rat. This was associated with a decreased expression of the fetal genes MYH7 and ANP, but not expression of ET-1, CT-1, or TGFβ-1.

Interleukin-1α stimulates proinflammatory cytokine expression in human cardiac myofibroblasts

Cardiac myofibroblasts (CMF) play a key role in infarct repair and scar formation following myocardial infarction (MI) and are also an important source of proinflammatory cytokines. We postulated that interleukin-1alpha (IL-1alpha), a potential early trigger of acute inflammation post-MI, could stimulate human CMF to express additional proinflammatory cytokines. Furthermore, we hypothesized that these effects may be modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). Human CMF were cultured from atrial biopsies from multiple patients. Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and cardiotrophin-1 (CT-1) mRNA expression and secretion were measured using quantitative real-time RT-PCR and enzyme-linked immunosorbent assay. IL-1alpha (0.001-10 ng/ml, 0-6 h) stimulated IL-1beta, TNF-alpha, and IL-6 mRNA expression with distinct temporal and concentration profiles, resulting in increased cytokine secretion. The response to IL-1alpha was much greater than with TNF-alpha. Neither IL-1alpha nor TNF-alpha treatment modulated CT-1 mRNA expression. Immunoblotting with phosphospecific antibodies revealed that IL-1alpha stimulated the extracellular signal-regulated kinase (ERK)-1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), and nuclear factor (NF)-kappaB signaling pathways. Pharmacological inhibitor studies indicated roles for PI 3-kinase/Akt and NF-kappaB pathways in mediating IL-1beta expression, and for NF-kappaB and p38 MAPK pathways in mediating TNF-alpha expression. IL-1alpha-induced IL-6 mRNA expression was reduced by p38 MAPK inhibition, but increased by ERK and JNK pathway inhibitors. IL-10 produced a consistent but modest reduction in IL-1alpha-induced IL-6 mRNA levels (not IL-1beta or TNF-alpha), but this was not reflected by reduced IL-6 protein secretion. In conclusion, IL-1alpha stimulates human CMF to express IL-1beta, TNF-alpha, and IL-6 via specific signaling pathways, responses that are unaffected by IL-10 exposure.

Aldosterone Induces Cardiotrophin-1 Expression in HL-1 Adult Cardiomyocytes

Aldosterone (ALDO) may induce cardiac hypertrophy by nonhemodynamic mechanisms that are not completely defined. Cardiotrophin-1 (CT-1) is a cytokine that exerts hypertrophic actions on isolated cardiomyocytes and promotes cardiac hypertrophy in vivo. We investigated whether ALDO induces CT-1 expression in HL-1 cardiomyocytes aiming at the possibility that the cytokine is involved in ALDO-induced cardiomyocyte hypertrophy. mRNA and protein expression were quantified by RT-PCR and Western blot. Cardiomyocyte area, as an index of hypertrophy, was assayed by image analysis in phalloidin-stained HL-1 cells. ALDO addition to adult HL-1 cardiomyocytes increased (P<0.01) CT-1 mRNA and protein expression in a concentration-dependent manner. This effect was abrogated by actinomycin D, the mineralocorticoid and glucocorticoid receptor antagonists spironolactone and RU486, respectively, and the p38 MAPK blocker SB203580. CT-1 signaling pathway blockade with specific antibodies against the cytokine and its two receptor subunits avoided (P<0.01) alpha-sarcomeric actin and c-fos protein overexpression as well as cell size increase induced by ALDO in HL-1 cells. In vivo, a single ALDO injection acutely increased (P<0.01) the myocardial expression of CT-1 in C57BJ6 wild-type mice but not CT-1-null mice. The bolus of the mineralocorticoid increased (P<0.01) ANP and c-fos mRNA expression in the myocardium of wild-type mice, whereas no changes were observed in CT-1-null mice. In summary, ALDO induces CT-1 expression in adult HL-1 cardiomyocytes via genomic and nongenomic mechanisms. CT-1 up-regulation could have relevance in the direct hypertrophic effects of ALDO in cardiomyocytes.

Differential hypertrophic effects of cardiotrophin-1 on adult cardiomyocytes from normotensive and spontaneously hypertensive rats

Cardiotrophin-1 (CT-1) produces longitudinal elongation of neonatal cardiomyocytes, but its effects in adult cardiomyocytes are not known. Recent observations indicate that CT-1 may be involved in pressure overload left ventricular hypertrophy (LVH). We investigated whether the hypertrophic effects of CT-1 are different in cardiomyocytes isolated from adult normotensive and spontaneously hypertensive rats (SHR). Hypertrophy was evaluated by planimetry and confocal microscopy, contractile proteins were quantified by Western blotting and real-time RT-PCR, and intracellular pathways were analyzed with specific chemical inhibitors. CT-1 increased c- fos and ANP expression ( p < 0.01) and cell area ( p < 0.01) in cardiomyocytes from both rat strains. In Wistar cells, CT-1 augmented cell length ( p < 0.01) but did not modify either the transverse diameter or cell depth. In SHR cells, CT-1 increased cell length ( p < 0.05), cell width ( p < 0.01) and cell depth, augmented the expression of myosin light chain-2v (MLC-2v) and skeletal α-actin ( p < 0.01) and enhanced MLC-2v phosphorylation ( p < 0.01). The blockade of gp130 or LIFR abolished CT-1-induced growth in the two cell types. All distinct effects observed in cardiomyocytes from SHR were mediated by STAT3. Baseline angiotensinogen expression was higher in SHR cells, and CT-1 induced a 1.7-fold and 3.2-fold increase of angiotensinogen mRNA in cardiomyocytes from Wistar rats and SHR respectively. In addition, AT1 blockade inhibited the specific effects of CT-1 in SHR cells. Finally, ex vivo determinations revealed that adult SHR exhibited enhanced myocardial CT-1 (mRNA and protein, p < 0.01), increased cell width ( p < 0.01) and concentric LVH compared with pre-hypertensive SHR. These findings reveal a specific cell-broadening effect of CT-1 in cardiomyocytes from adult SHR and suggest that the hypertensive phenotype of these cells may influence the hypertrophic effects of CT-1, probably by means of an exaggerated induction of angiotensinogen expression. We suggest that CT-1 might facilitate LVH in genetic hypertension through a cross-talk with the renin–angiotensin system.

Cardiotrophin: its importance as a pathogenetic factor and as a measure of left ventricular hypertrophy

Cardiotrophin: its importance as a pathogenetic factor and as a measure of left ventricular hypertrophy

Amlodipine at high dose increases preproendothelin-1 expression in the ventricles and aorta of normotensive rats.

High doses of dihydropyridine calcium channel blockers can activate the sympathetic nervous system and the renin-angiotensin system. Both noradrenaline and angiotensin II stimulate preproendothelin-1 gene expression, yet the effects of high doses of dihydropyridines on preproendothelin-1 expression in vivo remain unknown. To investigate the effects of high doses of dihydropyridines on preproendothelin-1 expression in the ventricles and aorta of normotensive rats. Sprague-Dawley rats were treated with amlodipine 5 or 20 mg/kg per day (Amlo 5 or Amlo 20) in drinking water for 5 days or 5 weeks. Systolic blood pressure and heart rate were measured by tail-cuff plethysmography. Gene expression was examined by reverse transcriptase polymerase chain reaction. Amlo 5 increased heart rate during the first week only and had no effect on blood pressure and ventricular weight and gene expression. Amlo 20 reduced blood pressure transiently and increased heart rate consistently. It did not change relative left ventricular weight (corrected for body weight) after 5 days, but increased it after 5 weeks; it increased relative right ventricular weight at both time points. Aorta weight (mg/mm) was decreased after 5 weeks of treatment with both dosages of amlodipine. Preproendothelin-1 mRNA levels were increased by Amlo 20 in the ventricles and aorta and, concomitantly, renin mRNA was increased in the kidney. Less consistently, interleukin-6 mRNA also increased in ventricles, whereas cardiotrophin-1 mRNA remained unchanged. The sensitivity of isolated aorta to the contractile effect of noradrenaline was decreased by Amlo 5, but not by Amlo 20. In Sprague-Dawley rats, high-dose amlodipine, while promoting neurohormonal activation, induced overexpression of preproendothelin-1 mRNA in the ventricles and aorta. Endothelin-1 overexpression could contribute to the lack of inhibitory effect of high-dose amlodipine on ventricular mass in normotensive rats.

Teratogenic effects of bis-diamine on the developing myocardium.

Bis-diamine induces conotruncal anomalies and disproportional ventricular development in rat embryos when administered to the mother. To evaluate the mechanisms of disproportional ventricular development in the anomalous heart, we analyzed the morphology of the embryonic heart and investigated cardiomyocytic DNA synthesis and apoptosis. A single dose of 200 mg of bis-diamine was administered to pregnant rats Wistar on day 9.5 of pregnancy. The embryos were removed on each embryonic day from 10.5 to 18.5. Expression of cardiotrophin-1 and hepatocyte growth factor was investigated on the sections, and cardiotrophin-1, hepatocyte growth factor and myocyte enhancer factor 2 mRNA expression was examined by reverse transcriptase-polymerase chain reaction. Myocardial DNA synthesis was investigated using 5-bromo-2'-deoxyuridine and the labeling index was calculated for each heart. Apoptosis was also analyzed using TUNEL reaction and electrophoresis of DNA fragmentation. The embryos treated with bis-diamine had conotruncal anomalies associated with thin left ventricular wall in the later stage. The labeling index on embryonic day 15.5 and 16.5 was significantly lower than those in the controls. Hepatocyte growth factor and cardiotrophin-1 mRNA expression was upregulated on embryonic day 12.5 and 15.5 in bis-diamine-treated hearts. Fewer apoptotic cells were detected in the hearts of bis-diamine-treated embryos than in control hearts from embryonic day 14.5 to 16.5. The ventricular disproportion in the bis-diamine-treated heart may be caused by the early myocardial differentiation delay and poor proliferation and reduced apoptosis associated with anomalous circulatory condition in the later stage.

Cardiotrophin-1 in choroid plexus and the cerebrospinal fluid circulatory system

There is a growing recognition of choroid plexus functioning as a source of neuropeptides, cytokines and growth factors in cerebrospinal fluid (CSF) with diffusional access into brain parenchyma. In this study, choroid plexus and other components of the CSF circulatory system were investigated by Western blotting, reverse transcriptase polymerase chain reaction and immunohistochemistry for production of interleukin-6-related cytokines characterized by neuroactivity [cardiotrophin-1 (CT-1), ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M] and signaling through the gp130/leukemia inhibitory factor receptor-β receptor heterodimer. Western blot analysis showed that CT-1 was the only cytokine family member detectable in adult rat choroid plexus, as in leptomeninges. The specificity of detection was verified with blots of the same tissues from CT-1-deficient mice. Levels of both CT-1 mRNA and protein were constitutively high in rat from birth through adulthood in choroid plexus, up-regulated postnatally in leptomeninges and undetectable in brain parenchyma. Using antigen retrieval, CT-1 immunolocalized to choroid epithelial cells in all choroid plexuses in addition to leptomeninges (arachnoid and pial–glial membranes). Ependymal cells lining the ventricular neuroaxis, unlike the central canal, were also CT-1-immunoreactive. Western blots indicated rat choroid epithelial cells express and release CT-1 immunoreactivity under defined culture conditions and also revealed the presence of a CT-1-like protein in human choroid plexus and CSF. Previously, CT-1 has been conceptualized to function as a target-derived factor for PNS neurons. Our study clearly demonstrates production of CT-1 in the postnatal and adult CNS, specifically by cell types comprising the blood–CSF barrier, and its accumulation in ventricular ependyma. This finding has broad implications for CT-1 functioning apart from other leukemia inhibitory factor receptor ligands as a CSF-borne signal of brain homeostasis, one possibly involving regulation of the barrier itself, the ependyma or target cells in the surrounding parenchyma, including the subventricular zone. A rationale for studies examining CT-1-deficient mice in these respects is provided by the data.

Ventricular cardiotrophin-1 activation precedes BNP in experimental heart failure

Both cardiotrophin-1 (CT-1) and B-type or brain natriuretic peptide (BNP) are activated by cardiomyocyte stretch, and gene expression of CT-1 and BNP are augmented in the heart in experimental and human congestive heart failure (CHF). The goal of this study was to define cardiac gene expression of CT-1 and BNP by Northern blot analysis in normal ( n=5), early left ventricular dysfunction (ELVD, n=5) and overt CHF dogs ( n=5), in which ventricular function is progressively decreased. CT-1 mRNA was detected in both atria and ventricles in normal dogs. Ventricular CT-1 mRNA production increased in ELVD, and it further increased in overt CHF. Ventricular BNP mRNA remained below or at the limit of detection in normal and ELVD models, and it markedly increased in overt CHF. This study reports differential regulation of gene expression of CT-1 and BNP in the heart during the progression of CHF, and demonstrates that ventricular CT-1 gene activation precedes ventricular BNP gene activation.

Augmented expression of cardiotrophin-1 in failing human hearts is accompanied by diminished glycoprotein 130 receptor protein abundance.

Cardiotrophin-1 (CT-1), a member of the interleukin-6 superfamily, is a potent inducer of cardiomyocyte hypertrophy that prolongs myocyte survival. Although cardiac CT-1 gene expression is known to be upregulated in some animal models of congestive heart failure, the activation state of the CT-1 system in patients with congestive heart failure is unknown. This study was designed to determine left ventricular expression of CT-1 and its glycoprotein 130 (gp130)/leukemia inhibitory factor receptor complex in human end-stage heart failure due to ischemic and dilated cardiomyopathy. In addition, we investigated the activation state of signal transducer and activator of transcription 3 (STAT3), the downstream effector of gp130 signaling. In the failing left ventricular myocardium, expression levels of CT-1 mRNA and protein were significantly increased by 142% and 68%, respectively, compared with non-failing donor hearts. Immunohistochemistry confirmed the increased expression of CT-1 in cardiac myocytes. Although gp130 gene expression was increased by 91% (P<0.001), gp130 protein abundance was significantly diminished by 34% in the failing myocardium. In contrast, leukemia inhibitory factor receptor and suppressor of cytokine signaling-3 protein concentrations were not changed. In addition, the ratio of activated tyrosine phosphorylated STAT3 to total STAT3 was not significantly altered in failing hearts compared with non-failing controls. Our data suggest that gp130 receptor downregulation balances enhanced CT-1 expression in human heart failure and thereby inhibits excessive activation of the gp130 signaling pathway.

Increased expression of cardiotrophin-1 during ventricular remodeling in hypertensive rats.

Cardiotrophin-1 (CT-1) stimulates longitudinal myocardial cell hypertrophy. We examined the expression of CT-1, leukemia inhibitory factor (LIF), and gp130 by competitive RT-PCR and Western blotting in Dahl salt-sensitive (DS) rats with a high-salt diet, which showed a distinct transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF). The expression levels of CT-1 mRNA and protein were significantly increased at the CHF stage compared with the LVH stage and age-matched Dahl salt-resistant (DR) rats (n = 6 for each group). mRNA expression of LIF was not changed in the left ventricle at any stage by RT-PCR. gp130 mRNA and protein levels of DS rats at 11 and 17 wk were significantly increased compared with age-matched DR rats. The isolated myocyte length of DS rats at 17 wk was the longest among the four groups of rats. The LV end-diastolic dimension (LVDd) of DS rats, determined by echocardiography, was significantly increased at the CHF stage. There was a significant correlation between the CT-1 protein level and LVDd. CT-1 may play a role in ventricular remodeling during transition from LVH to CHF in the rat hypertensive model.

Augmented Expression of Cardiotrophin-1 and its Receptor Component, gp130, in both Left and Right Ventricles after Myocardial Infarction in the Rat

Cardiotrophin-1 (CT-1) is a potent cytokine that stimulates the assembly of sarcomeric units in series in cardiomyocytes through gp130 signaling, resulting in myocardial cell hypertrophy. To clarify the role of CT-1 and the gp130-signaling pathway during ventricular remodeling after myocardial infarction, we examined the expression of CT-1 and gp130 in a rat model of myocardial infarction. At 1, 3, 7, 14, 28 and 56 days (n=12 for each group) after ligation of a coronary artery, tissue samples were obtained from infarct tissue, the ventricular septum and the right ventricle. All animals developed large myocardial infarctions, with infarct sizes ranging from 39.8% to 50.3%. Progressive left ventricular dilatation and inadequate hypertrophy of the surviving myocardium were confirmed by echocardiography. CT-1 and gp130 mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction using 1 or 5 μ g of total RNA followed by Southern blotting. The densitometric analysis of the Southern blots revealed a significant increase in CT-1 and gp130 mRNA levels (P<0.01) compared with those of the sham-operated rats at 1, 3, 7, 14, 28 and 56 days post-infarct in the infarct area, the ventricular septum (non-infarcted area) and right ventricle. The protein levels of CT-1 and gp130, determined by Western blot analysis, were significantly increased (P<0.05) compared with those of sham-operated rats, peaked during the acute stage and declined thereafter in the three regions described above. Immunohistochemical staining showed that CT-1 and gp130-immunoreactivities were detected in cardiomyocytes and fibroblast-like cells and that the intensity of staining was increased at 7 days post-infarct compared with that in sham-operated rats. An augmented CT-1 and gp130 system thus appears to play an important role during ventricular remodeling after myocardial infarction.

Interleukin-6 Family of Cytokines Mediate Angiotensin II-induced Cardiac Hypertrophy in Rodent Cardiomyocytes

This study was designed to investigate whether angiotensin II induces the interleukin (IL)-6 family of cytokines in cardiac fibroblasts and, if so, whether these cytokines can augment cardiac hypertrophy. Angiotensin II increased IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 mRNA by 6.5-, 10.2-, and 2.0-fold, respectively, but did not affect IL-11, ciliary neurotrophic factor, or oncostatin M in cardiac fibroblasts. Enzyme-linked immunosorbent assay revealed that angiotensin II-stimulated conditioned medium from cardiac fibroblasts contained 9.3 ng/ml IL-6 at 24 h, which was 24-fold higher than the control. It phosphorylated gp130 and STAT3 in cardiomyocytes, which was reduced with RX435 (anti-gp130 blocking antibody). It increased [(3)H]phenylalanine uptake and cell area by 44% and 86% in cardiomyocytes compared with mock medium. RX435 suppressed these increases by 26% and 38%, while TAK044 (endothelin-A/B-R blocker) suppressed them by 52% and 52%, respectively. Antisense oligonucleotides against LIF and cardiotrophin-1 blocked their up-regulation, and attenuated the conditioned medium-induced increase in [(3)H]phenylalanine uptake by 21% and 13%, respectively. The combination of antisense oligonucleotides to LIF and cardiotrophin-1 decreased their uptake by 33%. These results indicated that angiotensin II induced IL-6, LIF, and cardiotrophin-1 in cardiac fibroblasts, and that these cytokines, particularly LIF and cardiotrophin-1, activated gp130-linked signaling and contributed to angiotensin II-induced cardiomyocyte hypertrophy.

Isolation and Characterization of the Murine Cardiotrophin-1 Gene: Expression and Norepinephrine-Induced Transcriptional Activation

Cardiotrophin-1 (CT-1) is a novel cytokine capable of inducing hypertrophy in cardiac myocytes and belongs to the interleukin-6 family that exert their biological effects through gp130. To clarify the involvement and pathophysiological role of CT-1 in myocardial diseases, it is important to characterize the regulation of CT-1 gene expression. In this study, we isolated and characterized the mouse CT-1 gene and studied the expression of CT-1 mRNA under norepinephrine (NE) stimulation. The mouse CT-1 gene constitutes 5.4 kilobases (kb) in length and consists of three exons and two introns. When nucleotide sequences of the coding regions of exons were compared with those of human, exon 1, 2 and 3 share 96%, 84% and 81% homology, respectively. The 2.2 kb of 5 flanking lesion of the mouse CT-1 gene contains a variety of transcription factor binding motif (e.g. CREB, MyoD, NF-IL6, Nkx2.5, GATA). Fluorescent in situ hybridization (FISH) analysis demonstrated that the mouse CT-1 gene was located on chromosome 7F3. The expression of CT-1 mRNA in cardiac myocytes was markedly augmented by NE stimulation, both in vivo and in vitro. Promoter analysis using deletion constructs of the CT-1 gene indicated that the NE responsive element located between -2174/-1540 and this region contained the cAMP responsive element (CRE). Electrophoretic gel mobility shift assays showed enhanced binding activity to the CRE motif in the nuclear extracts from NE-stimulated cardiac myocytes. These studies indicate that CT-1 is abundantly expressed in the heart and that the CRE is a possible cis -acting element of the CT-1 gene under NE-stimulation. These data suggest that the CT-1 gene expression is regulated, at least partially, by transcriptional machinery.

Expressional Patterns of Cytokines in a Murine Model of Acute Myocarditis: Early Expression of Cardiotrophin-1

To determine the role of cytokines in acute myocarditis, we examined expressional patterns of cardiotrophin-1 (CT-1), TNF-α, and IL-1α in a murine model of acute myocarditis. Ten-day-old Institute of Cancer Research mice were injected with Coxsackievirus B3 and killed on Days 1, 2, 3, 4, 5, 7, 10, 14, and 28 of injection. TNF-α and IL-1α expressions were investigated on histological sections from each heart, and mRNA expression of TNF-α, IL-1α, and CT-1 in the heart was examined by reverse transcription-polymerase chain reaction and RNase protection assay. To determine myocardial regeneration, cardiomyocytic DNA synthesis was investigated using bromodeoxyuridine on Days 3, 5, 7, and 10, and the labeling index was calculated in each heart. Age-matched uninfected mice were used as controls. TNFα and IL-1α expression was first detected in the cardiomyocytes on Day 3 and reached the maximum level on Day 7, when inflammatory changes were most prominent. Although an increased expression of TNFα and IL-1α mRNA was also detected on Day 3, CT-1 mRNA expression was distinctly augmented on Day 2. The labeling indices in the hearts with myocarditis were significantly higher than in those of the controls in all of the time points examined. CT-1 expression preceded TNF-α and IL-1α expressions and active DNA synthesis in a murine model of acute myocarditis. All CVB3-infected mice with anti-glycoprotein-130 antibody treatment died within 6 days. CT-1 may exert a protective role by modulating cytokine production and by inducing cardiomyocytic proliferation in CVB3-infected murine hearts.