Peptides 24 (2003) 889–892
Ventricular cardiotrophin-1 activation precedes BNP in experimental heart failure Michihisa Jougasaki a,b,∗ , Hanna Leskinen b , Amy M. Larsen b , Andreas Luchner b , Alessandro Cataliotti b , Issei Tachibana b , John C. Burnett Jr. b a
Institute for Clinical Research, National Hospital Kyushu Cardiovascular Center, 8-1 Shiroyama-cho, Kagoshima 892-0853, Japan b Cardiorenal Research Laboratory, Mayo Clinic and Foundation, Rochester, MN 55905, USA Received 17 December 2002; accepted 19 May 2003
Abstract 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. © 2003 Elsevier Inc. All rights reserved. Keywords: BNP; Cardiotrophin-1; Congestive heart failure; Cytokines; Northern blot analysis
1. Introduction Cardiotrophin-1 (CT-1) is a newly identified cardiac hypertrophic factor discovered by expression cloning in a mouse embryonic stem cell-based system of cardiogenesis [9]. Mechanical stretch augments CT-1 gene expression in cultured neonatal rat cardiomyocytes [8]. Augmented expression of CT-1 and its receptor gp130 were observed in the ventricle after myocardial infarction in rats [1]. We have demonstrated that both atrial and ventricular CT-1 gene expressions are increased in experimental congestive heart failure (CHF) [3]. More recently, increased ventricular expression of CT-1 was reported in human end-stage CHF [11]. To date, however, the timing of cardiac CT-1 gene activation during the progression of CHF remains unknown. B-type or brain natriuretic peptide (BNP) is also activated by cardiomyocyte stretch [2], and the release of BNP from the heart has emerged as a sensitive and specific marker for CHF [7]. CT-1 increases BNP secretion from the cardiomyocytes in vitro, and the antibody against CT-1 suppresses this augmentation [4]. Although BNP synthesis and secre∗
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[email protected] (M. Jougasaki).
0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0196-9781(03)00163-3
tion are augmented in the failing heart [6], the relationship between cardiac BNP and CT-1 gene activation during the progression of CHF remains unclear. We have already established an animal model of pacinginduced experimental CHF that evolves from early left ventricular dysfunction (ELVD) to overt CHF [6,10], in which ventricular function progressively decreased. In the present study, we defined cardiac CT-1 gene expression in normal, ELVD and overt CHF dogs by Northern blot analysis and compared those results to cardiac BNP gene expression.
2. Materials and methods 2.1. Experimental canine model of CHF Three groups of male mongrel dogs weighing 19–24 kg (mean 21 kg) were studied with five dogs in each group (normal, ELVD and overt CHF). Experimental CHF was produced by rapid ventricular pacing as reported previously [6,10]. Five dogs were paced at 180 beats per minute (bpm) for 10 days and served as tissue donors for ELVD. Other five dogs were paced at 180 bpm for 10 days, and the pacing
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rate was increased weekly to 200, 210, 220 and 240 bpm to produce overt CHF. Lastly, five normal dogs served as cardiac tissue donors for normal control group. A short-axis echocardiogram was performed and left ventricular end-diastolic (LVDd) and end-systolic (LVDs) dimensions were determined. Left ventricular ejection fraction (EF) was calculated as previously described [6]. Mean arterial blood pressure in conscious state was measured via the femoral artery catheter. Each dog was sacrificed, and the cardiac tissues were rapidly harvested. This study was approved by IACUC of the Mayo Clinic and conducted in accordance with the Animal Welfare Act.
Table 1 Mean arterial pressure, left ventricular dimension and ejection fraction in normal, ELVD and overt CHF dogs
Mean arterial pressure (mmHg) LVDd (mm) LVDs (mm) Ejection fraction (%)
Normal
ELVD
Overt CHF
108 ± 3
108 ± 6
95 ± 7∗†
39 ± 1 27 ± 1 53 ± 1
42 ± 1∗ 35 ± 1∗ 31 ± 3∗
47 ± 1∗† 42 ± 1∗† 22 ± 2∗†
CHF, congestive heart failure; ELVD, early left ventricular dysfunction; LVDd, left ventricular end-diastolic diameter; LVDs, left ventricular end-systolic diameter. Data were obtained in the conscious animal and are expressed as mean ± S.E. ∗ P < 0.05 vs. normal, † P < 0.05 vs. ELVD.
2.2. Gene expression study Messenger RNA was isolated from the canine heart using the FastTrackTM 2.0 kit (Invitrogen, Carlsbad, CA) and Northern blot analysis was performed as previously reported [3]. Four micrograms of atrial and ventricular mRNA were transferred to a nylon membrane, and were hybridized with a canine CT-1 cDNA probe [3] and BNP cDNA probe [6] labeled using a random-priming labeling kit (Strip EZTM DNA, Ambion Inc., Austin, TX). Blots were stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. 2.3. Statistical analysis Results of the values are expressed as mean ± S.E.M. Statistical comparisons among the normal, ELVD and overt CHF groups were performed by analysis of variance followed by Fisher’s least significant difference test. The correlation analysis between EF and ventricular CT-1 mRNA was performed using linear regression analysis. Nonlinear regression analysis was used to generate the best-fit line illustrating the relationship between EF and ventricular BNP mRNA. Statistical significance was accepted for P < 0.05.
CHF dogs had cardiac dysfunction with signs of pulmonary and systemic congestion. 3.2. Northern blot analysis for CT-1 mRNA and BNP mRNA Fig. 1 illustrates Northern blot analysis for cardiac CT-1 mRNA and BNP mRNA in normal, ELVD and overt CHF dogs. CT-1 mRNA was detected in both atria and ventricles in normal dogs. Atrial CT-1 mRNA was already up-regulated in ELVD and remained at high levels in overt CHF. In contrast, ventricular CT-1 mRNA was increased in ELVD, and it further increased in overt CHF. BNP mRNA was detected principally in the atria and at very low levels in the ventricles in normal dogs. Atrial BNP mRNA was increased in ELVD, and it further increased in overt CHF. On the other hand, ventricular BNP mRNA remained below or at the limit of detection in ELVD, and markedly increased in overt CHF. There was an inverse linear correlation between EF and ventricular CT-1 mRNA (r = −0.81, P < 0.0001, Fig. 2A). Correlation between EF and ventricular BNP mRNA was not linear (Fig. 2B).
4. Discussion 3. Results 3.1. Cardiac diameters and functions Mean arterial blood pressure was unchanged in ELVD (not significant versus normal) and decreased in overt CHF (P < 0.05 versus normal and P < 0.05 versus ELVD). LVDd increased in ELVD (P < 0.05 versus normal) and further increased in overt CHF (P < 0.05 versus normal and P < 0.05 versus ELVD). LVDs also increased in ELVD (P < 0.05 versus normal) and further increased in overt CHF (P < 0.05 versus normal and P < 0.05 versus ELVD). EF decreased in ELVD (P < 0.05 versus normal) and further decreased in overt CHF (P < 0.05 versus normal and P < 0.05 versus ELVD) (Table 1). Although ELVD dogs were free of signs of CHF, all overt CHF dogs had pulmonary edema, pleural effusion and ascites, indicating that overt
The present study was designed to define the timing of cardiac CT-1 gene activation during the progression of experimental CHF in comparison with cardiac BNP gene activation. Three major findings were observed in the present study. First, CT-1 and BNP gene expressions were regulated differently in the atria and ventricles during the progression of CHF. Secondly, ventricular CT-1 gene activation preceded BNP gene activation in ELVD. Lastly, overt CHF was characterized by over-expression of CT-1 and BNP in both atria and ventricles. Both CT-1 and BNP gene expressions are augmented by cardiomyocyte stretch [2,8], and both factors are increased in CHF [3,6,11]. Although BNP is accepted as a sensitive and specific marker for CHF, the role of CT-1 in the pathophysiology of CHF or as a marker for CHF remains unclear. In the present study, ventricular CT-1 gene activation
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Fig. 1. (A) Representative Northern blot analysis for cardiac CT-1 mRNA and BNP mRNA in normal (N), ELVD (E) and overt CHF dogs (C). The expression of GAPDH mRNA was used as an internal control. (B) Bar graphs showing the results of densitometric analysis of Northern blotting for CT-1 mRNA (left panel) and BNP mRNA (right panel) in normal, ELVD and overt CHF dogs. Data are mean ± S.E.M. for the CT-1 mRNA and BNP mRNA levels corrected by GAPDH mRNA levels and expressed as arbitrary units (AU). ∗ P < 0.05 vs. normal control, and † P < 0.05 vs. ELVD.
precedes BNP gene activation in CHF. In ELVD, an early stage of CHF which has many similarities with asymptomatic human CHF [10], ventricular CT-1 gene expression was augmented, while ventricular BNP gene expression was
not increased. These findings raise the possibility that ventricular CT-1 is a biomarker for detecting early ventricular dysfunction in CHF as compared to BNP, which is a marker for overt CHF.
Fig. 2. Graphs showing correlation between EF and ventricular CT-1 gene expression (A), and correlation between EF and ventricular BNP gene expression (B).
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CT-1 was originally discovered by the activity of cardiomyocyte hypertrophy as judged by cell enlargement, myosin light chain organization and natriuretic peptide induction [9], indicating that induction of natriuretic peptide in the cardiomyocyte is one of the key properties of CT-1. In addition, previous reports demonstrated that CT-1 significantly increased BNP secretion, and the augmentation was completely suppressed in the presence of antibody against CT-1 [4,5]. Therefore, initially activated CT-1 might stimulate BNP gene expression in the failing heart in an autocrine and/or paracrine fashion during the progression of CHF in the present study. Further studies are needed to determine if increased cardiac CT-1 production might cause reactivation of embryonic genes such as BNP in CHF. Although mechanical stretch in cardiomyocytes induces activation of both CT-1 and BNP [2,8], the temporal pattern of activation of these two genes was different during the progression of experimental CHF in the present study. If mechanical stretch itself is the only cause of gene activation of CT-1 and BNP, the activating pattern of these two genes should be same. Therefore, the factors other than mechanical stretch are associated with the different gene activation pattern between CT-1 and BNP. Our preliminary studies showed that gene activation pattern of BNP rather than that of CT-1 mimicked the pattern of changes in ventricular hypertrophy during the progression of CHF (data not shown), indicating that cardiomyocyte hypertrophic process might be associated with the different gene activation pattern between CT-1 and BNP. Further studies are needed to clarify this issue. In summary, this study reports differential regulation of CT-1 and BNP gene expressions in the heart during the progression of CHF, and demonstrates that ventricular CT-1 gene activation precedes BNP gene activation during the progression of CHF. This study strongly supports a potential role for CT-1 in the pathophysiology of CHF. CT-1 is an early ventricular genetic biomarker for left ventricular dysfunction with significant diagnostic potential. Acknowledgments We acknowledge the technical assistance of Gail J. Harty, Denise M. Heublein and Sharon M. Sandberg. This work
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