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A novel cardiac hypertrophic factor, neurotrophin-3, is paradoxically downregulated in cardiac hypertrophy
Life Sciences 81 (2007) 385 – 392 www.elsevier.com/locate/lifescie
A novel cardiac hypertrophic factor, neurotrophin-3, is paradoxically downregulated in cardiac hypertrophy Haruko Kawaguchi-Manabe a,c,1 , Masaki Ieda a,b,1 , Kensuke Kimura a,b , Tomohiro Manabe a,b , Satoru Miyatake a,d , Hideaki Kanazawa a,b , Takashi Kawakami a,b , Satoshi Ogawa b , Makoto Suematsu c , Keiichi Fukuda a,⁎ a
c
Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b Cardiology Division, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Biochemistry and Integrative Medical Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan d Department of Emergency and Critical Care Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Received 5 February 2007; accepted 29 May 2007
Abstract The neurotrophin family plays pivotal roles in the development of the nervous system. Recently, the role of the neurotrophin in non-neural tissue has been extensively investigated. Among them, neurotrophin-3 and its receptor TrkC are critical for embryonic heart development, though little is known about neurotrophin-3/TrkC function in adult heart. Moreover, the expressions of other neurotrophin and Trk families in the cardiovascular system have not been fully determined. In adult and neonatal rats, only TrkC mRNA was expressed more abundantly in heart than aorta among the neurotrophin receptors, while all neurotrophins were equally expressed in the cardiovascular system. Immunohistochemistry confirmed the protein expressions of neurotrophin-3/TrkC in rat ventricles. In primary-cultured rat cardiomyocytes, neurotrophin-3 strongly activated p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Jun N-terminal kinase pathways in Western blot analysis. In Northern blot analysis, neurotrophin-3 strongly increased mRNA expressions of cardiac hypertrophic markers (skeletal α-actin and atrial natriuretic peptide) in cardiomocytes. [3H]-phenylalanine uptake into cardiomyocytes, myofilament reorganization, and cardiomyocyte size were also augmented with neurotrophin-3 stimulation, indicating that neurotrophin-3 is a novel cardiac hypertrophic factor. Unexpectedly, neurotrophin-3 was downregulated in cardiac hypertrophy induced by pressure overload (in vivo), and in cardiomyocyte hypertrophy evoked by endothelin-1 stimulation (in vitro). Interestingly, the cell size and BNP mRNA expression level (markers of hypertrophy) were greater in cardiomyocytes treated with both neurotrophin-3 and endothelin-1 than in those stimulated with endothelin-1 alone. These findings demonstrate that neurotrophin-3 is a unique hypertrophic factor, which is paradoxically downregulated in cardiac hypertrophy and might counteract hypertrophic change. © 2007 Elsevier Inc. All rights reserved. Keywords: Neurotrophin-3; TrkC; Cardiomyocyte; Heart; Cardiac hypertrophy
Introduction The neurotrophin family of growth factors plays pivotal roles in the development of the central and peripheral nervous system
⁎ Corresponding author. Tel.: +81 3 5363 3874; fax: +81 3 5363 3875. E-mail address: [email protected] (K. Fukuda). 1 These authors contributed equally to this study. 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.05.024
(Snider, 1994). This family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, and NT-4/5 (Snider, 1994). Neurotrophins selectively bind and activate distinct members of the Trk receptor tyrosine kinase family, which are highly expressed in the central and peripheral nervous systems (NGF activates TrkA, BDNF and NT-4/5 activate TrkB, and NT-3 activates TrkC) (Segal, 2003). We recently found that NGF is abundantly expressed in the heart, where it regulates cardiac sympathetic and sensory
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innervation (Ieda et al., 2004, 2006). Downregulation of cardiac NGF causes sensory denervation in diabetic hearts, and NGF gene therapy rescues this neuropathy (Ieda et al., 2006). BDNF/TrkB signaling is critical for embryonic vascular development, and it also promotes atherosclerosis in ApoE-null mutant mice (Donovan et al., 2000; Kraemer et al., 2005). In embryos, NT-3/ TrkC signaling promotes cardiomyocyte proliferation and is required for ventricular trabeculation (Lin et al., 2000). NT-3 and TrkC gene-targeted mice exhibit early postnatal mortality and have defects in cardiac septation, valvulogenesis, and conotruncal formation (Donovan et al., 1996; Tessarollo et al., 1997). Although these results demonstrate the critical roles of the neurotrophin family in cardiovascular regulation during embryogenesis and adulthood, the expression levels of the neurotrophin and Trk families in the cardiovascular system have not been fully characterized. Moreover, the functions of NT-3 and TrkC in adult hearts and their regulation in pathological hearts remain unknown. Cardiac hypertrophy is a pathological process that can lead to congestive heart failure. Progression towards heart failure is influenced by the balance of hypertrophic and cardioprotective factors. Sustained overexpression of hypertrophic factors in the heart results in cardiac remodeling and ventricular dysfunction (Kim et al., 2001; Sakai et al., 1996; Willenheimer et al., 2005). Despite their importance, our knowledge about cardiac hypertrophic factors remains limited. In this study we found that NGF, BDNF, and NT-3 were highly expressed in the rat heart and vasculature. In contrast, TrkC (but not TrkA or TrkB) was expressed more abundantly in hearts than in blood vessels. NT-3 strongly induced cardiomyocyte hypertrophy via TrkC, but unexpectedly, NT-3 expression was paradoxically reduced in cardiac hypertrophy models both in vivo and in vitro. NT-3 supplementation augmented endothelin-1-induced cardiomyocyte hypertrophy, suggesting that NT-3 downregulation may counteract cardiac hypertrophy. Materials and methods Cell culture Primary cultures of cardiomyocytes were prepared from the ventricles of 1-day-old neonatal Wistar rats (Japan CLEA) as described previously (Pan et al., 1999). Cardiomyocytes were isolated from other cell types by differential adhesion. Cells were seeded at a density of 5 × 105 cells/cm2, and were incubated in media containing serum for 24 h. The media was then replaced with fresh serum-free media, and the cells were stimulated with human recombinant NT-3 (Calbiochem) or 10− 7 M endothelin-1 (ET-1, Sigma) (Ieda et al., 2004). RNA extraction and quantitative RT-PCR RNA extraction and quantitative RT-PCR were performed as described previously (Ieda et al., 2004). The primers and probes for NGF were as used before (Ieda et al., 2004). Quantitative RT-PCR was performed using TaqMan probes (Applied Biosystems): BDNF (Rn01484928_m1), NT-3 (Rn00589280_m1), TrkA (Rn00572130_m1), TrkB (Rn00820626_m1), TrkC
(Rn00570389_m1), B-type natriuretic peptide (BNP, Rn00580641_m1), ET-1 (Rn00561129_m1), and leukemia inhibitory factor (LIF, Rn00573491_m1). The mRNA levels were normalized by comparison to GAPDH mRNA. Northern blot and poly(A)+-Northern blot analysis For Northern blot analysis, 20 μg of total RNAwas used, and for poly(A)+-Northern blot analysis, 2 μg of poly(A)+RNA was used, as described previously (Ieda et al., 2004, 2006). Rat NT-3, skeletal α-actin, atrial natriuretic peptide (ANP), BNP, and GAPDH cDNA were obtained from the heart by RT-PCR. In some experiments, K252a (20 μmol/l; Calbiochem), a specific inhibitor of TrkB and TrkC, was administered prior to NT-3 administration. Western blot analysis After 24 h of serum depletion, cardiomyocytes were stimulated with 10 ng/ml NT-3 for the times specified. To detect phosphorylation, the cell lysates were separated by 12.5% SDSPAGE as described previously (Pan et al., 1999). After transfer to nitrocellulose membranes, rabbit polyclonal antibodies against the phosphorylated forms of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, and Jun N-terminal kinase (JNK; New England Biolabs) were used. Signals were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Immunohistochemistry of hearts Hearts were perfused from the apex with 4% paraformaldehyde, fixed overnight, and then embedded in OCT compound and frozen in liquid nitrogen. Cryostat sections were stained with mouse monoclonal anti-α-actinin (Sigma Aldrich), rabbit polyclonal anti-NT-3 (Chemicon), and rabbit polyclonal anti-TrkC (Santa Cruz) antibodies. The sections were incubated with secondary antibodies conjugated with Alexa 488 and 594 (Molecular Probes) and the nuclei were stained with TOTO-3 (Molecular Probes). All confocal microscopy was carried out on a LSM 510 META (Carl Zeiss) (Ieda et al., 2006). Incorporation of [ 3 H]-phenylalanine into rat neonatal cardiomyocytes Cardiomyocytes were cultured on gelatin-coated 24-well plates, serum depleted for 24 h, and stimulated with various concentrations of NT-3 for 24 h. [3H]-phenylalanine was added at the same time as NT-3. After washing, [3H]-phenylalanine uptake was measured with a liquid scintillation counter, as described previously (Sano et al., 2000). Cell-sizing protocol After 24 h of serum depletion, primary-cultured cardiomyocytes were stimulated with NT-3 (10 ng/ml) and ET-1 (10− 7 M) for 48 h on glass coverslips, as described previously (Kodama et al., 2000). Cells were fixed in 4% paraformaldehyde for
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Fig. 1. The expression of neurotrophin and Trk family in the cardiovascular system. (A) Quantitative RT-PCR analysis of the neurotrophin family expression in the adult rat cardiovascular system (n = 5). (B) Quantitative RT-PCR analysis of the Trk family expression in the cardiovascular system. The same reverse transcription products used in A were analyzed (n = 5). (C) Triple-immunofluorescence staining for α-actinin, NT-3 and TOTO-3 for adult rat ventricles. (D) Tripleimmunofluorescence staining for α-actinin, TrkC and TOTO-3 for adult rat ventricles. Note that TrkC was exclusively expressed in cardiomyocytes but not in vasculature (arrows). Representative data are shown in each panel. ⁎P b.01; ns, not significant vs. relative control.
Fig. 2. NT-3 induces phosphorylation of ERK1/2, p38MAPK and JNK, and augments hypertrophy marker gene expressions in cardiomyocytes. (A) Western blot analysis of phosphorylated ERK1/2 of the cells stimulated with NT-3 for the indicated time. Note that NT-3 induced ERK phosphorylation. Each lane contained equal amounts of ERK1/2. (B) Western blot analysis of phosphorylated p38MAPK in cells stimulated with NT-3 for the indicated times. (C) Western blot analysis of phosphorylated JNK in cells stimulated with NT-3 for the indicated times. (D) Quantitative data of each Western blot after 30 min stimulation with NT-3 were shown (n = 4). NT-3-induced activation of ERK, p38MAPK, and JNK pathways were blocked by pretreatment with K252a (a specific inhibitor of TrkB and TrkC) (n = 4). (E) Cardiomyocytes were stimulated with NT-3 for 24 h, and Northern blot analysis for α-sk-actin and ANP was performed. Note that NT-3 increased mRNA expression of α-sk-actin and ANP in cardiomyocytes, and that K-252a pretreatment inhibited this augmentation. The lowest panel shows 18S RNA, an internal control for RNA loading. Representative data are shown in each panel. ⁎P b.01 vs. relative control.
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Fig. 3. NT-3 induces cardiomyocyte hypertrophy via TrkC. (A) [3H]-phenylalanine uptake was increased with NT-3 in a dose-dependent manner (n = 5). (B) Cardiomyocytes were stimulated with NT-3 in the presence or absence of K-252a. The NT-3-induced increase in [3H]-phenylalanine uptake was completely inhibited by K-252a pretreatment (n = 5). (C) Immunohistochemistry for α-actinin and TOTO-3 in cardiomyocytes. Note that the exposure to NT-3 for 48 h led to myofilament reorganization (NT-3). Preincubation of cells with K-252a strongly inhibited NT-3-induced reorganization of myofilaments (NT-3 + K-252a), and the cells were unaffected by K-252a treatment itself (K-252a). (D) Cell area and (E) cell perimeter of cardiomyocytes were quantitated by NIH image. NT-3 increased cell area and perimeter. Results are means ± SEM of 200 cells, and are representative of three separate experiments. ⁎P b.01; ⁎⁎P b.05; ns, not significant vs. relative control.
30 min, and were stained with mouse monoclonal antibody against α-actinin (Sigma Aldrich). Cells were incubated with secondary antibody conjugated with Alexa 594 (Molecular Probes) and the nuclei were stained with TOTO-3 (Molecular
Probes). The sizes (cell area and perimeter) of cardiomyocytes were determined using confocal microscopy (LSM 510 META, Carl Zeiss) and NIH image, as described previously (Ieda et al., 2006; Kodama et al., 2000).
Fig. 4. NT-3 expression is decreased in cardiac hypertrophy induced by pressure overload. (A) The ratios of heart weight to body weight were significantly increased at 1 and 7 days following the TAC operation. (B) The expression of NT-3 mRNA was determined by quantitative RT-PCR (n = 5). (C–E) The expression of mRNA for BNP (C), ET-1 (D) and LIF (E) were determined by quantitative RT-PCR. The analysis used the same reverse transcription products as in (B) (n = 5). ⁎P b.01; ⁎⁎P b.05 vs. relative control.
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Fig. 5. NT-3 supplementation augments ET-1-induced cardiomyocyte hypertrophy. (A) Cardiomyocytes were stimulated with ET-1 for the times indicated, and NT-3, BNP and GAPDH expression were analyzed by Northern blotting. ET-1 strongly inhibited NT-3 expression but augmented BNP expression. (B) Quantitative Northern blot data for NT-3 (n = 4). (C) Cardiomyocytes were stimulated with NT-3, ET-1 alone, and ET-1 plus two different concentrations of NT-3. NT(1) indicates 1 ng/ml NT3; NT(10), 10 ng/ml. BNP levels were determined by quantitative RT-PCR (n = 5). NT-3 supplementation significantly increased BNP expression in ET-1-stimulated cardiomyocytes. (D) Immunohistochemistry for α-actinin and TOTO-3 in cardiomyocytes treated with ET-1 alone or ET-1 plus NT-3 (10 ng/ml). Representative images are shown. Note that NT-3 supplementation led to further cardiomyocyte hypertrophy. (E and F) Cardiomyocyte area (E) and perimeter (F) were quantitated using NIH image. Cell area and perimeter were significantly greater in the ET-1 plus NT-3 (NT) group. Results are means ± SEM of 200 cells, and 3 separate experiments were performed. ⁎P b.01 vs. relative control (B); ⁎P b.01, ⁎⁎P b.05 (others).
Generation of left ventricular hypertrophy by transverse aortic constriction Eight-week-old Wistar rats were subjected to left ventricular pressure overload produced by transverse aortic constriction (TAC), as described previously (Butler et al., 2006). Control rats were subjected to sham operations, and the animals were studied 1 and 7 days following surgery. All experimental procedures and protocols were approved by the Animal Care and Use Committee of Keio University, Japan. Statistical analysis Values are presented as means ± SEM. Differences between groups were examined for statistical significance using the Student t test or ANOVA. P b.05 was considered significant. Results The expression of neurotrophin and Trk family in the rat cardiovascular system We first investigated the expression of neurotrophin and Trk mRNAs in the adult rat heart and thoracic aorta using quantitative RT-PCR. There were no significant differences between the aorta and heart in NGF, BDNF and NT-3 mRNA expression (Fig. 1A).
In contrast, Trk family members showed distinct expression patterns. Expression of TrkA and TrkB was strong in the aorta, but negligible in the heart. Expression of TrkC mRNA, however, was 10-fold more abundant in the heart than the aorta (Fig. 1B). The expression patterns of neurotrophin and Trk mRNAs in the neonatal rat cardiovascular system are similar to those of the adult rat (data not shown). To examine the expression patterns of NT-3 and TrkC, we performed triple immunostaining with mouse monoclonal αactinin (a marker of cardiomyocytes), rabbit polyclonal NT-3 or TrkC antibodies, and TOTO-3 (nuclei) in adult rat ventricles. NT-3 was expressed in both cardiomyocytes and vessels, but TrkC was exclusively expressed in cardiomyocytes (Fig. 1C,D). These findings indicated that NT-3 and TrkC were abundantly expressed in the rat heart. NT-3 induces MAPK activation and mRNA expression of cardiac hypertrophy markers in cardiomyocytes To investigate the intracellular signaling of NT-3/TrkC in cardiomyocytes, we stimulated primary-cultured cardiomyocytes with NT-3, and analyzed the activation of mitogenactivated protein kinase (MAPK) pathways. NT-3 significantly induced phosphorylation of ERK1/2, p38MAPK, and JNK in cardiomyocytes, with the peak in each case being reached after 15 to 30 min (Fig. 2A–D). Preincubation with K-252a
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completely inhibited MAPK phosphorylation after 30 min of NT-3 stimulation (Fig. 2D), suggesting that TrkC mediates NT3-induced MAPK activation in cardiomyocytes. Northern blot analysis demonstrated that NT-3 increased the expression in cardiomyocytes of the hypertrophy marker genes, skeletal αactin and ANP, and that K-252a pretreatment inhibited this augmentation (Fig. 2E). These findings suggested that NT-3/ TrkC activates signal transduction pathways involved in cardiac hypertrophy. NT-3 induces cardiomyocyte hypertrophy via TrkC To determine the effects of NT-3 on cardiomyocytes, we first performed [3H]-phenylalanine uptake analysis. NT-3 increased [3H]-phenylalanine uptake into primary-cultured cardiomyocytes in a dose-dependent manner (Fig. 3A). The maximal increase in uptake was 47% over control at an NT-3 concentration of 10 ng/ml. Preincubation with K-252a (a specific inhibitor of TrkB and TrkC) inhibited NT-3-induced [3H]-phenylalanine uptake (Fig. 3B). Given that TrkB expression in primary-cultured cardiomyocytes was below the level detectable by quantitative RT-PCR (data not shown), these results suggested that TrkC mediates NT-3-induced protein synthesis in cardiomyocytes. Myofilament reorganization is another marker of cardiac hypertrophy. Immunohistochemistry for sarcomeric actin clearly showed that the exposure to NT-3 for 48 h led to myofilament reorganization in cardiomyocytes. Preincubation of cells with K-252a inhibited NT-3-induced reorganization of myofilaments, and the cells were unaffected by K-252a treatment itself (Fig. 3C). NT-3 increased the surface area of cardiomyocytes by 62% and the perimeter by 37%; these effects were specifically blocked by K-252a preincubation (Fig. 3D,E). These results strongly suggested that NT-3 induces cardiomyocyte hypertrophy via TrkC. NT-3 expression is downregulated in cardiac hypertrophy induced by pressure overload Cardiac hypertrophic factors (e.g., ET-1, LIF, and angiotensin II) are usually upregulated in cardiac hypertrophy, leading to a vicious cycle of deterioration in cardiac function (Kim et al., 2001; Sakai et al., 1996; Willenheimer et al., 2005). Given the apparent role of NT-3 as a cardiac hypertrophic factor, we postulated that it would also be increased in cardiac hypertrophy. To address this, we induced left ventricular hypertrophy in 8-week-old rats by TAC, and analyzed cardiac NT-3 expression at 1 and 7 days following surgery. The ratio of heart weight to body weight was significantly increased in a time-dependent manner (Fig. 4A). Unexpectedly, quantitative RT-PCR analysis demonstrated that NT-3 expression was significantly reduced (by 71%) in pressure-overloaded hearts after 7 days of TAC (Fig. 4B). In contrast, BNP, ET-1 and LIF mRNA expression was increased by TAC (Fig. 4C–E). These results showed that NT-3, unlike other hypertrophic factors, is paradoxically decreased in cardiac hypertrophy induced by pressure overload.
NT-3 supplementation exaggerates cardiomyocyte hypertrophy induced by ET-1 To determine whether NT-3 expression is reduced following other hypertrophic stimuli, we used Northern blot analysis to determine NT-3 expression in primary-cultured cardiomyocytes treated with ET-1. NT-3 mRNA expression was strongly suppressed by ET-1. The suppression was apparent with 2 h of exposure and was maintained at 24 h (Fig. 5A, B). In contrast, BNP mRNA expression was increased in ET-1stimulated cardiomyocytes (Fig. 5A). We then investigated whether application of NT-3 augmented the hypertrophy induced by ET-1 in cardiomyocytes. Cardiomyocytes were treated with NT-3, ET-1 alone, ET-1 plus 1 ng/ml or 10 ng/ml NT-3. BNP expression was significantly greater in cardiomyocytes treated with both NT-3 and ET-1 than in those stimulated with ET-1 alone, and the relationship was dose-dependent (Fig. 5C). In cardiomyocytes that received combined treatment (ET-1 plus 10 ng/ml NT-3), the cell surface area was 121% and the perimeter was 113% that of the cardiomyocytes treated with ET-1 alone (Fig. 5D–F). These results suggest that cardiac hypertrophy would be aggravated by any increase in expression of NT-3. The suppression of endogenous NT-3 expression in ET-1-stimulated cardiomyocytes might therefore represent a response that counteracts hypertrophy. Discussion In this study we found that NT-3/TrkC was highly expressed in the rat heart, and that it acted as a novel ligand/receptor system to induce cardiomyocyte hypertrophy. Interestingly, cardiac NT-3 expression was paradoxically reduced in cardiac hypertrophy induced by either pressure overload or ET-1. Moreover, NT-3 supplementation in ET-1-stimulated cardiomyocytes had a further negative impact on cardiac hypertrophy. TrkC was expressed more strongly in heart than in blood vessels, and our results strongly suggest it mediated the cardiomyocyte hypertrophy induced by NT-3. In contrast, TrkA and TrkB were more abundantly expressed in the aorta. Consistent with our findings, TrkA and TrkB are highly expressed in vascular smooth muscle cells, and NGF and BDNF (their ligands) are aberrantly upregulated in intimal lesions and promote vascular smooth muscle cell migration in atherosclerosis (Kraemer et al., 1999, 2005). Together, these findings suggest that NT-3/TrkC plays a critical role in cardiac performance, whereas NGF/TrkA and BDNF/TrkB are important in the vasculature. Trk is a member of the tyrosine kinase receptor family, which includes epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) receptors. We and others previously reported that the expression of their ligands, the EGF family and IGF-1, is increased in cardiac hypertrophy, and that the consequent activation of MAPK and phosphoinositide 3-kinase (PI3K) pathways in cardiomyocytes leads to further cardiac hypertrophy (Fujino et al., 1998; Kodama et al., 2002; Takahashi et al., 1999). Consistent with these results, we found in this study that
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NT-3/TrkC signaling stimulated MAPK pathways and induced cardiomyocyte hypertrophy in vitro. Given that cardiac NT-3 expression increases with development after birth (Ieda et al., 2006), and that NT-3 is a potent cardiac hypertrophic factor, NT3/TrkC signaling might be involved in physiological cardiac growth. By comparison, IGF-1 and its downstream signals, PI3K and Akt, promote physiological cardiac hypertrophy and determine the heart size (DeBosch et al., 2006; Shioi et al., 2000). In cardiac hypertrophy, the expression of G protein-coupled receptor ligands (e.g., ET-1, angiotensin II, and catecholamines) is increased, in addition to the changes in tyrosine kinase receptor ligands. Cardioprotective factors such as ANP and BNP are also elevated under pathological conditions. In clinical medicine, angiotensin II receptor antagonists and β-blocker therapy are known to improve the prognosis of heart failure by preventing cardiac remodeling and ventricular dysfunction (Kim et al., 2001; Sakai et al., 1996; Willenheimer et al., 2005). ANP and BNP are also used to treat congestive heart failure (Woods, 2004). Our new findings that a potent cardiac hypertrophic factor, NT-3, was reduced in cardiac hypertrophy, and that NT-3 supplementation further exaggerated ET-1induced cardiomyocyte hypertrophy, indicates the potential for downregulation of NT-3/TrkC signaling to be protective against cardiac hypertrophy. In compensated cardiac hypertrophy, as occurs in hypertensive heart disease, hypertrophic factors are upregulated to counteract pressure overload. Therefore, it is also possible that NT-3 downregulation might adversely affect cardiac function in the early phases of cardiac hypertrophy. Further in vivo experiments are required to address the functional significance of NT-3 downregulation in pathological hearts. To our knowledge, this is the first report demonstrating that a cardiac hypertrophic factor is paradoxically downregulated in cardiac hypertrophy. It will be also intriguing to determine the regulatory mechanism of NT-3 expression in the heart. Expression of mRNA for NT-3, but not NGF or BDNF, is downregulated in primary-cultured vascular smooth muscle cells treated with serum or phorbor ester (Nemoto et al., 1998). Activating protein-1 (AP-1), a downstream target of serum and phorbor ester, is critically involved in this transcriptional suppression (Nemoto et al., 1998). Given that AP-1 is an immediate early gene and is strongly upregulated in various types of cardiac hypertrophy (Osaki et al., 1997), AP-1 upregulation might mediate NT-3 reduction in cardiac hypertrophy. In conclusion, our results indicate that NT-3/TrkC signaling plays a key role in cardiac hypertrophy. Knowledge of this novel cardiac hypertrophic factor might represent a new step toward potential therapies for heart failure. Acknowledgments This study was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation.
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Tessarollo, L., Tsoulfas, P., Donovan, M.J., Palko, M.E., Blair-Flynn, J., Hempstead, B.L., Parada, L.F., 1997. Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis. Proceedings of the National Academy of Sciences of the United States of America 94, 14776–14781. Willenheimer, R., van Veldhuisen, D.J., Silke, B., Erdmann, E., Follath, F., Krum, H., Ponikowski, P., Skene, A., van de Ven, L., Verkenne, P., Lechat, P., 2005. Effect on survival and hospitalization of initiating treatment for chronic heart failure with bisoprolol followed by enalapril, as compared with the opposite sequence: results of the randomized Cardiac Insufficiency Bisoprolol Study (CIBIS) III. Circulation 112, 2426–2435. Woods, R.L., 2004. Cardioprotective functions of atrial natriuretic peptide and B-type natriuretic peptide: a brief review. Clinical and Experimental Pharmacology and Physiology 31, 791–794.
A novel cardiac hypertrophic factor, neurotrophin-3, is paradoxically downregulated in cardiac hypertrophy Haruko Kawaguchi-Manabe a,c,1 , Masaki Ieda a,b,1 , Kensuke Kimura a,b , Tomohiro Manabe a,b , Satoru Miyatake a,d , Hideaki Kanazawa a,b , Takashi Kawakami a,b , Satoshi Ogawa b , Makoto Suematsu c , Keiichi Fukuda a,⁎ a
c
Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b Cardiology Division, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Biochemistry and Integrative Medical Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan d Department of Emergency and Critical Care Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Received 5 February 2007; accepted 29 May 2007
Abstract The neurotrophin family plays pivotal roles in the development of the nervous system. Recently, the role of the neurotrophin in non-neural tissue has been extensively investigated. Among them, neurotrophin-3 and its receptor TrkC are critical for embryonic heart development, though little is known about neurotrophin-3/TrkC function in adult heart. Moreover, the expressions of other neurotrophin and Trk families in the cardiovascular system have not been fully determined. In adult and neonatal rats, only TrkC mRNA was expressed more abundantly in heart than aorta among the neurotrophin receptors, while all neurotrophins were equally expressed in the cardiovascular system. Immunohistochemistry confirmed the protein expressions of neurotrophin-3/TrkC in rat ventricles. In primary-cultured rat cardiomyocytes, neurotrophin-3 strongly activated p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Jun N-terminal kinase pathways in Western blot analysis. In Northern blot analysis, neurotrophin-3 strongly increased mRNA expressions of cardiac hypertrophic markers (skeletal α-actin and atrial natriuretic peptide) in cardiomocytes. [3H]-phenylalanine uptake into cardiomyocytes, myofilament reorganization, and cardiomyocyte size were also augmented with neurotrophin-3 stimulation, indicating that neurotrophin-3 is a novel cardiac hypertrophic factor. Unexpectedly, neurotrophin-3 was downregulated in cardiac hypertrophy induced by pressure overload (in vivo), and in cardiomyocyte hypertrophy evoked by endothelin-1 stimulation (in vitro). Interestingly, the cell size and BNP mRNA expression level (markers of hypertrophy) were greater in cardiomyocytes treated with both neurotrophin-3 and endothelin-1 than in those stimulated with endothelin-1 alone. These findings demonstrate that neurotrophin-3 is a unique hypertrophic factor, which is paradoxically downregulated in cardiac hypertrophy and might counteract hypertrophic change. © 2007 Elsevier Inc. All rights reserved. Keywords: Neurotrophin-3; TrkC; Cardiomyocyte; Heart; Cardiac hypertrophy
Introduction The neurotrophin family of growth factors plays pivotal roles in the development of the central and peripheral nervous system
⁎ Corresponding author. Tel.: +81 3 5363 3874; fax: +81 3 5363 3875. E-mail address: [email protected] (K. Fukuda). 1 These authors contributed equally to this study. 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.05.024
(Snider, 1994). This family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, and NT-4/5 (Snider, 1994). Neurotrophins selectively bind and activate distinct members of the Trk receptor tyrosine kinase family, which are highly expressed in the central and peripheral nervous systems (NGF activates TrkA, BDNF and NT-4/5 activate TrkB, and NT-3 activates TrkC) (Segal, 2003). We recently found that NGF is abundantly expressed in the heart, where it regulates cardiac sympathetic and sensory
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innervation (Ieda et al., 2004, 2006). Downregulation of cardiac NGF causes sensory denervation in diabetic hearts, and NGF gene therapy rescues this neuropathy (Ieda et al., 2006). BDNF/TrkB signaling is critical for embryonic vascular development, and it also promotes atherosclerosis in ApoE-null mutant mice (Donovan et al., 2000; Kraemer et al., 2005). In embryos, NT-3/ TrkC signaling promotes cardiomyocyte proliferation and is required for ventricular trabeculation (Lin et al., 2000). NT-3 and TrkC gene-targeted mice exhibit early postnatal mortality and have defects in cardiac septation, valvulogenesis, and conotruncal formation (Donovan et al., 1996; Tessarollo et al., 1997). Although these results demonstrate the critical roles of the neurotrophin family in cardiovascular regulation during embryogenesis and adulthood, the expression levels of the neurotrophin and Trk families in the cardiovascular system have not been fully characterized. Moreover, the functions of NT-3 and TrkC in adult hearts and their regulation in pathological hearts remain unknown. Cardiac hypertrophy is a pathological process that can lead to congestive heart failure. Progression towards heart failure is influenced by the balance of hypertrophic and cardioprotective factors. Sustained overexpression of hypertrophic factors in the heart results in cardiac remodeling and ventricular dysfunction (Kim et al., 2001; Sakai et al., 1996; Willenheimer et al., 2005). Despite their importance, our knowledge about cardiac hypertrophic factors remains limited. In this study we found that NGF, BDNF, and NT-3 were highly expressed in the rat heart and vasculature. In contrast, TrkC (but not TrkA or TrkB) was expressed more abundantly in hearts than in blood vessels. NT-3 strongly induced cardiomyocyte hypertrophy via TrkC, but unexpectedly, NT-3 expression was paradoxically reduced in cardiac hypertrophy models both in vivo and in vitro. NT-3 supplementation augmented endothelin-1-induced cardiomyocyte hypertrophy, suggesting that NT-3 downregulation may counteract cardiac hypertrophy. Materials and methods Cell culture Primary cultures of cardiomyocytes were prepared from the ventricles of 1-day-old neonatal Wistar rats (Japan CLEA) as described previously (Pan et al., 1999). Cardiomyocytes were isolated from other cell types by differential adhesion. Cells were seeded at a density of 5 × 105 cells/cm2, and were incubated in media containing serum for 24 h. The media was then replaced with fresh serum-free media, and the cells were stimulated with human recombinant NT-3 (Calbiochem) or 10− 7 M endothelin-1 (ET-1, Sigma) (Ieda et al., 2004). RNA extraction and quantitative RT-PCR RNA extraction and quantitative RT-PCR were performed as described previously (Ieda et al., 2004). The primers and probes for NGF were as used before (Ieda et al., 2004). Quantitative RT-PCR was performed using TaqMan probes (Applied Biosystems): BDNF (Rn01484928_m1), NT-3 (Rn00589280_m1), TrkA (Rn00572130_m1), TrkB (Rn00820626_m1), TrkC
(Rn00570389_m1), B-type natriuretic peptide (BNP, Rn00580641_m1), ET-1 (Rn00561129_m1), and leukemia inhibitory factor (LIF, Rn00573491_m1). The mRNA levels were normalized by comparison to GAPDH mRNA. Northern blot and poly(A)+-Northern blot analysis For Northern blot analysis, 20 μg of total RNAwas used, and for poly(A)+-Northern blot analysis, 2 μg of poly(A)+RNA was used, as described previously (Ieda et al., 2004, 2006). Rat NT-3, skeletal α-actin, atrial natriuretic peptide (ANP), BNP, and GAPDH cDNA were obtained from the heart by RT-PCR. In some experiments, K252a (20 μmol/l; Calbiochem), a specific inhibitor of TrkB and TrkC, was administered prior to NT-3 administration. Western blot analysis After 24 h of serum depletion, cardiomyocytes were stimulated with 10 ng/ml NT-3 for the times specified. To detect phosphorylation, the cell lysates were separated by 12.5% SDSPAGE as described previously (Pan et al., 1999). After transfer to nitrocellulose membranes, rabbit polyclonal antibodies against the phosphorylated forms of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, and Jun N-terminal kinase (JNK; New England Biolabs) were used. Signals were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Immunohistochemistry of hearts Hearts were perfused from the apex with 4% paraformaldehyde, fixed overnight, and then embedded in OCT compound and frozen in liquid nitrogen. Cryostat sections were stained with mouse monoclonal anti-α-actinin (Sigma Aldrich), rabbit polyclonal anti-NT-3 (Chemicon), and rabbit polyclonal anti-TrkC (Santa Cruz) antibodies. The sections were incubated with secondary antibodies conjugated with Alexa 488 and 594 (Molecular Probes) and the nuclei were stained with TOTO-3 (Molecular Probes). All confocal microscopy was carried out on a LSM 510 META (Carl Zeiss) (Ieda et al., 2006). Incorporation of [ 3 H]-phenylalanine into rat neonatal cardiomyocytes Cardiomyocytes were cultured on gelatin-coated 24-well plates, serum depleted for 24 h, and stimulated with various concentrations of NT-3 for 24 h. [3H]-phenylalanine was added at the same time as NT-3. After washing, [3H]-phenylalanine uptake was measured with a liquid scintillation counter, as described previously (Sano et al., 2000). Cell-sizing protocol After 24 h of serum depletion, primary-cultured cardiomyocytes were stimulated with NT-3 (10 ng/ml) and ET-1 (10− 7 M) for 48 h on glass coverslips, as described previously (Kodama et al., 2000). Cells were fixed in 4% paraformaldehyde for
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Fig. 1. The expression of neurotrophin and Trk family in the cardiovascular system. (A) Quantitative RT-PCR analysis of the neurotrophin family expression in the adult rat cardiovascular system (n = 5). (B) Quantitative RT-PCR analysis of the Trk family expression in the cardiovascular system. The same reverse transcription products used in A were analyzed (n = 5). (C) Triple-immunofluorescence staining for α-actinin, NT-3 and TOTO-3 for adult rat ventricles. (D) Tripleimmunofluorescence staining for α-actinin, TrkC and TOTO-3 for adult rat ventricles. Note that TrkC was exclusively expressed in cardiomyocytes but not in vasculature (arrows). Representative data are shown in each panel. ⁎P b.01; ns, not significant vs. relative control.
Fig. 2. NT-3 induces phosphorylation of ERK1/2, p38MAPK and JNK, and augments hypertrophy marker gene expressions in cardiomyocytes. (A) Western blot analysis of phosphorylated ERK1/2 of the cells stimulated with NT-3 for the indicated time. Note that NT-3 induced ERK phosphorylation. Each lane contained equal amounts of ERK1/2. (B) Western blot analysis of phosphorylated p38MAPK in cells stimulated with NT-3 for the indicated times. (C) Western blot analysis of phosphorylated JNK in cells stimulated with NT-3 for the indicated times. (D) Quantitative data of each Western blot after 30 min stimulation with NT-3 were shown (n = 4). NT-3-induced activation of ERK, p38MAPK, and JNK pathways were blocked by pretreatment with K252a (a specific inhibitor of TrkB and TrkC) (n = 4). (E) Cardiomyocytes were stimulated with NT-3 for 24 h, and Northern blot analysis for α-sk-actin and ANP was performed. Note that NT-3 increased mRNA expression of α-sk-actin and ANP in cardiomyocytes, and that K-252a pretreatment inhibited this augmentation. The lowest panel shows 18S RNA, an internal control for RNA loading. Representative data are shown in each panel. ⁎P b.01 vs. relative control.
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Fig. 3. NT-3 induces cardiomyocyte hypertrophy via TrkC. (A) [3H]-phenylalanine uptake was increased with NT-3 in a dose-dependent manner (n = 5). (B) Cardiomyocytes were stimulated with NT-3 in the presence or absence of K-252a. The NT-3-induced increase in [3H]-phenylalanine uptake was completely inhibited by K-252a pretreatment (n = 5). (C) Immunohistochemistry for α-actinin and TOTO-3 in cardiomyocytes. Note that the exposure to NT-3 for 48 h led to myofilament reorganization (NT-3). Preincubation of cells with K-252a strongly inhibited NT-3-induced reorganization of myofilaments (NT-3 + K-252a), and the cells were unaffected by K-252a treatment itself (K-252a). (D) Cell area and (E) cell perimeter of cardiomyocytes were quantitated by NIH image. NT-3 increased cell area and perimeter. Results are means ± SEM of 200 cells, and are representative of three separate experiments. ⁎P b.01; ⁎⁎P b.05; ns, not significant vs. relative control.
30 min, and were stained with mouse monoclonal antibody against α-actinin (Sigma Aldrich). Cells were incubated with secondary antibody conjugated with Alexa 594 (Molecular Probes) and the nuclei were stained with TOTO-3 (Molecular
Probes). The sizes (cell area and perimeter) of cardiomyocytes were determined using confocal microscopy (LSM 510 META, Carl Zeiss) and NIH image, as described previously (Ieda et al., 2006; Kodama et al., 2000).
Fig. 4. NT-3 expression is decreased in cardiac hypertrophy induced by pressure overload. (A) The ratios of heart weight to body weight were significantly increased at 1 and 7 days following the TAC operation. (B) The expression of NT-3 mRNA was determined by quantitative RT-PCR (n = 5). (C–E) The expression of mRNA for BNP (C), ET-1 (D) and LIF (E) were determined by quantitative RT-PCR. The analysis used the same reverse transcription products as in (B) (n = 5). ⁎P b.01; ⁎⁎P b.05 vs. relative control.
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Fig. 5. NT-3 supplementation augments ET-1-induced cardiomyocyte hypertrophy. (A) Cardiomyocytes were stimulated with ET-1 for the times indicated, and NT-3, BNP and GAPDH expression were analyzed by Northern blotting. ET-1 strongly inhibited NT-3 expression but augmented BNP expression. (B) Quantitative Northern blot data for NT-3 (n = 4). (C) Cardiomyocytes were stimulated with NT-3, ET-1 alone, and ET-1 plus two different concentrations of NT-3. NT(1) indicates 1 ng/ml NT3; NT(10), 10 ng/ml. BNP levels were determined by quantitative RT-PCR (n = 5). NT-3 supplementation significantly increased BNP expression in ET-1-stimulated cardiomyocytes. (D) Immunohistochemistry for α-actinin and TOTO-3 in cardiomyocytes treated with ET-1 alone or ET-1 plus NT-3 (10 ng/ml). Representative images are shown. Note that NT-3 supplementation led to further cardiomyocyte hypertrophy. (E and F) Cardiomyocyte area (E) and perimeter (F) were quantitated using NIH image. Cell area and perimeter were significantly greater in the ET-1 plus NT-3 (NT) group. Results are means ± SEM of 200 cells, and 3 separate experiments were performed. ⁎P b.01 vs. relative control (B); ⁎P b.01, ⁎⁎P b.05 (others).
Generation of left ventricular hypertrophy by transverse aortic constriction Eight-week-old Wistar rats were subjected to left ventricular pressure overload produced by transverse aortic constriction (TAC), as described previously (Butler et al., 2006). Control rats were subjected to sham operations, and the animals were studied 1 and 7 days following surgery. All experimental procedures and protocols were approved by the Animal Care and Use Committee of Keio University, Japan. Statistical analysis Values are presented as means ± SEM. Differences between groups were examined for statistical significance using the Student t test or ANOVA. P b.05 was considered significant. Results The expression of neurotrophin and Trk family in the rat cardiovascular system We first investigated the expression of neurotrophin and Trk mRNAs in the adult rat heart and thoracic aorta using quantitative RT-PCR. There were no significant differences between the aorta and heart in NGF, BDNF and NT-3 mRNA expression (Fig. 1A).
In contrast, Trk family members showed distinct expression patterns. Expression of TrkA and TrkB was strong in the aorta, but negligible in the heart. Expression of TrkC mRNA, however, was 10-fold more abundant in the heart than the aorta (Fig. 1B). The expression patterns of neurotrophin and Trk mRNAs in the neonatal rat cardiovascular system are similar to those of the adult rat (data not shown). To examine the expression patterns of NT-3 and TrkC, we performed triple immunostaining with mouse monoclonal αactinin (a marker of cardiomyocytes), rabbit polyclonal NT-3 or TrkC antibodies, and TOTO-3 (nuclei) in adult rat ventricles. NT-3 was expressed in both cardiomyocytes and vessels, but TrkC was exclusively expressed in cardiomyocytes (Fig. 1C,D). These findings indicated that NT-3 and TrkC were abundantly expressed in the rat heart. NT-3 induces MAPK activation and mRNA expression of cardiac hypertrophy markers in cardiomyocytes To investigate the intracellular signaling of NT-3/TrkC in cardiomyocytes, we stimulated primary-cultured cardiomyocytes with NT-3, and analyzed the activation of mitogenactivated protein kinase (MAPK) pathways. NT-3 significantly induced phosphorylation of ERK1/2, p38MAPK, and JNK in cardiomyocytes, with the peak in each case being reached after 15 to 30 min (Fig. 2A–D). Preincubation with K-252a
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completely inhibited MAPK phosphorylation after 30 min of NT-3 stimulation (Fig. 2D), suggesting that TrkC mediates NT3-induced MAPK activation in cardiomyocytes. Northern blot analysis demonstrated that NT-3 increased the expression in cardiomyocytes of the hypertrophy marker genes, skeletal αactin and ANP, and that K-252a pretreatment inhibited this augmentation (Fig. 2E). These findings suggested that NT-3/ TrkC activates signal transduction pathways involved in cardiac hypertrophy. NT-3 induces cardiomyocyte hypertrophy via TrkC To determine the effects of NT-3 on cardiomyocytes, we first performed [3H]-phenylalanine uptake analysis. NT-3 increased [3H]-phenylalanine uptake into primary-cultured cardiomyocytes in a dose-dependent manner (Fig. 3A). The maximal increase in uptake was 47% over control at an NT-3 concentration of 10 ng/ml. Preincubation with K-252a (a specific inhibitor of TrkB and TrkC) inhibited NT-3-induced [3H]-phenylalanine uptake (Fig. 3B). Given that TrkB expression in primary-cultured cardiomyocytes was below the level detectable by quantitative RT-PCR (data not shown), these results suggested that TrkC mediates NT-3-induced protein synthesis in cardiomyocytes. Myofilament reorganization is another marker of cardiac hypertrophy. Immunohistochemistry for sarcomeric actin clearly showed that the exposure to NT-3 for 48 h led to myofilament reorganization in cardiomyocytes. Preincubation of cells with K-252a inhibited NT-3-induced reorganization of myofilaments, and the cells were unaffected by K-252a treatment itself (Fig. 3C). NT-3 increased the surface area of cardiomyocytes by 62% and the perimeter by 37%; these effects were specifically blocked by K-252a preincubation (Fig. 3D,E). These results strongly suggested that NT-3 induces cardiomyocyte hypertrophy via TrkC. NT-3 expression is downregulated in cardiac hypertrophy induced by pressure overload Cardiac hypertrophic factors (e.g., ET-1, LIF, and angiotensin II) are usually upregulated in cardiac hypertrophy, leading to a vicious cycle of deterioration in cardiac function (Kim et al., 2001; Sakai et al., 1996; Willenheimer et al., 2005). Given the apparent role of NT-3 as a cardiac hypertrophic factor, we postulated that it would also be increased in cardiac hypertrophy. To address this, we induced left ventricular hypertrophy in 8-week-old rats by TAC, and analyzed cardiac NT-3 expression at 1 and 7 days following surgery. The ratio of heart weight to body weight was significantly increased in a time-dependent manner (Fig. 4A). Unexpectedly, quantitative RT-PCR analysis demonstrated that NT-3 expression was significantly reduced (by 71%) in pressure-overloaded hearts after 7 days of TAC (Fig. 4B). In contrast, BNP, ET-1 and LIF mRNA expression was increased by TAC (Fig. 4C–E). These results showed that NT-3, unlike other hypertrophic factors, is paradoxically decreased in cardiac hypertrophy induced by pressure overload.
NT-3 supplementation exaggerates cardiomyocyte hypertrophy induced by ET-1 To determine whether NT-3 expression is reduced following other hypertrophic stimuli, we used Northern blot analysis to determine NT-3 expression in primary-cultured cardiomyocytes treated with ET-1. NT-3 mRNA expression was strongly suppressed by ET-1. The suppression was apparent with 2 h of exposure and was maintained at 24 h (Fig. 5A, B). In contrast, BNP mRNA expression was increased in ET-1stimulated cardiomyocytes (Fig. 5A). We then investigated whether application of NT-3 augmented the hypertrophy induced by ET-1 in cardiomyocytes. Cardiomyocytes were treated with NT-3, ET-1 alone, ET-1 plus 1 ng/ml or 10 ng/ml NT-3. BNP expression was significantly greater in cardiomyocytes treated with both NT-3 and ET-1 than in those stimulated with ET-1 alone, and the relationship was dose-dependent (Fig. 5C). In cardiomyocytes that received combined treatment (ET-1 plus 10 ng/ml NT-3), the cell surface area was 121% and the perimeter was 113% that of the cardiomyocytes treated with ET-1 alone (Fig. 5D–F). These results suggest that cardiac hypertrophy would be aggravated by any increase in expression of NT-3. The suppression of endogenous NT-3 expression in ET-1-stimulated cardiomyocytes might therefore represent a response that counteracts hypertrophy. Discussion In this study we found that NT-3/TrkC was highly expressed in the rat heart, and that it acted as a novel ligand/receptor system to induce cardiomyocyte hypertrophy. Interestingly, cardiac NT-3 expression was paradoxically reduced in cardiac hypertrophy induced by either pressure overload or ET-1. Moreover, NT-3 supplementation in ET-1-stimulated cardiomyocytes had a further negative impact on cardiac hypertrophy. TrkC was expressed more strongly in heart than in blood vessels, and our results strongly suggest it mediated the cardiomyocyte hypertrophy induced by NT-3. In contrast, TrkA and TrkB were more abundantly expressed in the aorta. Consistent with our findings, TrkA and TrkB are highly expressed in vascular smooth muscle cells, and NGF and BDNF (their ligands) are aberrantly upregulated in intimal lesions and promote vascular smooth muscle cell migration in atherosclerosis (Kraemer et al., 1999, 2005). Together, these findings suggest that NT-3/TrkC plays a critical role in cardiac performance, whereas NGF/TrkA and BDNF/TrkB are important in the vasculature. Trk is a member of the tyrosine kinase receptor family, which includes epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) receptors. We and others previously reported that the expression of their ligands, the EGF family and IGF-1, is increased in cardiac hypertrophy, and that the consequent activation of MAPK and phosphoinositide 3-kinase (PI3K) pathways in cardiomyocytes leads to further cardiac hypertrophy (Fujino et al., 1998; Kodama et al., 2002; Takahashi et al., 1999). Consistent with these results, we found in this study that
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NT-3/TrkC signaling stimulated MAPK pathways and induced cardiomyocyte hypertrophy in vitro. Given that cardiac NT-3 expression increases with development after birth (Ieda et al., 2006), and that NT-3 is a potent cardiac hypertrophic factor, NT3/TrkC signaling might be involved in physiological cardiac growth. By comparison, IGF-1 and its downstream signals, PI3K and Akt, promote physiological cardiac hypertrophy and determine the heart size (DeBosch et al., 2006; Shioi et al., 2000). In cardiac hypertrophy, the expression of G protein-coupled receptor ligands (e.g., ET-1, angiotensin II, and catecholamines) is increased, in addition to the changes in tyrosine kinase receptor ligands. Cardioprotective factors such as ANP and BNP are also elevated under pathological conditions. In clinical medicine, angiotensin II receptor antagonists and β-blocker therapy are known to improve the prognosis of heart failure by preventing cardiac remodeling and ventricular dysfunction (Kim et al., 2001; Sakai et al., 1996; Willenheimer et al., 2005). ANP and BNP are also used to treat congestive heart failure (Woods, 2004). Our new findings that a potent cardiac hypertrophic factor, NT-3, was reduced in cardiac hypertrophy, and that NT-3 supplementation further exaggerated ET-1induced cardiomyocyte hypertrophy, indicates the potential for downregulation of NT-3/TrkC signaling to be protective against cardiac hypertrophy. In compensated cardiac hypertrophy, as occurs in hypertensive heart disease, hypertrophic factors are upregulated to counteract pressure overload. Therefore, it is also possible that NT-3 downregulation might adversely affect cardiac function in the early phases of cardiac hypertrophy. Further in vivo experiments are required to address the functional significance of NT-3 downregulation in pathological hearts. To our knowledge, this is the first report demonstrating that a cardiac hypertrophic factor is paradoxically downregulated in cardiac hypertrophy. It will be also intriguing to determine the regulatory mechanism of NT-3 expression in the heart. Expression of mRNA for NT-3, but not NGF or BDNF, is downregulated in primary-cultured vascular smooth muscle cells treated with serum or phorbor ester (Nemoto et al., 1998). Activating protein-1 (AP-1), a downstream target of serum and phorbor ester, is critically involved in this transcriptional suppression (Nemoto et al., 1998). Given that AP-1 is an immediate early gene and is strongly upregulated in various types of cardiac hypertrophy (Osaki et al., 1997), AP-1 upregulation might mediate NT-3 reduction in cardiac hypertrophy. In conclusion, our results indicate that NT-3/TrkC signaling plays a key role in cardiac hypertrophy. Knowledge of this novel cardiac hypertrophic factor might represent a new step toward potential therapies for heart failure. Acknowledgments This study was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation.
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