Published online before print November 20, 2008, doi: 10.1161/CIRCRESAHA.108.180976
© 2009 American Heart Association, Inc.
Integrative Physiology |
Serotonin and Angiotensin Receptors in Cardiac Fibroblasts Coregulate Adrenergic-Dependent Cardiac Hypertrophy
From Institut National de la Santé et de la Recherche Médicale, U839 (F.J., V.S., S.D., L.M.), Paris, France; Unité Mixte de Recherche S0839 (F.J., V.S., S.D., L.M.), Université Pierre et Marie Curie Paris 6, France; Institut du Fer à Moulin (F.J., V.S., S.D., L.M.), Paris, France; Service de Physiologie–Explorations Fonctionnelles (P.B., H.D.), Assistance Publique–Hôpitaux de Paris, Hôpital Lariboisière, Université Denis Diderot Paris 7, France; Institut National de la Santé et de la Recherche Médicale, U689 (P.B., H.D.), Centre de Recherche Cardiovasculaire, Hôpital Lariboisière, Paris, France; Service de Biochimie (J.C., J.-M.L.), Assistance Publique–Hôpitaux de Paris, Hôpital Lariboisière, France; EA3621 (J.C., J.-M.L.), IFR71, Paris, France; Institut National de la Santé et de la Recherche Médicale, U715 (L.M.), Faculté de Médecine, Strasbourg, France; Service de Cardiologie (B.M.), Hôpital de Colmar, France; and Cardiovascular Research Institute (B.C.B.), Aab Cardiovascular Institute, University of Rochester School of Medicine, New York.
Correspondence to Luc Maroteaux INSERM, U839, Institut du Fer à Moulin, 17 rue du Fer à Moulin, 75005 Paris, France. E-mail luc.maroteaux{at}chups.jussieu.fr
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Abstract |
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By mimicking sympathetic stimulation in vivo, we previously reported that mice globally lacking serotonin 5-HT2B receptors did not develop isoproterenol-induced left ventricular hypertrophy. However, the exact cardiac cell type(s) expressing 5-HT2B receptors (cardiomyocytes versus noncardiomyocytes) involved in pathological heart hypertrophy was never addressed in vivo. We report here that mice expressing the 5-HT2B receptor solely in cardiomyocytes, like global 5-HT2B receptor–null mice, are resistant to isoproterenol-induced cardiac hypertrophy and dysfunction, as well as to isoproterenol-induced increases in cytokine plasma-levels. These data reveal a key role of noncardiomyocytes in isoproterenol-induced hypertrophy in vivo. Interestingly, we show that primary cultures of angiotensinogen null adult cardiac fibroblasts are releasing cytokines on stimulation with either angiotensin II or serotonin, but not in response to isoproterenol stimulation, demonstrating a critical role of angiotensinogen in adrenergic-dependent cytokine production. We then show a functional interdependence between AT1Rs and 5-HT2B receptors in fibroblasts by revealing a transinhibition mechanism that may involve heterodimeric receptor complexes. Both serotonin- and angiotensin II–dependent cytokine production occur via a Src/heparin-binding epidermal growth factor–dependent transactivation of epidermal growth factor receptors in cardiac fibroblasts, supporting a common signaling pathway. Finally, we demonstrate that 5-HT2B receptors are overexpressed in hearts from patients with congestive heart failure, this overexpression being positively correlated with cytokine and norepinephrine plasma levels. Collectively, these results reveal for the first time that interactions between AT1 and 5-HT2B receptors coexpressed by noncardiomyocytes are limiting key events in adrenergic agonist-induced, angiotensin-dependent cardiac hypertrophy. Accordingly, antagonists of 5-HT2B receptors might represent novel therapeutics for sympathetic overstimulation-dependent heart failure.
Key Words: fibroblast • heart failure • hypertrophy • interleukins • sympathetic nervous system
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Introduction |
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Cardiac hypertrophy is a physiological adaptation of the heart to increased workload. Recent data challenged the widely held belief that cardiac hypertrophy is a necessary compensatory mechanism to maintain normal heart function.1,2 When sustained and extensive, cardiac hypertrophy can lead to maladaptation and progressive dysfunction leading to heart failure secondary to cardiomyocyte apoptosis and fibrosis.3 In addition to biomechanical stress, several neurohumoral factors acting via G protein–coupled receptors (GPCRs), including β-adrenergic (β-AR), endothelin and angiotensin (Ang) II type 1 (AT1R) receptors have been identified as potent inducers of cardiac hypertrophy.4–7
The chronic adrenergic stimulation experienced by patients with congestive heart failure (CHF) is a strong predictor of morbidity and mortality. Norepinephrine, through stimulation of β-ARs, is a well-known trigger of cardiac hypertrophy. The extent of left ventricular dysfunction in human pathology correlates to plasma norepinephrine concentration independently of arterial blood pressure.8 Plasma levels of cytokines such as tumor necrosis factor (TNF)-
and interleukin (IL)-69 or transforming growth factor (TGF)-β110 are also significantly increased in primary idiopathic hypertrophic cardiomyopathy. Noncardiomyocyte (cardiac fibroblast) stimulation by adrenergic agonists or Ang II can release growth factors,11 endothelin-1, and cytokines including TNF-, IL-6, IL-1β, and TGF-β1.12–14 Interestingly, several authors have demonstrated marked in vitro release of these hypertrophic factors by cardiac fibroblasts and suggested a causal link between this release and cardiomyocyte hypertrophy. The hypertrophic capacity of cytokines was also validated in vivo by the observation that mice with cardiac-restricted overexpression of TNF-, IL-6, or IL-1 exhibited cardiac hypertrophy.15–17
The question of whether β-AR stimulation promotes pathological cardiac hypertrophy by a direct effect on myocytes and/or nonmyocytes remains debatable. In vitro, most of the investigators used neonatal rat cardiomyocytes and found that the β-AR agonist, isoproterenol (ISO), leads to a mild hypertrophy of these cells, the hypertrophy being stronger in presence of cardiac fibroblast-conditioned medium.18 However, it was reported that ISO had no hypertrophic effects on adult rat myocytes,19,20 suggesting that noncardiomyocytes could participate in β-adrenergic–dependent cardiac hypertrophy in an in vivo adult context.
Using a genetic approach, we previously showed that serotonin 5-HT2B receptors (5-HT2BRs) have a trophic action on newborn cardiomyocytes in vitro21 and by -myosin heavy chain (-MHC)-dependent 5-HT2BR overexpression in cardiomyocytes in vivo.22 The initial cardiomyopathy of 5-HT2BR mutant mice is compensated over time in the absence of hypertrophic stage.23 Thus, we studied their response to a pathological hypertrophic stimulus using chronic ISO infusion as a model of sympathetic stimulation in vivo. We reported that either total genetic (5-HT2BR mutant mice) or pharmacological (SB206553 or SB215505, 5-HT2BR antagonists) blockade of 5-HT2BR function completely prevented ISO-induced cardiac hypertrophy.13 Recently, 5-HT2BRs were shown to be required for left ventricular hypertrophy in another model of cardiac hypertrophy (Ang II chronic infusion).24 Nevertheless, neither the exact cardiac cells requiring 5-HT2BRs (cardiomyocytes versus fibroblasts), the receptor crosstalk nor their transduction pathway has been addressed in in vivo models of pathological cardiac hypertrophy.
The purpose of this study was to determine: (1) whether 5-HT2BR expression in cardiomyocytes is required for ISO-induced left ventricular hypertrophy; (2) whether Ang II participates in β-AR-dependent cardiac hypertrophy in vivo; (3) which epistatic relationships exist between β-AR–, AT1R–, and 5-HT2BR–dependent hypertrophic factor release; and (4) whether similar mechanisms could be found in human CHF.
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Materials and Methods |
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Generation of 5-HT2BR Transgenic Mice and Genotyping
Generation of
-MHC-5-HT2B+/– (Tg) mice and 5-HT2B–/– mice has been described previously.22,25 All animal experiments were performed in accordance with institutional guidelines and European regulations.
Induction of Cardiac Hypertrophy by ISO
In 11-week-old male mice, ISO (30 mg/kg per day), was delivered for 7 days by miniosmotic pumps (1007D, Alzet Corp) implanted subcutaneously under anesthesia (0.75% isoflurane).
Cardiovascular Phenotyping by Echocardiography
Left ventricular dimension and heart rate were assessed before and after ISO infusion under isoflurane anesthesia (0.75%) by echocardiography.13 After echocardiographic analysis, mice were euthanized by CO2 and weighed.
Analysis of 5-HT2R and AT1R Expression by Binding Assays
Membrane proteins prepared from heart ventricles or from adult mouse cardiac fibroblast or myocytes primary cultures were analyzed by binding studies to assess receptor expression.23
Patients
Cardiac samples were obtained from explant grafts except for normal controls, the tissue of which was obtained from donors without recipient. ELISAs for plasma concentration for cytokines were performed. The local ethical committee (Comité Consultatif de Protection des Personnes se Prétant à la Recherche Biomédicale, CCPPRB d’Alsace) approved the study, and all patients gave their informed consent before tissue collection and plasma collection. For frozen human heart sections, all harvest and use of human tissue was performed in accordance with NIH and University of Rochester Medical Center institutional review board guidelines.
Adult Cardiac Fibroblasts Primary Culture
Cultures of ventricular noncardiomyocytes were obtained by differential plating from dissociated heart of male adults mice (10 to 12 weeks) or from neonatal rat hearts (3 to 4 days). Cardiac fibroblasts used during early passages were identified by characteristic morphology and positive staining with antibody to vimentin (>90%)13 and negative staining for macrophage marker F4/8026 (<1%) (Figure II in the online data supplement). One day before the experiments, the cells were serum-starved.
Measurement of Cytokines in Plasma and Culture Supernatants
Concentrations of Ang II, IL-6, IL-1β, TNF-, and TGF-β1 were measured in plasma and culture supernatants by ELISA kits (Bertin, DY 406, DY 401, DY 410 and DY 1679, R&D systems).13
Confocal Imaging
Cells or tissues were observed after 4% paraformaldehyde fixation and revealed using either a mouse monoclonal anti–FLAG M2 (Sigma, 1:100), a rabbit anti-GFP antibody (Santa Cruz Biotechnology, 1:100), a monoclonal anti-5-HT2BR antibody (Pharmingen,1:100), a rabbit anti-AT1R (N-10, Santa Cruz Biotechnology, 1:100) or a rabbit anti-Vimentin (Santa Cruz Biotechnology, 1:200).
Immunoprecipitation and Western Blotting
Serum-starved cells were homogenized at 4°C in RIPA buffer, centrifuged at 10 000g, and incubated with either anti-FLAG affinity matrix (40 µL, Sigma) overnight at 4°C or a monoclonal anti–5-HT2BR antibody (Pharmingen, 2 µg). Western blot analysis of immunoprecipitated samples was performed on SDS-PAGE 10% gels and revealed using either a rabbit anti-GFP antibody (Santa Cruz Biotechnology, 1:1000) or a rabbit anti-AT1R (N-10, Santa Cruz Biotechnology, 1:1000).
Data Analysis and Statistics
All results are expressed as means±SEM. Different groups were compared through 1-way ANOVA, followed by Newman–Keuls test. All calculations were performed using the GraphPad Prism 4.0 program.
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Results |
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Expression of 5-HT2BRs by Noncardiomyocytes Is Required for ISO-Induced Cardiac Hypertrophy
We first verified the expression of 5-HT2BRs and AT1Rs either by immunohistochemistry of human adult left ventricles (Figure 1a and 1b) or by binding assays on primary cultures of wild-type and 5-HT2BR–/– mice (Figure 1d and 1e). Interestingly, 5-HT2BRs were found mainly colocalized with AT1Rs and vimentin (a marker of cardiac fibroblasts) in human heart sections. To assess whether 5-HT2BR expression in cardiomyocytes is required for left ventricular hypertrophy, global 5-HT2BR–/– mice were crossed with transgenic mice expressing 5-HT2BRs selectively in cardiomyocytes via the cardiomyocyte-specific
-MHC promoter (Tg) (Figure 2a).22 After genotyping, the 4 resulting strains (5-HT2B+/+, Tg; 5-HT2B+/+, Tg; 5-HT2B+/–, Tg; 5-HT2B–/–, and 5-HT2B–/– [Figure 2b]) were assessed for cardiac 5-HT2BR expression (Figure 2c). As expected after ISO infusion, a similar heart rate increase was observed in all 4 genotypes (+25%) (supplemental Table I). By echocardiography, ISO infusion led to cardiac hypertrophy in Tg; 5-HT2B+/+ and in Tg; 5-HT2B+/– mice as shown by increased left ventricular mass-to-body weight ratio (+30%) (Figure 3a and supplemental Table I). However, Tg; 5-HT2B–/– mice expressing 5-HT2BRs solely in cardiomyocytes were, like global 5-HT2B–/– mice, resistant to ISO-induced cardiac hypertrophy. ISO-induced impairment of left ventricle contractility was observed in the Tg; 5-HT2B+/+ and in the Tg; 5-HT2B+/– mice, as demonstrated by a decrease of both fractional shortening and systolic ejection volume in these 2 groups. Conversely, ISO did not modify ventricular functions in Tg; 5-HT2B–/– or in global 5-HT2B–/– mice (Figure 3b and 3c). Importantly, Ang II plasma level was not increased at 7 days of ISO-infusion in any mice (405±32, versus 354±61 fg/mL, P>0.05, n=6 per genotype each in triplicate). However, ISO infusion led to significant increases in plasma concentrations of TNF- (1.4-fold over basal; supplemental Figure IV), IL-6 (2.5-fold over basal), IL-1β (2.8-fold over basal), and TGF-β (2.5-fold over basal) in Tg; 5-HT2B+/+ and in Tg; 5-HT2B+/– mice (Figure 3d and 3f). Furthermore, Tg; 5-HT2B–/– mice expressing 5-HT2BRs only in cardiomyocytes were, like global 5-HT2B–/– mice, resistant to ISO-induced increase in plasma cytokines.
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Angiotensinogen, AT1Rs, and 5-HT2BRs Are Required for ISO-Induced Cytokine Release in Noncardiomyocytes
Interestingly, primary cultures of adult noncardiomyocytes isolated from Angiotensinogen mutant mice (Agt–/–), which are unable to generate Ang II,27 did not exhibit any increase in cytokine release after ISO stimulation, whereas Ang II (100 nmol/L) significantly increased concentrations of IL-6 (4.7-fold over basal), IL-1β (4.1-fold), and TNF- (1.6-fold) at 4 hours, as did 5-HT (1 µmol/L) stimulation (Figure 4a and 4d and supplemental Figure IV, a). Furthermore, ISO stimulation of wild-type cardiac fibroblasts elicited a significant increase in the release of Ang II (883±24 fg/mL at 4 hours versus 190±7 fg/mL at 0 hour; n=4 independent determinations in triplicate; P<0.05) but not of 5-HT (<1 nmol/L). The potent and selective AT1R antagonist ZD7155 at 100 nmol/L (supplemental Figure I, A and C) significantly reduced ISO-induced cytokine release (IL-6, 4-, IL-1β, 1.8-, and TNF-; 2.3-fold,) at 8 hours (Figure 4b through 4e and supplemental Figure IV, b) but not basal cytokine levels. Either genetic or pharmacological (using the potent and selective antagonist SB206553; supplemental Figure I, B and D) blockade of 5-HT2BRs inhibited Ang II–induced cytokine release in adult cardiac fibroblasts (Figure 4c and 4f and supplemental Figure IV, c). Moreover, we also verified that 5-HT2BRs were required for cytokine production by newborn cardiac fibroblasts (supplemental Figure III, E and F).
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) but was observed after a 4-hour Ang II (100 nmol/L) (
) or 5-HT (1 µmol/L) (
) stimulation. Similarly, IL-6 (b) and IL-1β (e) cytokines were significantly increased in adult cardiac fibroblasts isolated from wild-type (WT) (
) or from global mutant for 5-HT2BRs (5-HT2B–/–) (–/–; 
). Results are means±SEM expressed in pg/mL of cardiac fibroblasts supernatants (n=3 independent fibroblast cultures each determination in triplicate). *P<0.05 vs antagonist-treated.
5-HT2BRs and AT1Rs Share a Common Epidermal Growth Factor Receptor Transactivation Pathway–Mediating Cytokine Release
Similar to the effects of 5-HT2BR antagonists on Ang II action, ZD7155 (100 nmol/L) significantly reduced 5-HT-induced cytokine release (Figure 5a through 5c and supplemental Figure IV, d). In noncardiac cells, activation of AT1Rs or β-ARs has been shown to induce shedding of heparin-binding epidermal growth factor (HB-EGF) through activation of matrix metalloproteinases (MMPs) and subsequent activation of the epidermal growth factor receptor (EGF-R), a phenomenon called transactivation.28,29 In adult cardiac fibroblasts in the presence of AG1478 (an EGF-R blocker, 100 nmol/L), either Ang II– or 5-HT–induced release of IL-6, TNF-, and IL-1β was totally prevented and that of TGF-β only partially (Figure 5a through 5c and supplemental Figure IV, d). Cytokine release stimulated by either Ang II or 5-HT was totally abrogated in adult cardiac fibroblasts prepared from mice lacking HB-EGF, although EGF stimulation (10 ng/mL) led to normal cytokine release (Figure 5a through 5c and supplemental Figure IV, d). GM6001 (an MMP inhibitor, 100 nmol/L) (supplemental Figure 3, C and D) or PP2 (a Src inhibitor, 200 nmol/L) totally prevented Ang II– and 5-HT–induced cytokine release by cardiac fibroblasts (Figure 5a through 5c and supplemental Figure IV, d). Interestingly, a strong reduction of Ang II– and complete reduction of 5-HT–induced cytokine release were induced by the p38 inhibitor SB203580 (10 µmol/L). Conversely, the extracellular signal-regulated kinase (ERK)1/2 inhibitor PD098059 (10 µmol/L) did not affect Ang II– or 5-HT-induced IL-6, IL-1β, or TGF-β release and only slightly reduced TNF- cytokine release (Figure 5a through 5c and supplemental Figure IV, d).
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5-HT2BRs and AT1Rs Colocalize and Coimmunoprecipitate
By confocal microscopy, we first observed membrane colocalization of 5-HT2BRs and AT1Rs in rat cardiac fibroblasts (Figure 6a). After immunoprecipitation of neonatal rat cardiac fibroblast extracts with an anti-5-HT2BR antibody, we detected bands of 41 to 43 kDa (expected molecular mass for AT1Rs) similar to those observed by direct Western blot analysis (Figure 6a) with an anti-AT1R antibody. We further confirm these putative interactions, using HEK293 transfected cells with FLAG-tagged human AT1Rs and CFP-tagged human 5-HT2BRs, that showed membrane colocalization (Figure 6b). After immunoprecipitation with an anti-FLAG antibody, we probed Western blots of immunoprecipitations with an anti-GFP antibody. We detected a single band of 80 kDa (the expected molecular mass of the CFP-5-HT2BR) only in cotransfected cells (Figure 6b). These results strongly suggest that AT1Rs and 5-HT2BRs colocalize and may directly interact in common signaling complexes in transfected cells.
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5-HT2BR Overexpression in Human Heart Failure
Because 5-HT2BRs were also expressed in both human cardiac fibroblasts and cardiomyocytes (Figure 1), we assessed a putative contribution of 5-HT2BRs in human CHF. We looked for 5-HT2BR expression in left ventricular biopsies of 16 CHF patients, compared to 7 normal control subjects. Expression of 5-HT2BRs, obtained from biopsies of left ventricles, were found to be significantly elevated in samples from failing hearts (Figure 7a). This increase appears to be independent of cardiomyopathy etiology, severity of the disease, or treatments (supplemental Table II). However, significant correlations were found between cardiac 5-HT2BR expression levels and plasma concentrations of either norepinephrine, IL-6, TGF-β, or TNF- in CHF patients (Figure 7b, 7c, and 7e and supplemental Figure IV, e) and reciprocally (Figure 7d and 7f and supplemental Figure IV, f) but not in controls (supplemental Table II). Taken together, these data support the notion that 5-HT2BR expression is linked to cardiac cytokine production during the sympathetic overactivity associated with CHF.
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Discussion |
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In light of our previous reports, 5-HT2BRs participate in trophic responses of the myocardium by acting directly on cardiomyocytes21,22 or indirectly on noncardiomyocytes through the release of paracrine factors induced by chronic ISO stimulation.13 By reexpressing the 5-HT2BR selectively into cardiomyocytes in a 5-HT2BR–null background, we describe here for the first time that its expression by noncardiomyocytes is absolutely required for ISO-induced cardiac hypertrophy. The ISO-mediated increases in TNF-
, TGF-β, IL-6, and IL-1β plasma levels observed in the wild-type mice also require 5-HT2BR expression in noncardiomyocytes. No increase in plasma Ang II could be detected after ISO infusion in mice but Ang II can be produced in the heart through a local renin-angiotensin system.30 Interestingly, angiotensinogen mRNA and protein levels are increased by β-AR stimulation in neonatal cardiac fibroblasts,31 which were shown, as opposed to cardiomyocytes, to serve as the predominant source of IL-6 after ISO stimulation in mouse myocardium. In our study, we uncover the Ang II/AT1R axis critical role for ISO-induced cytokine release in adult cardiac fibroblasts as validated by several observations. (1) A significant increase in Ang II, but not in 5-HT, was detected in supernatants of adult mouse cardiac fibroblast culture after ISO stimulation. (2) This peak of Ang II release occurs at 4 hours of ISO stimulation, similar to that of cytokines after direct Ang II stimulation. (3) The cytokine release peak following ISO stimulation occurred only after 8 hours, suggesting a multistep process. (4) Consistent with these results, Agt–/– cardiac fibroblasts did not release cytokines on ISO stimulation, but cytokine release in these cells was similar to wild-type cells when stimulated with Ang II or 5-HT. (5) Finally, using ZD7155, we show that AT1Rs are also required for ISO-induced cytokine release. Together, these data reveal, for the first time, that ISO-dependent Ang II production by cardiac fibroblasts leading to the autocrine AT1R stimulation is absolutely required for hypertrophic cytokine release in heart.
The present report addresses unknowns regarding the AT1R and 5-HT2BR signaling pathway(s) controlling cytokine release in cardiac fibroblasts. In the present work, we demonstrate that expression of HB-EGF and Src activity are critical for either an Ang II– or a 5-HT–dependent cytokine release process. We show that MMPs are responsible for HB-EGF shedding and subsequent EGF-R transactivation that is induced by GPCR agonists such as Ang II or 5-HT.32,33 TNF-–converting enzyme (TACE) (ADAM-17) was found to control HB-EGF shedding in fibroblasts,34 and a recent report indicated that 5-HT2BRs can directly regulate this enzyme activity in neuronal cells.35 Our work also highlights the importance of p38 but not ERK1/2 pathway for cytokine release. In summary, our data support the following epistatic relationships (Figure 8 and online data and video): ISO
Ang II5-HT2B+AT1RsSrcMMPsHB-EGFErbB-1/4-Rsp38IL-6, TNF-, TGF-β, and IL-1β release. All of these findings support that AT1Rs and 5-HT2BRs share common EGF-R-dependent signaling pathways in adult cardiac fibroblasts.
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Blockade of 1 of the 2 receptors prevents cytokine release induced by the other receptor, supporting interactions between 5-HT2BRs and AT1Rs. Using coimmunolocalization and a pull-down assay, we show that the 2 receptors interact in a common cell compartment. Recently, reports have suggested that GPCRs exist in heterodimeric complexes that may play a key role in receptor maturation and trafficking to the plasma membrane and/or signaling (for review, see Bulenger et al36). The protein network associated with the C terminus of the 5-HT2Rs includes scaffolding proteins containing 1 or several PDZ domains, signaling proteins and proteins of the cytoskeleton that may be involved in signaling complexes.37 More work will be necessary to conclude whether in vivo interactions between AT1Rs and 5-HT2BRs are direct or mediated by adaptor proteins. Only a few reports have described an inhibitory mechanism in trans between 2 GPCRs. To our knowledge, this process was first described between AT1Rs and β2-ARs in COS-7 cells that express equal endogenous levels of AT1Rs and β2-ARs and was also shown to occur in adult cardiomyocytes.38 Together, our findings are consistent with the hypothesis that AT1Rs and 5-HT2BRs exist in common signaling complexes and that they may interact together.
The increase of 5-HT2BR sites in biopsies of left ventricles from CHF patients that we observed appears independent of the type of cardiopathy, its duration, or the treatments (including β blockers or ACE inhibitors). In recent cardiac transcriptome analysis, an increase in 5-HT2BR mRNA was also reported in human39 or rat40 failing heart tissue, during the functional recovery of end-stage human heart failure,41 and in rats after banding of the ascending aorta.42,43 The significant correlation between 5-HT2BR expression and cytokines IL-6, TNF-, or TGF-β plasma levels supports, in humans, our findings in mice. Interestingly, the significant correlations with sympathetic activity found in patients indicate that 5-HT2BRs are as well required for adrenergic-dependent cytokines production in humans. A cardiac hypertrophy–associated switch of adult to fetal genes has been reported. It is tempting to speculate that similar mechanisms might be operative at 5-HT2B receptor expression, which is expressed in embryonic heart. Sympathetic overstimulation may also participate via cAMP-dependent regulation as an autocrine regulatory loop.35
In summary, our data indicate that a selective 5-HT2BR antagonist blocks both Ang II and adrenergic adverse effects in pathological conditions with no alterations of hemodynamics or blood pressure.
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Acknowledgments |
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We thank K. Boutourlinsky for excellent technical assistance. We acknowledge the generous gift of Agt mutant mice from Dr Michael Bader. We also acknowledge Laure-Anne Helis for the schematic and the 3D animation model (http://www.razhed.com/Transactivation.html).
Sources of Funding
This work was supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université Pierre et Marie Curie, and the Université Louis Pasteur and by grants from the Fondation de France, the Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer, the French Ministry of Research (Agence Nationale pour la Recherche), and the European Union. The laboratory of L.M. is an "Equipe Fondation pour la Recherche Médicale." F.J. is supported by a fellowship of Fondation pour la Recherche Médicale. Work in the laboratory of B.C.B. is supported by NIH grant HL084087.
Disclosures
None.
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Footnotes |
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Original received June 6, 2008; revision received October 15, 2008; accepted November 6, 2008.
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References |
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1. Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, Rockman HA. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation. 2002; 105: 85–92.
2. Hill JA, Karimi M, Kutschke W, Davisson RL, Zimmerman K, Wang Z, Kerber RE, Weiss RM. Cardiac hypertrophy is not a required compensatory response to short-term pressure overload. Circulation. 2000; 101: 2863–2869.
3. Frey N, Olson EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol. 2003; 65: 45–79.[CrossRef][Medline] [Order article via Infotrieve]
4. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol. 2006; 7: 589–600.[CrossRef][Medline] [Order article via Infotrieve]
5. Molkentin JD, Dorn GWn. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu Rev Physiol. 2001; 63: 391–426.[CrossRef][Medline] [Order article via Infotrieve]
6. Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of beta-adrenergic signaling in heart failure? Circ Res. 2003; 93: 896–906.
7. Salazar NC, Chen J, Rockman HA. Cardiac GPCRs: GPCR signaling in healthy and failing hearts. Biochim Biophys Acta. 2007; 1768: 1006–1018.[Medline] [Order article via Infotrieve]
8. Kelm M, Schafer S, Mingers S, Heydthausen M, Vogt M, Motz W, Strauer BE. Left ventricular mass is linked to cardiac noradrenaline in normotensive and hypertensive patients. J Hypertens. 1996; 14: 1357–1364.[CrossRef][Medline] [Order article via Infotrieve]
9. Zen K, Irie H, Doue T, Takamiya M, Yamano T, Sawada T, Azuma A, Matsubara H. Analysis of circulating apoptosis mediators and proinflammatory cytokines in patients with idiopathic hypertrophic cardiomyopathy: comparison between nonobstructive and dilated-phase hypertrophic cardiomyopathy. Int Heart J. 2005; 46: 231–244.[CrossRef][Medline] [Order article via Infotrieve]
10. Li G, Li RK, Mickle DA, Weisel RD, Merante F, Ball WT, Christakis GT, Cusimano RJ, Williams WG. Elevated insulin-like growth factor-I and transforming growth factor-beta 1 and their receptors in patients with idiopathic hypertrophic obstructive cardiomyopathy. A possible mechanism. Circulation. 1998; 98: 144–149.
11. Ushikoshi H, Takahashi T, Chen X, Khai NC, Esaki M, Goto K, Takemura G, Maruyama R, Minatoguchi S, Fujiwara T, Nagano S, Yuge K, Kawai T, Murofushi Y, Fujiwara H, Kosai K. Local overexpression of HB-EGF exacerbates remodeling following myocardial infarction by activating noncardiomyocytes. Lab Invest. 2005; 85: 862–873.[CrossRef][Medline] [Order article via Infotrieve]
12. Burger A, Benicke M, Deten A, Zimmer HG. Catecholamines stimulate interleukin-6 synthesis in rat cardiac fibroblasts. Am J Physiol Heart Circ Physiol. 2001; 281: H14–H21.
13. Jaffré F, Callebert J, Sarre A, Etienne N, Nebigil CG, Launay JM, Maroteaux L, Monassier L. Involvement of the serotonin 5-HT2B receptor in cardiac hypertrophy linked to sympathetic stimulation: control of interleukin-6, interleukin-1 beta, and tumor necrosis factor-alpha cytokine production by ventricular fibroblasts. Circulation. 2004; 110: 969–974.
14. Sano M, Fukuda K, Kodama H, Pan J, Saito M, Matsuzaki J, Takahashi T, Makino S, Kato T, Ogawa S. Interleukin-6 family of cytokines mediate angiotensin II-induced cardiac hypertrophy in rodent cardiomyocytes. J Biol Chem. 2000; 275: 29717–29723.
15. Hirota H, Yoshida K, Kishimoto T, Taga T. Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice. Proc Natl Acad Sci U S A. 1995; 92: 4862–4866.
16. Sivasubramanian N, Coker ML, Kurrelmeyer KM, MacLellan WR, DeMayo FJ, Spinale FG, Mann DL. Left ventricular remodeling in transgenic mice with cardiac restricted overexpression of tumor necrosis factor. Circulation. 2001; 104: 826–831.
17. Nishikawa K, Yoshida M, Kusuhara M, Ishigami N, Isoda K, Miyazaki K, Ohsuzu F. Left ventricular hypertrophy in mice with a cardiac-specific overexpression of interleukin-1. Am J Physiol Heart Circ Physiol. 2006; 291: H176–H183.
18. Long CS, Hartogensis WE, Simpson PC. Beta-adrenergic stimulation of cardiac non-myocytes augments the growth-promoting activity of non-myocyte conditioned medium. J Mol Cell Cardiol. 1993; 25: 915–925.[CrossRef][Medline] [Order article via Infotrieve]
19. Schafer M, Frischkopf K, Taimor G, Piper HM, Schluter KD. Hypertrophic effect of selective beta(1)-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat. Am J Physiol Cell Physiol. 2000; 279: C495–C503.
20. Schluter KD, Piper HM. Trophic effects of catecholamines and parathyroid hormone on adult ventricular cardiomyocytes. Am J Physiol. 1992; 263: H1739–H1746.[Medline] [Order article via Infotrieve]
21. Nebigil CG, Etienne N, Messaddeq N, Maroteaux L. Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT2B-receptor signaling. FASEB J. 2003; 17: 1373–1375.
22. Nebigil CG, Jaffré F, Messaddeq N, Hickel P, Monassier L, Launay JM, Maroteaux L. Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy. Circulation. 2003; 107: 3223–3229.
23. Nebigil CG, Hickel P, Messaddeq N, Vonesch J-L, Douchet M-P, Monassier L, György K, Martz R, Andriantsitohaina R, Manivet P, Launay J-M, Maroteaux L. Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function. Circulation. 2001; 103: 2973–2979.
24. Monassier L, Laplante MA, Jaffre F, Bousquet P, Maroteaux L, de Champlain J. Serotonin 5-HT2B receptor blockade prevents reactive oxygen species-induced cardiac hypertrophy in mice. Hypertension. 2008; 52: 301–307.
25. Nebigil CG, Choi D-S, Dierich A, Hickel P, Le Meur M, Messaddeq N, Launay J-M, Maroteaux L. Serotonin 2B receptor is required for heart development. Proc Natl Acad Sci U S A. 2000; 97: 9508–9513.
26. Landeen LK, Aroonsakool N, Haga JH, Hu BS, Giles WR. Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions. Am J Physiol Heart Circ Physiol. 2007; 292: H2698–H2711.
27. Tanimoto K, Sugiyama F, Goto Y, Ishida J, Takimoto E, Yagami K, Fukamizu A, Murakami K. Angiotensinogen-deficient mice with hypotension. J Biol Chem. 1994; 269: 31334–31337.
28. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, Ullrich A. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 1999; 402: 884–888.[Medline] [Order article via Infotrieve]
29. Pierce KL, Luttrell LM, Lefkowitz RJ. New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene. 2001; 20: 1532–1539.[CrossRef][Medline] [Order article via Infotrieve]
30. Bader M. Role of the local renin-angiotensin system in cardiac damage: a minireview focussing on transgenic animal models. J Mol Cell Cardiol. 2002; 34: 1455–1462.[CrossRef][Medline] [Order article via Infotrieve]
31. Dostal DE, Booz GW, Baker KM. Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and beta-adrenergic stimulation. Basic Res Cardiol. 2000; 95: 485–490.[CrossRef][Medline] [Order article via Infotrieve]
32. Ohtsu H, Dempsey PJ, Frank GD, Brailoiu E, Higuchi S, Suzuki H, Nakashima H, Eguchi K, Eguchi S. ADAM17 mediates epidermal growth factor receptor transactivation and vascular smooth muscle cell hypertrophy induced by angiotensin II. Arterioscler Thromb Vasc Biol. 2006; 26: e133–137.
33. Gooz M, Gooz P, Luttrell LM, Raymond JR. 5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells. J Biol Chem. 2006; 281: 21004–21012.
34. Hinkle CL, Sunnarborg SW, Loiselle D, Parker CE, Stevenson M, Russell WE, Lee DC. Selective roles for tumor necrosis factor alpha-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family: the juxtamembrane stalk determines cleavage efficiency. J Biol Chem. 2004; 279: 24179–24188.
35. Pietri M, Schneider B, Mouillet-Richard S, Ermonval M, Mutel V, Launay J-M, Kellermann O. Reactive oxygen species-dependent TNF-alpha converting enzyme activation trough stimulation of 5-HT2B and alpha1D autoreceptors in neuronal cells. FASEB J. 2005; 19: 1078–1087.
36. Bulenger S, Marullo S, Bouvier M. Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation. Trends Pharmacol Sci. 2005; 26: 131–137.[CrossRef][Medline] [Order article via Infotrieve]
37. Gavarini S, Becamel C, Chanrion B, Bockaert J, Marin P. Molecular and functional characterization of proteins interacting with the C-terminal domains of 5-HT2 receptors: emergence of 5-HT2 "receptosomes". Biol Cell. 2004; 96: 373–381.[CrossRef][Medline] [Order article via Infotrieve]
38. Barki-Harrington L, Luttrell LM, Rockman HA. Dual inhibition of beta-adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation. 2003; 108: 1611–1618.
39. Thum T, Galuppo P, Wolf C, Fiedler J, Kneitz S, van Laake LW, Doevendans PA, Mummery CL, Borlak J, Haverich A, Gross C, Engelhardt S, Ertl G, Bauersachs J. MicroRNAs in the human heart: a clue to fetal gene reprogramming in heart failure. Circulation. 2007; 116: 258–267.
40. Koyanagi T, Wong LY, Inagaki K, Petrauskene OV, Mochly-Rosen D. Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats. Am J Physiol Heart Circ Physiol. 2008; 295: H222–H226.
41. Hall JL, Birks EJ, Grindle S, Cullen ME, Barton PJ, Rider JE, Lee S, Harwalker S, Mariash A, Adhikari N, Charles NJ, Felkin LE, Polster S, George RS, Miller LW, Yacoub MH. Molecular signature of recovery following combination left ventricular assist device (LVAD) support and pharmacologic therapy. Eur Heart J. 2007; 28: 613–627.
42. Brattelid T, Qvigstad E, Birkeland JA, Swift F, Bekkevold SV, Krobert KA, Sejersted OM, Skomedal T, Osnes JB, Levy FO, Sjaastad I. Serotonin responsiveness through 5-HT(2A) and 5-HT(4) receptors is differentially regulated in hypertrophic and failing rat cardiac ventricle. J Mol Cell Cardiol. 2007; 43: 767–779.[Medline] [Order article via Infotrieve]
43. Liang YJ, Lai LP, Wang BW, Juang SJ, Chang CM, Leu JG, Shyu KG. Mechanical stress enhances serotonin 2B receptor modulating brain natriuretic peptide through nuclear factor-kappaB in cardiomyocytes. Cardiovasc Res. 2006; 72: 303–312.
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