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Published as doi: 10.1096/fj.07-100743.
 (The FASEB Journal. 2008;22:2340-2349.)
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Maternal serotonin influences cardiac function in adult offspring

Cécile Fligny*, Yves Fromes{dagger}, Philippe Bonnin§, Michèle Darmon{ddagger}, Elisa Bayard*, Jean-Marie Launay||, Francine Côté*, Jacques Mallet*,1 and Guilan Vodjdani*,1

* Unité Mixte de Recherche 7091 Centre National de la Recherche Scientifique/Université Pierre et Marie Curie, Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs (LGN),

{dagger} Institut de Myologie, U582 Institut National de la Santé et de la Recherche Médicale, and

{ddagger} Unité Mixte de Recherche 677 Institut National de la Santé et de la Recherche Médicale/Université Pierre et Marie Curie, Laboratoire de Neuropsychopharmacologie, Hôpital de la Pitié-Salpêtrière, Paris, France; and

§ Centre de Recherche Cardiovasculaire, Physiologic Explorations Fonctionnelles, U689 Institut National de la Santé et de la Recherche Médicale, Université Denis Diderot, and

|| Service de Biochimie, Hôpital Lariboisière, APHP, Paris, France

1Correspondence: UMR 7091 CNRS/UPMC, LGN, Hôpital de la Pitié-Salpêtrière, Bâtiment CERVI, 83 boulevard de l’Hôpital, 75013 Paris, France. E-mail: J.M., mallet{at}chups.jussieu.fr; G.V., vodjdani{at}chups.jussieu.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using the Tph1-invalidated mouse line, in which blood is depleted in serotonin (5-hydroxytryptamine, 5-HT), we have demonstrated previously that maternal 5-HT is required for normal embryonic development. Here, we address the issue of the influence of the maternal 5-HT concentration on the cardiac function of the offspring as adults. We investigated the cardiac phenotype of Tph1-invalidated mice born to Tph1 heterozygous and null mothers. Functionally, all mutants display a significant decrease of cardiac contractility, indicative of impaired left ventricular function. They exhibit progressive dilated cardiomyopathy and are unable to adapt appropriately to a pharmacological stress. Moreover, we show that the cardiopathy is more severe in adult Tph1–/– mice born to homozygous mothers than to heterozygous mothers. Importantly, the severity of the cardiac phenotype is inversely correlated with the plasma 5-HT concentration but not the whole-blood 5-HT concentration. Thus, plasma 5-HT concentration may be a useful index of heart failure. These findings show that cardiac function, through the plasma 5-HT concentration, is influenced by the maternal serotonergic status.—Fligny, C., Fromes, Y., Bonnin, P., Darmon, M., Bayard, E., Launay, J.-M., Côté, F., Mallet, J., Vodjdani, G. Maternal serotonin influences cardiac function in adult offspring.


Key Words: maternal effect • tryptophan hydroxylase 1-invalidated mouse • heart failure animal model • dilated cardiomyopathy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEROTONIN (5-HYDROXYTRYPTAMINE, 5-HT) was first identified in blood as a vasoconstrictor agent of large vessels (1 , 2) and was subsequently shown to be a neurotransmitter in the central nervous system (3) . The first step in the biosynthesis of 5-HT is catalyzed by tryptophan hydroxylase (TPH), which is the rate-limiting enzyme of the pathway. It is encoded by two genes: the non-neuronal Tph1 gene and the neuronal Tph2 gene (4 , 5) . Most of the serotonin found throughout the body is synthesized by TPH1 in enterochromaffin cells of the gastrointestinal tract (6 , 7) . It is secreted into the bloodstream and is taken up and accumulates in platelet-dense granules (>99%, at the micromolar range), which constitute the main serotonin reservoir in the organism (8) .

The major physiological role of circulating serotonin is regulation of hemostasis, vascular tone, and cardiac function (9 10 11) . Serotonin is involved in cardiovascular responses including bradycardia or tachycardia, hypotension or hypertension, and vasodilatation or vasoconstriction. At the periphery, these responses may involve various 5-HT receptors (5-HTRs) expressed on vascular smooth muscle and endothelium (12) , as well as on cardiac tissue (13 14 15) . In rodents, serotonin directly stimulates the heart by activating either 5-HT2BR (16 17 18) or 5-HT2A/4R (19 20 21) . We previously generated a mouse line defective for the Tph1 gene and, thus, defective for peripheral 5-HT synthesis (4) . In these Tph1-mutant mice, the 5-HT concentration in blood is 3–15% of that in wild-type (wt) mice. Null mutants (Tph1–/–) born to heterozygous mothers [Tph1–/–(+/–)], exposed to sufficient 5-HT during embryogenesis, are viable and display no gross anatomical abnormalities. When adult, these mice develop progressive loss of heart contractility that may lead to lethal heart failure (HF), indicating that normal cardiac activity requires an appropriate circulating 5-HT concentration.

Recently, we have shown that normal embryonic development requires suitable circulating 5-HT levels that are provided by the mother (22) . Analysis of the phenotype of Tph1 embryos born to heterozygous or null mothers revealed that Tph1–/–(–/–) animals, exposed to only very low levels of circulating 5-HT (3–15%) during development, display major alterations. Maternal serotonin is thus involved in the control of morphogenesis and in particular that of the brain during the developmental stages that precede the appearance of endogenous serotonin. These findings established the concept that the genotype of the mother may, in some circumstances, supersede that of the fetuses.

How variations in the level of circulating 5-HT contribute to the onset and/or progression of cardiac dysfunction and failure is still unclear. In particular, the influence of the level of the 5-HT in the mother during development on cardiac function in adult offspring is unknown. Our model can be used to address these issues. Here, we show that the cardiopathy is more severe in surviving adult Tph1–/– mice born to homozygous mothers than to heterozygous mothers. These mice exhibit progressive dilated cardiomyopathy and are unable to adapt appropriately to a pharmacological stress. Importantly, the severity of the cardiac phenotype correlated inversely with the plasma 5-HT concentration but not the whole-blood 5-HT concentration. These findings show that the maternal serotonergic status influences cardiac function and the plasma 5-HT concentration of the offspring.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal model
Targeted mutagenesis of the Tph1 gene was described previously (4) . Briefly, exon 2 of the Tph1 locus has been substituted by the nlslacZneopolyA cassette. To study the influence of the maternal 5-HT level on the adult offspring cardiac function, we have examined Tph1-invalidated mice derived either from heterozygous or homozygous mothers. In the mutant mouse population born to Tph1–/– mothers [Tph1–/–(–/–) mice], 80% embryonic death was observed (22) , and only 20% of offspring survived to adulthood. Therefore, extensive hemodynamic analyses were performed on Tph1–/–(+/–) mice and then on Tph1–/–(–/–) animals. Age-matched transgenic and commercial wild-type animals were derived from pure C57BL/6 genetic backgrounds in our animal care facility. Animals were maintained on a standard rodent diet with 12-h light and dark cycles. To eliminate the potential effect of sex, we studied only male mice of both genotypes. The mean age of mice analyzed was 5 months. All animal experimentation was performed in accordance with institutional guidelines. Protocols were approved by the French Animal Care Committee in accordance with European regulations. The animals were weighted before each experiment.

Electrocardiographic recording and analysis
Restrained electrocardiographic (ECG) monitoring of conscious mice was performed during the daytime with the EasyCG tools system (EMKA Technologies, Paris, France), as described previously (23) . The system consists of a four-sensor ecgTUNNEL system platform, connected to a wireless transmitter and amplifier system (EMKAPACK). Three independent sessions of 30-min recording were performed for each animal. Dedicated software (Ecg-auto) was used to analyze the data collected.

Hemodynamic evaluation
Cardiac hemodynamics were evaluated for both Tph1–/– (+/–) and Tph1–/–(–/–) mice. Mice were anesthetized by intraperitoneal injection of KXM (50 mg/kg ketamine, 8 mg/kg xylazine, and 0.1 mg/kg midazolam). Spontaneous ventilation was maintained. Basal conductance catheterization was performed with a 1.4-French high-fidelity Millar conductance catheter (model SPR-839; Millar Instruments, Houston, TX, USA) as described previously (4) . In brief, the pressure catheter was inserted via the right carotid artery into the left ventricle. After left ventricular (LV) function and heart rate (HR) had stabilized, developed pressure, end-diastolic volume, cardiac output, and maximal positive (dP/dtmax) or negative (dP/dtmin) pressure development were recorded. For acute stress hemodynamic evaluation, once baseline measurements were recorded, a low dose of dobutamine (5 µg/kg/min), a β-adrenergic receptor (AR) agonist, was continuously infused through the right jugular vein for 5 min. Conductance catheter-derived pressure-volume data were monitored during infusion and the 10 min after the end of dobutamine administration. Calibration of the Millar catheter was verified before each measurement. At the end of the experiment, the hearts of the animals were removed, weighed, fixed in 4% paraformaldehyde in PBS, and then embedded in paraffin. Transverse sections (5 µm thick) were obtained from each heart, and the gross morphology was assessed by staining with hematoxylin and eosin (H&E)/saffron.

Echocardiographic analyses
Cardiac ultrasound imaging was performed under isoflurane anesthesia, administered with a vaporizer (model 100-F; OH Medical Instruments, Cincinnati, OH, USA). Isoflurane induction was performed over 1 min in an isolation chamber with 0.50% isoflurane in 100% O2, and anesthesia was maintained during spontaneous breathing with the same mixture via a small nose cone as described previously (24) . This procedure resulted in sedation without cardiorespiratory depression during the ultrasound study. Transthoracic echocardiography was performed using an echocardiograph (Vivid 7; GE Medical Systems Ultrasound, Horten, Norway) equipped with a 12-MHz linear transducer (12-lead) connected to an image workstation for subsequent analysis (PC EchoPAC; GE Medical Systems Ultrasound). The frame rate was ~50 frames/s when zoom was applied. Two-dimensional echocardiographic loops and parasternal long-axis motion-mode (M-mode) tracings were recorded. The M-mode recordings were used for all calculations of cardiac structure and function.

Radioligand binding experiments
β-AR binding
Crude ventricular membrane preparation and incubation with [125I]iodocyanopindolol ([125I]CYP) (2000 Ci/mmol; Amersham Biosciences Corp., Piscataway, NJ, USA) were adapted from Brouri et al. (25) . Briefly, left and right ventricles from wt and Tph1–/–(+/–) mice were dissected free from atria and homogenized in ice-cold 50 mM Tris-HCl, pH 7.4. Homogenates were centrifuged at 1000 g for 10 min, and supernatants were collected for a second centrifugation at 40,000 g for 10 min. The resulting pellets were resuspended in 50 mM Tris-HCl, pH 7.4, and centrifuged again at 40,000 g for 10 min. The final pellet was suspended in 1x binding buffer (50 mM Tris-HCl, 10 mM MgCl2, and 150 mM NaCl, pH 7.4), and protein concentration was determined in triplicate according to the method of Lowry et al. (26) . For the determination of the radioligand binding, 100 µg of protein extract was used in reaction mixtures of 500 µl containing various concentrations (10–200 pM) of [125I]CYP and either CGP 20712A (selective β1-AR antagonist) or ICI 118551 (selective β2-AR antagonist) and DL-propanolol (nonspecific ligand). All binding reactions were performed at 37°C for 1 h and then stopped by rapid vacuum filtration through Whatman GF-B filters and three 3-ml washes of the filters with ice-cold incubation buffer. Membrane-bound [125I]CYP was measured by gamma emission. Saturation and competition data were analyzed with Prism software. Specific binding is reported as fentomoles of receptor per milligram of membrane protein.

Binding to 5-HT receptors and monoamine transporter
Cardiac fibroblasts are known to express 5-HT2A and 5-HT2B receptors and the 5-HT transporter (16 , 18 , 27) . To investigate the serotonergic phenotype of the cells responsible for cardiac contraction, the binding study was performed on purified ventricular cardiomyocytes. For this experiment, adult wt and Tph1–/–(+/–) mice were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). The heart was quickly removed and transferred to ice-cold Tyrode’s solution (10 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 10 mM Hepes, and 10 mM glucose, pH 7.35). The ventricles, free from atria, were minced, transferred to Ca2+-free Krebs-Ringer solution (35 mM NaCl, 7.75 mM KCl, 1.18 mM KH2PO4, 10 mM Hepes, 67 mM sucrose, and 25 mM NaHCO3, pH 7.4), and incubated at 37°C in Ca2+-free Krebs-Ringer solution supplemented with 1% BSA (Sigma-Aldrich Corp., St. Quentin Fallavier, France), 250 U/ml collagenase type II (Worthington Biochemicals Corp., Lakewood, NJ, USA), and 0.6 mg/ml pancreatin (Sigma-Aldrich). After a 6-min digestion, the enzyme solution was replaced by the same fresh solution containing only collagenase type II (250 U/ml). After 15 min of digestion, the small pieces were gently stirred in a solution containing 130 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 5 mM glucose, and 25 mM Hepes (pH 7.4), supplemented with 2% BSA. The isolated cells were filtered on a 150-µm diameter nylon filter and maintained for 1 h at 37°C in the same solution. The cardiomyocytes that were concentrated in the pellet were collected and gradually suspended in M199 medium (Gibco-BRL, Gaithersburg, MD, USA). To eliminate the eventual contaminant cardiac fibroblasts, the cells were seeded on a nonpretreated dish. Two hours later, the cell suspension containing only cardiomyocytes was centrifuged for 10 min at 2,000 g, and the pellet was frozen and kept at –80°C until the binding study was performed.

Proteins from isolated ventricular cardiomyocyte membranes were prepared from wt and Tph1–/–(+/–) mice by Polytron homogenization of the tissue pellets in imidazole buffer (pH 7.3) containing 4 mM EDTA, 1 mM EGTA, and 0.1 mM PMSF. Nuclei and tissue debris were separated by centrifugation at 5,000 g for 10 min, and the supernatant was collected and centrifuged at 100,000 g for 90 min on a 20% sucrose gradient. The resulting pellet was suspended in imidazole buffer (pH 7.3) containing 75 mM KCl, 5 mM MgCl2, and 1 mM EGTA, and the protein concentration was determined with the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL, USA) (28) . For the determination of radioligand binding, 20 µg of protein extract was used in a 50-µl reaction mixture containing a 1 nM concentration of radiolabeled ligand and a 100 nM concentration of nonlabeled ligand. Ligands used in the experiments were [3H]MDL100907 for the 5-HT2AR, [3H]LY266097 for the 5-HT2BR, [3H]mesulergine for the 5-HT2CR, and [125I]RTI55 for the monoamine transporters (dopamine, norepinephrine, and serotonin transporters). Membrane-bound radiolabeled ligand was measured by beta and gamma emission. Specific binding is reported as picomoles of receptor per milligram of membrane protein.

Biochemical and molecular measurements
Wild-type, Tph1–/–(+/–), and Tph1–/–(–/–) adult mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). Blood samples were obtained after thoracotomy by intracardiac sampling and were collected in tubes containing 0.106 M citrate solution to prevent blood coagulation. For whole-blood 5-HT level determination, 50-µl blood samples were stored at –80°C. The remaining whole-blood samples were centrifuged for 15 min at 1900 g at room temperature, which prevents platelet degranulation. The supernatant constituted the platelet-free plasma fraction. Whole-blood and plasma 5-HT levels were measured by radioenzymology (29) and norepinephrine levels were determined by HPLC. The same plasma samples were used for the determination of the creatine kinase muscle-brain isoenzyme (CK-MB), brain natriuretic peptide (BNP), and cardiac troponin I (troponin Ic) levels by enzyme immunoassays.

Statistical analyses
Data are expressed as mean ± SD. Statistics on heart rate variability (HRV) parameters were analyzed with ANOVA tests for group comparisons; multiple comparisons vs. controls were performed with the Bonferroni t test. A pairwise comparison independent Student’s t test was used to determine P values. The nonparametric Mann-Whitney test was used to compare hemodynamic parameters. For all analyses, P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We investigated various features and markers of cardiac function in vivo to identify and compare the primary cause of HF in Tph1–/–(+/–) and Tph1–/–(–/–) mice. This could result either from a defective electrical command of the heart contraction or from an impaired mechanical response to this command. The cardiac electrical activity was assessed by electrocardiography, and the geometry of the heart and the mechanics of the contracting ventricles were investigated by echocardiography and conductance catheterization. Twenty percent of the embryos from a mutant mother survived to adulthood; thus extensive hemodynamic analyses were carried out on Tph1–/–(+/–) mice and then on Tph1–/–(–/–) animals.

Cardiac phenotype of Tph1–/–(+/–) mice
We reported previously that in a mixed 129SvJ-C57BL/6 background, Tph1–/–(+/–) mice exhibit progressive HF, with heterogeneous symptoms (4) . We first investigated the origin of the HF by assessing the electrical cardiac activity of Tph1–/–(+/–) mice in a purified C57BL/6 genetic background (10th backcross) and C57BL/6 wt controls. Alert animals were tested in a restrained ECG monitoring system, and basal HR and beat-to-beat variability were analyzed. Tph1 mutants had a slower HR than wt mice (Table 1 ), because of a significantly longer mean interval (Table 2 ) between two consecutive beats (RR interval) (83.476±2.605 ms, relative to 80.100±2.459 ms). The SD of total RR intervals and the SD of averaged means of normal RR intervals did not differ between mutant and control mice (Table 2) . Both instantaneous HR and RR intervals fluctuate with various time scales, known as HRV. The quantitative indicator HRV (HRV index) was significantly higher in Tph1–/–(+/–) mice (5.000±1.305 ms) than in wt mice (3.567±0.805 ms) (Table 2) . Thus, the HR of Tph1–/–(+/–) mice is slower and more variable than that of wt animals, implicating circulating serotonin in the regulation of the cardiac cycle.


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  		Table 1. Basal cardiac mechanical variables as assessed by in vivo cardiac catheterization


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  		Table 2. Time-domain measures of HRV by continuous ECG recording and analysis

To establish the link between the modified electrical command observed in Tph1–/–(+/–) hearts and mechanical and anatomical cardiac parameters, we assessed LV function and the heart anatomy of the mutant mice by conductance catheterization. The hemodynamic variables and baseline contraction were measured in KXM-anesthetized mice (Table 2) . The HR of Tph1–/–(+/–) mice was significantly lower than that of wt animals (476±67 vs. 366±71 bpm). Cardiac output was 60% lower and the developed pressure was 30% lower in the mutants than in the controls. The LV dP/dtmax (a measure of the force of cardiac contraction) was significantly lower in Tph1–/– (+/–) than in wt mice (Table 2) . These results demonstrate that circulating serotonin also contributes to the regulation of cardiac contraction.

We then studied the geometry of the heart in isoflurane-anesthetized 5-month-old Tph1–/–(+/–) mice (the mean age of mice analyzed by conductance catheterization) by echocardiographic analysis. The thickness of the LV wall and interventricular septum and the intraventricular diameter were all normal (Table 3 ). Histological analyses of transverse sections of the mutant hearts by H&E staining similarly did not reveal any myocardial disarray or interstitial fibrosis (Fig. 1 A). However, the ratio of Tph1–/–(+/–) and Tph1–/– (–/–) heart weight to body weight, which is high in many types of cardiac structural abnormalities, was slightly but significantly greater than that in wt mice (Fig. 1B and data not shown).


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  		Table 3. Anatomical and functional cardiac variables in Tph1 mutants monitored by transthoracic echocardiography


Figure 1
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  		Figure 1. Basal cardiac anatomical variables. A) Top panels: low-power views after H&E staining of transverse section from Tph1+/+ and Tph1–/–(+/–) hearts; scale bars = 1 mm. Bottom panels: high magnification of H&E staining from the same sections as in the top panels. B) Body weight, heart weight, and ratios of heart weight to body weight from the mice analyzed in Table 2 . Gray and black histograms correspond, respectively, to Tph1–/–(+/–) and Tph1+/+ mice. Values are means ± SD from n = 15 Tph1–/–(+/–) and n = 10 Tph1+/+ mice. *P < 0.05; **P < 0.01, relative to wt values.

These findings indicate that Tph1–/–(+/–) mice exhibit HF associated with both impaired cardiac electric commands and mechanical responses. The structural integrity of the Tph1–/–(+/–) hearts appeared unaffected, implying that the origin of HF is functional rather than structural.

Exacerbated cardiac pathology in Tph1–/–(–/–) animals
Considering that 20% of the Tph1–/–(–/–) mice survive to adulthood, we assessed cardiac anatomy and contractile performance in these surviving animals by the noninvasive echocardiographic method. At 2 months of age, the cardiac anatomical and functional parameters of both Tph1–/–(–/–) and Tph1–/– (+/–) mice were normal, compared with those of wt mice (data not shown), indicating the absence of overt cardiac developmental defects in Tph1–/–(–/–) mice. In contrast, at 5 months of age, the left ventricle interior diameter, both at end-diastole and at end-systole, was larger in Tph1–/–(–/–) mice than in either wt or Tph1–/–(+/–) mice. The interventricular septum and the LV posterior wall at end-systole were thinner; both of these are anatomical signs of dilated cardiomyopathy (DCM) (Table 3) . The DCM was concomitant with a significant reduction of the shortening fraction, indicative of a defect in cardiac contractility (Table 3) . The echocardiographic features of Tph1–/– (+/–) mice at 5 months of age appeared to be normal (Table 3) , indicating that the cardiac dysfunction is more pronounced in Tph1–/–(–/–) than in Tph1–/–(+/–) animals.

Tph1 mutant mice are devoid of cardiac contractile reserve
Heart function is characterized by its capacity to adapt and survive in constantly changing environments, and this is reflected by the rapid modulation of HR. We tested the capacity of both Tph1–/–(+/–) and Tph1–/–(–/–) mouse hearts to adapt to a stressful condition. We applied a pharmacological stress by stimulating the cardiac β-AR with dobutamine (a β-AR agonist) to force cardiac overactivity. Baseline contraction and acute contractile responses to β-adrenergic stimulation were measured by LV conductance catheterization. In response to dobutamine infusion, the cardiac contractility and the HR of wt mice increased rapidly to their maximum thresholds, which were maintained during dobutamine infusion, and progressively decreased after the infusion was stopped (Fig. 2 ). In sharp contrast with the known HF animal models, most of the Tph1 mutant mice died from sudden cardiac arrest after the β-adrenergic stimulation. The increase in cardiac contractility of Tph1–/–(+/–) mice was almost 60% lower than that in the wt mice, and 75% of the mutants died within 20 min of the start of the adrenergic stimulation; their mean survival time was 11.4 ± 2.8 min (Fig. 2) . Thus, the hearts of the Tph1 mutant mice are unable to sustain an effort. Moreover, there was no correlation between the individual basal hemodynamic variables before dobutamine injection and the contractile reserve (data not shown). The adult Tph1–/–(–/–) mice that were examined displayed the same responses as Tph1–/–(+/–) mice to dobutamine infusion (data not shown). In conclusion, all of the mutant animals lack a contractile reserve when challenged by a stressful condition. Tph1–/– mice thus constitute a unique mouse model of HF in which pharmacological stress leads to death. Therefore, circulating 5-HT is required, not only for normal basal cardiac function, but also for cardiac adaptation to challenge.


Figure 2
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  		Figure 2. Cardiac contractile performance of mice under dobutamine stimulation. Contractile performance from Tph1–/–(+/–) and Tph1+/+ hearts measured by in vivo conductance catheterization. Cardiac variables were recorded for 5 min under basal conditions (values reported in Table 2 ). Hearts were then challenged with dobutamine infusion (5 µg/kg/min) for 5 min and then allowed to recover for another 10 min. dP/dtmax is the maximum rate of the increase of LV pressure. Values are means ± SD from n = 15 Tph1–/–(+/–) and n = 10 Tph1+/+ mice.

In most mammals, HF is associated with modified β1 and/or β2 subtype β-AR expression (30 , 31) . Consequently, we performed binding studies to investigate the expression of both β1- and β2-ARs in total ventricle crude membranes from Tph1–/–(+/–) and wt mice (Table 4 ). There were no significant differences in either β1-AR or β2-AR density and affinity (β1 Bmax and β1 Kd) (Table 4 and data not shown) or the β1-AR/β2-AR ratio (Table 4) . The incapability of Tph1–/– hearts to increase cardiac rate and contractility in response to dobutamine is therefore not associated with a modification in β-AR expression and is unlikely to be associated with a signaling defect. We also found that the level of norepinephrine in the Tph1–/–(+/–) heart was normal (Table 4) .


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  		Table 4. β-Adrenergic receptor status in the Tph1–/– mice with failing hearts

Serotonin mediates its actions either by entering the cell via the specific serotonin transporter SERT (32) as well as via other monoamine transporters (33) or by interacting with specific receptors (34 , 35) . In mice, 5-HT2AR, 5-HT2BR, and SERT are all expressed in cardiovascular tissue (10) . We analyzed the expression of these cardiac 5-HTRs and of the monoamine transporter in failing Tph1 mutant hearts by binding studies with adult isolated ventricular cardiomyocytes as exemplified in Table 5 . 5-HT2BR expression in Tph1–/–(+/–) mice was double that in wt mice, whereas 5-HT2AR expression was normal. Furthermore, the expression of monoamine transporter in Tph1–/–(+/–) mice was 4x higher than that in wt mice. The overexpression of 5-HT2BR and the transporter in the myocardium of Tph1–/–(+/–) mice may result from a compensatory mechanism in response to the low circulating 5-HT concentrations.


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  		Table 5. Serotonergic receptor and monoamine transporter status in the Tph1–/– mice with failing hearts

Severity of cardiopathy is correlated to low plasma 5-HT levels
Serum concentrations of troponin Ic, CK-MB, and BNP are markers of myocardial defects that lead to HF. We assayed these three plasma markers in 5-month-old Tph1–/–(+/–), Tph1–/–(–/–), and wt mice analyzed previously by echocardiography. The concentrations of all three markers were significantly higher in plasma from Tph1–/–(–/–) and/or in Tph1–/–(+/–) mice than in wt mice (Fig. 3 ). This result is consistent with the cardiac functional phenotype of the Tph1–/– (–/–) mice being more severe than that of Tph1–/– (+/–) mice. These findings provide further evidence that the severity of the cardiopathy is, in part, dependent on the genotype of the mother.


Figure 3
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  		Figure 3. Tph1–/–(–/–) mice develop a more pronounced cardiac phenotype than Tph1–/–(+/–) mice. Early markers of HF as determined in plasma samples of mice analyzed in Table 3 . Values are means ± SD from n = 6 Tph1–/–(+/–), n = 4 Tph1–/–(–/–), and n = 8 Tph1+/+ mice. *P < 0.05; **P < 0.01; ***P < 0.001.

Finally, we have tested whether a direct correlation exists between the severity of the cardiopathy sufferedby Tph1–/– animals and their individual circulating 5-HT levels. We determined individual circulating serotonin concentrations in both whole-blood and plasma compartments of both the Tph1–/–(+/–) and Tph1–/– (–/–) mice that were assayed above. In agreement with our previous report (4) , the whole-blood 5-HT level of the Tph1–/–(+/–) mice was ~6% of that of wt mice (Fig. 4 A). No differences in whole-blood 5-HT were observed between Tph1–/–(+/–) and Tph1–/– (–/–) mice, so the severity of the cardiopathy was not correlated with the whole-blood 5-HT levels.


Figure 4
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  		Figure 4. The severity of cardiopathy is inversely correlated with the plasma 5-HT concentration. A, B) Whole-blood (A) (expressed in µmol/L) and plasma (B) (expressed in nmol/L) 5-HT concentrations measured in samples from mice analyzed in Table 3 . Values are means ± SD from n = 4 Tph1–/–(+/–), n = 4 Tph1–/–(–/–), and n = 6 Tph1+/+ mice. *P < 0.05; **P < 0.01; ***P < 0.001. C) A highly significant inverse correlation exists between individual values of plasma markers of HF, as reported in Fig. 3 , and individual values of plasma 5-HT, reported here in B. Figure 4, Tph1–/–(+/–) mice; {circ}, Tph1–/–(–/–) mice; •, Tph1+/+ mice.

Plasma 5-HT levels are in the nanomolar range. We thus used a sensitive method for 5-HT measurement, which prevents platelet degranulation. Consistent with the low 5-HT level in platelets, plasma 5-HT concentrations in Tph1–/–(+/–) animals (6.15 nmol/L) were substantially lower than those in wt animals (15.29 nmol/L) (Fig. 4B ). Strikingly, the 5-HT concentration in the plasma of Tph1–/–(–/–) mice was also significantly lower (3.05 nmol/L) than that in Tph1–/–(+/–) mice (Fig. 4B ). Most interestingly, a strong and significant negative correlation between the individual plasma 5-HT values and the markers of HF was observed (Fig. 4C ). These results support the notions that 1) the severity of the cardiopathy is inversely correlated with the plasma 5-HT concentration and 2) the plasma 5-HT level in the offspring is dependent on the maternal genotype.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The rate-limiting enzyme of serotonin synthesis, tryptophan hydroxylase, is encoded by two genes designated Tph1 and Tph2 (4 , 5) . Tph1 gene disruption leads to a dramatic drop in serotonin concentrations in the periphery owing to the absence of the enzyme in enterochromaffin cells. This initial report linked the loss of Tph1 gene expression and thus of peripheral 5-HT to a cardiac dysfunction phenotype. The abnormal cardiac activity, ultimately led to HF in the Tph1–/– mice (4) .

To investigate whether these defects result from altered electrical control of heart contraction or from an impaired mechanical response to this control, we characterized the HF in Tph1–/– mutant mice using histological, physiological, and biochemical approaches, including electrocardiography, echocardiography, and conductance catheterization. From the ECG data, we conclude that the low level of circulating 5-HT in these animals has shifted the balance of the cardiac rhythm. Because HRV can be considered as an index of the interactions between vagal (reducing HR) and sympathetic (increasing HR) regulation, the increased variability in the HR dynamic in Tph1–/–(+/–) mice might result from modifications of the sympathetic/parasympathetic tonus. Serotonin thus appears to be a regulatory element of the cardiac cycle. We have shown that it is further involved in the control of cardiac contraction. Tph1–/–(+/–) mice display a significant decrease in both shortening fraction and cardiac contractility, indicative of impaired LV function. In contrast, the structural integrity of their hearts appeared to be unaffected, indicating that the origin of HF is functional rather than structural. Remarkably, we show that the cardiac phenotype of Tph1–/–(–/–) mice is greatly exacerbated. In addition to the pathological features observed in Tph1–/–(+/–) mice, their left ventricles are larger and their interventricular septum and LV posterior walls are thinner. The subtype of cardiopathy suffered by Tph1–/– animals is a progressive DCM, and its phenotype varied between Tph1–/– (+/–) and Tph1–/–(–/–) animals. Maternal 5-HT status can therefore be a determining factor of cardiac function of their offspring as adults.

Another important finding here is the direct correlation between the 5-HT concentration in the plasma and the severity of the cardiac phenotype. No such phenomenon was found for whole-blood concentrations of 5-HT. The 5-HT concentration in plasma in Tph1–/–(–/–) mice is half that in Tph1–/–(+/–) mice, pinpointing the influence of maternal 5-HT. There was a significant inverse correlation between individual concentrations of plasma 5-HT and markers of HF including BNP, CK-MK, and troponin Ic. The plasma 5-HT concentration should thus serve as an index of a developing cardiac alteration in mutant Tph1 mice. If this were also true in humans, it would provide a useful index for diagnosis and therapeutics.

Most investigations have considered the plasma 5-HT concentration to be negligible and thus have only focused on the whole-blood 5-HT level. The whole-blood 5-HT level reflects that contained within the blood platelets, because these cells contain more than 99% of all circulating 5-HT. The platelet 5-HT concentration is in the micromolar range, whereas the concentration of 5-HT in plasma is in the nanomolar range (8 , 28 , 36 37 38) . As we show, the total amount of 5-HT in blood does not correlate with the concentration of physiologically active 5-HT. As a matter of fact, free plasma 5-HT is likely to be the only physiologically active fraction, acting as a messenger to control the physiological processes and homeostatic mechanisms in various peripheral tissues including the heart, blood vessels, lungs, and intestine. Consequently, studies that refer to the role of peripheral 5-HT may need to be reconsidered. It is noteworthy that various diseases including chronic systemic and pulmonary hypertension (36 , 39 , 40) , asthma attacks (41 , 42) , and autism (43) have been associated with abnormal plasma 5-HT concentrations.

The mechanism by which 5-HT affects cardiac function at the molecular level is unclear. Several pathways may be involved, either directly or indirectly. The monoamine transporters and a number of 5-HT receptors are present on cardiomyocytes, smooth muscle, and endothelial cells of blood vessels and nerve endings, and they participate in controlling blood pressure (for review, see ref. 13 ). Impairment of multiple pathways that regulate cardiac function could explain why the hearts of Tph1–/– mice are defective in the compensatory mechanisms used by wt animals to adapt to stressful events. In particular, the Tph1–/– mice are unable to cope with the pharmacological stress imposed by dobutamine infusion. Interestingly, the expressions of 5HT2BR and monoamine transporter were 2x and 4x those in wt animals, respectively. 5HT2BR has been implicated in the development and function of the heart (44 , 45) . Mice in which the 5HT2BR gene has been inactivated do not survive in 30% of cases, and the surviving animals show cardiomyocyte cytoarchitecture defects and impaired heart contractility (16) . In wt mice, the plasma concentration of circulating 5-HT (~15 nM) is sufficient for the activation of 5-HT2BR (Ki 10 nM) on both cardiac fibroblasts (18) and cardiomyocytes. Our work indicates that the residual plasma 5-HT concentrations in Tph1–/– mice, born either to Tph1 heterozygous (~6 nM) or to homozygous (~3 nM) mothers, are theoretically insufficient to activate 5-HT2BR. In Tph1–/–(+/–) cardiomyocytes, the overexpression of 5HT2BR and of monoamine transporter(s) may be compensatory responses allowing survival of the Tph1 null mice. It has been reported that SERT is not expressed in adult heart (27 , 46) . Whether the overexpressed transporter corresponds to SERT and whether its level is further enhanced in Tph1–/–(–/–) cardiomyocytes remain to be established. Other targets and mechanisms are likely to be involved. A global systems biology approach should help clarify the functions of peripheral 5-HT in physiological and pathophysiological conditions. In particular, an understanding of the role of 5-HT in cardiac function may lead to novel therapeutic possibilities. Tph1–/– mice are a useful model for such endeavors.

The cause of the 2-fold lower plasma 5-HT concentration in the Tph1–/–(–/–) than Tph1–/–(+/–) mice requires further investigation. It cannot be the result of decreased 5-HT production from the gut and/or an increased 5-HT catabolism because the concentration of whole-blood 5-HT between the two Tph1–/– animal groups was not modified. A modified 5-HT storage ability of platelets may account for an impaired platelet function in which uptake of serotonin in Tph1–/–(–/–) mice could be more affected compared with Tph1–/–(+/–). Tph1–/–(–/–) mice most likely have impaired platelet function and/or structure, which alters the mechanism responsible for controlling the exchange of serotonin. Serotonin exchange between platelets and plasma must be controlled by finely tuned mechanisms. Similarly, Brenner et al. (40) also proposed that plasma 5-HT controls its own concentration levels by modulating the uptake properties of platelet SERT although the mechanism by which it occurs has not yet been clarified. Obvious candidates are the 5-HT transporter SERT and the 5HT2AR and the components of their corresponding transduction pathways.

In conclusion, our study provides evidence that the low plasma serotonin level in the systemic circulation of the Tph1-invalidated mice leads to the progressive development of a dilated cardiopathy in which animals are unable to cope with a stressful challenge. This work has highlighted the important role of serotonin as a peripheral neuroendocrine factor, which is now to be considered as a fully active contributor to cardiac function and pathophysiology. Furthermore, we have extended our previous finding about the requirement of maternal 5-HT for normal development (22) to show that the plasma 5-HT level in adult Tph1–/– mice also depends on maternal serotonergic status and, most likely, influences the cardiac function of offspring as adults. Given the diversity of action of 5-HT in a variety of situations, the normal and pathological functioning of numerous systems may be subject to maternal effects. Consideration of the genotype of the mother in addition to that of affected individuals may help unravel the genetic intricacies of complex diseases and heart defects in particular. As suggested previously, it would be important to uncover other hormones that could be involved.


   ACKNOWLEDGMENTS
 
We thank Luc Maroteaux, Fabrice Jaffré, and Michel Hamon for helpful discussions. C.F. was supported by the Ministère de l'Enseignement Supérieur et de la Recherche and by the Association Française contre les Myopathies (AFM). This work was supported by the Centre National de la Recherche Scientifique, the INSERM, the Université Pierre et Marie Curie, the Agence Nationale pour la Recherche, and the AFM.

Received for publication October 22, 2007. Accepted for publication January 10, 2008.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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