HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/6/641 04-2518fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bianchi, P. Articles by Parini, A. Search for Related Content PubMed PubMed Citation Articles by Bianchi, P. Articles by Parini, A.

A new hypertrophic mechanism of serotonin in cardiac myocytes: receptor-independent ROS generation

Pascale Bianchi^*, David R. Pimentel^*, Michael P. Murphy, Wilson S. Colucci^* and Angelo Parini^,1

^* Cardiovascular Medicine Section, Boston University Medical Center, Boston, Massachusetts, USA;
Medical Research Council Dunn Human Nutrition Unit, Cambridge, UK; and
INSERM U388, CHU Rangueil, Toulouse, France

¹Correspondence: INSERM U388, Institut Louis Bugnard-CHU Rangueil-Bât. L3 TSA 50032, Toulouse 31059, France. E-mail: parini{at}toulouse.inserm.fr

SPECIFIC AIMS

Reactive oxygen species (ROS) play a critical role in cardiac hypertrophy. We have recently shown that the serotonin-degrading enzyme monoamine oxidase A (MAO A) is an important source of hydrogen peroxide in rat heart. The aim of the present study was to define whether hydrogen peroxide generated by MAO A contributes to the hypertrophic effects of serotonin in cardiac myocytes.

PRINCIPAL FINDINGS

1. Serotonin stimulates cardiac myocyte hypertrophy by receptor independent and receptor-dependent mechanisms
Exposure of myocytes to serotonin for 48 h increased ³H-leucine incorporation and total cellular protein content. The effect was observed at 1 µM and reached a maximum at 5 µM (³H-leucine incorporation: +43%; total protein content: +22%) (Fig. 1 A). To define the mechanism responsible for myocyte hypertrophy by serotonin, we investigated the ability of various compounds to modulate the serotonin effect. The effect of serotonin on both ³H-leucine incorporation and total protein content was prevented by pretreatment with the amine transporter inhibitor, imipramine, or the irreversible MAO inhibitor, pargyline (Fig. 1B ). Previous studies showed that hypertrophic effect of serotonin requires stimulation of 5-HT_2B receptors. In accordance with these results, we observed that SB 206553, a 5-HT_2B receptor antagonist, reduced the serotonin stimulated ³H-leucine incorporation only by 30% (P<0.001). Similar results were observed using the 5HT₂ receptor antagonist ketanserin (data not shown). These results show that, in addition to the receptor-mediated hypertrophic effect, serotonin induces cardiomyocyte hypertrophy by a mechanism requiring its internalization into myocytes and degradation by MAOs.

View larger version (26K): [in this window] [in a new window]   Figure 1. Serotonin induces cardiac myocyte hypertrophy via MAO-dependent ROS generation. A) Effect of indicated concentrations of serotonin for 48 h on hypertrophy of ARVM. B) Cells were pretreated 30 min with pargyline (10 µM), imipramine (25 µM), SB 206553 (1 µM), deferoxamine (50 µM) or infected for 30 h with adenovirus leading to overexpression of catalase before 5-HT (5 µM) exposure. Adenovirus vector expressing ß-galactosidase enzyme (LacZ) was used as control. Protein synthesis was assessed by [3H] leucine uptake and total protein content. Data are expressed as mean ± SEM of 3–5 experiments, each performed in triplicate. *P< 0.05; ***P < 0.001 for difference from control values.

2. ROS mediate the MAO-dependent hypertrophic effect of 5-HT
Based on our previous studies showing that H₂O₂ production by MAOs mediates the proliferative effect of dopamine in renal epithelial cells, we next investigated the involvement of ROS in MAO-dependent hypertrophic effect of serotonin. We first quantified hydrogen peroxide in intact cardiomyocytes using the fluorescent probe DCF. Incubation of cardiac myocytes with 5 µM 5-HT induced a significant increase in fluorescence intensity (+30%) (Fig. 2 A). This effect was prevented by pargyline and catalase suggesting that serotonin induces hydrogen peroxide production by a MAO-dependent mechanism. Next, we investigated the ability of serotonin to generate an intracellular oxidative stress. Incubation of cardiomyocytes with serotonin (5 µM, 48 h) decreased GSH/GSSG ratio, an index of intracellular oxidative stress. This effect was prevented by MAO inhibitor, pargyline, indicating that H₂O₂ generated by MAO may mediate the hypertrophic effect of serotonin (Fig. 2B ). This possibility was supported by the results obtained in cardiomyocytes infected with an adenoviral vector encoding rat catalase. As shown in Fig. 1B , serotonin-stimulated increases in ³H-leucine incorporation and protein content were significantly inhibited by overexpression of catalase (vs. Lac-Z), indicating involvement of H₂O₂ in serotonin-stimulated hypertrophy. In the presence of Fe²⁺, H₂O₂ may lead to the generation of hydroxyl radical (OH^–) by Fenton or Haber-Weiss reactions. Pretreatment with the iron chelator, deferoxamine, fully prevented the hypertrophic effect of serotonin, suggesting an important role for OH^–.

View larger version (13K): [in this window] [in a new window]   Figure 2. Serotonin induces H2O2 production and oxidative stress generation in cardiomyocytes. A) Effect of 5-HT (5 µM, 5 min) on H2O2 production (DCF generation) in the presence or in the absence of pargyline (10 µM) or overexpressed catalase. B) Effect of pargyline (10 µM) on reduced (GSH) and oxidized (GSSH) glutathione concentrations by 5-HT (5 µM) treatment of cardiac myocytes for 48 h. Data represent the means ± SEM of 3–5 experiments, each performed in triplicate. **P < 0.01; ***P < 0.001 for difference from control values.

3. Role of ROS in MAO-dependent MAPK activation
Since MAPKs may mediate the effects of ROS, we next investigated the potential role of ERK1/2 in mediating the MAO-dependent effects of serotonin in cardiac myocytes. The MEK1/2 inhibitor PD98059 fully prevented the serotonin-stimulated increases in ³H-leucine incorporation and total protein (–106 and –79.5%, respectively, compared with serotonin). Exposure to serotonin (5 µM) increased ERK1/2 phosphorylation. As with hypertrophy, ERK1/2 phosphorylation was inhibited by pargyline, imipramine, PD98059, deferoxamine, or catalase overexpression. In contrast, ERK1/2 phosphorylation was unaffected by the 5-HT_2B antagonist SB 206553.

MAOs are intrinsic proteins of the outer mitochondrial membrane. A previous study has shown that mitochondria treatment with different MAO substrates decreased mitochondrial respiration and induced GSSG formation. Therefore, we considered the possible role of mitochondria in mediating the ROS-dependent effects of 5-HT. Cardiac myocytes treatment with the mitochondrial-specific antioxidant, MitoQ, decreased 5-HT-stimulated ERK1/2 activation. This finding suggests that intramitochondrial ROS are involved for ERK1/2 activation.

CONCLUSIONS AND SIGNIFICANCE

In the present study, we identified a novel mechanism of cardiac myocyte hypertrophy by serotonin. This mechanism is independent of receptor stimulation and requires serotonin uptake into cardiomyocytes, its degradation by the mitochondrial enzyme monoamine oxidase A, hydrogen peroxide production, and ERKs phosphorylation.

The demonstration that serotonin is involved in cardiac hypertrophy is relatively recent and it has been supplied by studies on genetically modified mice. These studies showed that overexpression of 5-HT_2B receptors in heart leads to hypertrophic cardiomyopathy associated with mitochondrial dysfunction. We showed that ventricular cardiomyocyte hypertrophy by serotonin was in part mediated by 5-HT receptors. The additional mechanism of hypertrophic serotonin effect we have identified requires serotonin uptake into cardiomyocyte and therefore seems to be independent of membrane receptor stimulation. The coexistence of receptor-dependent and receptor-independent effect of serotonin within the same cell type is not limited to cardiomyocyte as it has been described in pulmonary vascular smooth muscle cells. In these cells, the mitogenic effect of serotonin is mediated by serotonin transporter, whereas its constricting effect requires 5-HT receptor stimulation. This transporter-dependent serotonin effect has been shown to involve reactive oxygen species. However, the intracellular sources of these ROS remain uncertain.

Monoamine oxidase A represents a likely source of ROS by serotonin. Monoamine oxidases are mitochondrial enzymes involved in the oxidative deamination of catecholamine, serotonin, and exogenous amines. They have been subdivided in two major forms, A and B, based on genetic criteria, substrate specificity, and inhibition by synthetic compounds. Monoamine oxydase A represent the major metabolic pathway for serotonin degradation. This enzyme has been particularly investigated in the central nervous system where it has been involved in mood disorders. We and others have shown that MAO-A is the predominant isoenzyme expressed in cardiac ventricle. The major role of this enzyme in heart has been mainly investigated in relation to its ability to regulate the substrate concentration. However, we have recently shown that MAO A is an important source of ROS in rat cardiac ventricle and its ability to produce ROS increases with aging, a situation frequently associated with cardiac failure. Furthermore, it has been reported that MAO activity is higher in the hearts of hypertensive compared with normotensive rats.

The results obtained in the present study allowed to identify MAO A as the intracellular source of ROS mediating the receptor-independent hypertrophic effects of serotonin. We showed that myocyte hypertrophy was prevented by the irreversible MAO inhibitor pargyline. Adenoviral overexpression of catalase or cell treatment with deferoxamine markedly inhibited protein synthesis and leucine incorporation, indicating that H₂O₂ and H₂O₂-derived hydroxyl radicals are involved in serotonin-mediated cardiomyocyte hypertrophy.

We previously showed that adrenergic receptors stimulate hypertrophy in rat cardiac myocytes, and that this effect is mediated via a ROS-dependent activation of ERK1/2. In the present study we found that the hypertrophic effect of serotonin is also associated with the potent activation of ERK1/2. However, this effect seems to be independent of serotonin receptor stimulation as it was fully prevented by inhibition of amine transporter or MAO-A. ERK1/2 activation was prevented by the mitochondrial antioxidant Mito-Q, indicating that ROS production by MAO-A during serotonin degradation may induce a mitochondrial dysfunction and additional ROS generation. These results are in agreement with previous findings showing that treatment of isolated mitochondria from brain with a MAO substrate, tyramine, induced a decrease in mitochondrial respiration concomitant with GSSG formation. Activation of ERK1/2 by serotonin has been previously associated with cardiomyocyte protection against apoptosis after serum deprivation. In contrast with that observed in our study, this effect appeared to be related to the stimulation of 5-HT_2B receptors. Based on this and our results, it is conceivable that receptor-dependent and receptor-independent ERK activation by serotonin may lead to different effects on cardiomyocytes.

It is now accepted that ROS are involved in the pathophysiology of myocardial hypertrophy and failure. There is evidence for increased oxidative stress in patients with heart failure, and in animal models of hemodynamic overload, including postmyocardial infarction and pressure overload. Several stimuli have been shown capable of causing ROS-dependent hypertrophy in cardiac myocytes, including mechanical strain, angiotensin II, tumor necrosis factor-, and -adrenergic receptor stimulation. Our data indicate that serotonin should be added to the list of stimuli for ROS-mediated myocyte hypertrophy. However, unlike other hormones acting on seven transmembrane domain receptors, serotonin seems to induce ROS production in cardiomyocytes mainly by a mechanism independent of receptor stimulation.

We have identified a novel mechanism of action of serotonin and MAOs leading to ROS-mediated cardiac myocyte hypertrophy. These results open a new perspective for the study of the role of serotonin and MAOs in the pathophysiology of myocardial hypertrophy and failure, and thus, may offer new therapeutic approaches to the prevention of myocardial disease.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic diagram. Proposed mechanisms of cardiomyocyte hypertrophy by serotonin.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2518fje;

This article has been cited by other articles:

				D. Pchejetski, O. Kunduzova, A. Dayon, D. Calise, M.-H. Seguelas, N. Leducq, I. Seif, A. Parini, and O. Cuvillier Oxidative Stress-Dependent Sphingosine Kinase-1 Inhibition Mediates Monoamine Oxidase A-Associated Cardiac Cell Apoptosis Circ. Res., January 5, 2007; 100(1): 41 - 49. [Abstract] [Full Text] [PDF]

				N. Ishizaka, K. Saito, I. Mori, G. Matsuzaki, M. Ohno, and R. Nagai Iron Chelation Suppresses Ferritin Upregulation and Attenuates Vascular Dysfunction in the Aorta of Angiotensin II-Infused Rats Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2282 - 2288. [Abstract] [Full Text] [PDF]

				A. Lawrie, E. Spiekerkoetter, E. C. Martinez, N. Ambartsumian, W. J. Sheward, M. R. MacLean, A. J. Harmar, A.-M. Schmidt, E. Lukanidin, and M. Rabinovitch Interdependent Serotonin Transporter and Receptor Pathways Regulate S100A4/Mts1, a Gene Associated With Pulmonary Vascular Disease Circ. Res., August 5, 2005; 97(3): 227 - 235. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/6/641 04-2518fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bianchi, P. Articles by Parini, A. Search for Related Content PubMed PubMed Citation Articles by Bianchi, P. Articles by Parini, A.