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Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT_2B receptor signaling¹

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, Université L. Pasteur de Strasbourg, BP 10142, 67404 Illkirch Cedex, France

²Correspondence: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, Université L. Pasteur de Strasbourg, BP 10142, 67404 Illkirch Cedex, France. E-mail: lucm{at}igbmc.u-strasbg.fr

SPECIFIC AIMS

Congenital heart disease is a major cause of disability and morbidity. Relatively little is known about the molecular mechanism of cardiac adaptation (hypertrophy) and maladaptation (apoptosis) underlying cardiac pathogenesis. Several lines of evidence suggest that serotonin (5-hydroxytryptamine, 5-HT) is a neurohormone that regulates cardiovascular functions. We have recently shown that inactivation of the Gq-coupled 5-HT_2BR gene leads to partial embryonic lethality due to trabeculae defects. Newborn 5-HT_2BR mutant mice exhibit cardiac dilation resulting from contractility deficits and structural deficits at the intercellular junctions between cardiomyocytes. We used cultured cardiomyocytes and 5-HT_2B receptor knockout mice as an animal model of dilated cardiomyopathy to identify the molecular mechanism of cardiac functions triggered by serotonin. These findings identify 5-HT as a novel survival factor targeting mitochondria in cardiomyocytes. Modulation of 5-HT_2B receptor signaling may have applications in the treatment of acute myocardial infarction and congestive heart failure.

PRINCIPAL FINDINGS

1. 5-HT via 5-HT_2BR inhibits apoptosis induced by serum deprivation in isolated cardiomyocytes
5-HT protects cardiomyocytes from apoptosis after serum deprivation as manifested with TUNEL and Hoechst staining (Fig. 1 ). In the presence of 5-HT, the number of TUNEL-positive cardiomyocytes declined from 38% to 8 ± 5%. Conversely, the 5-HT_2BR knockout cardiomyocytes exhibited an 45% apoptotic cells 4–6 days after serum deprivation and still displayed 46 ± 5% apoptotic cells in the presence of 5-HT. These data indicate that 5-HT via 5-HT_2BR protects cardiomyocytes from apoptosis.

View larger version (39K): [in this window] [in a new window]   Figure 1. 5-HT via 5-HT2BR acts as a survival factor in cardiomyocytes through activation of ERK1/2 and Akt kinases. For MAPK, cardiomyocytes were treated in serum-free (SF) conditions with ±1 µM 5-HT and ± 50 µM PD-098059 (PD) or with mouse p42 and p44MAPK sense (MAPK-S) or antisense (MAPK-AS) fluorescent-labeled synthetic phosphorothioate oligodeoxynucleotides. For PI3K/Akt activity, cardiomyocytes were incubated with ±1 µM 5-HT and ±10 µM LY-294002 (LY) or with adenovirus construct encoding a dominant negative Akt (d3A-AKT) with a K179A/T308A/S473A mutation. The number of TUNEL-positive cells in each microscopic field was determined. Bars: 500 µm. Each value represents the mean of 10 separate fields (150 cells/field) ± SE. M indicates mock transfection with the virus.

2. PI3K/Akt and ERK1/2 activities cross-talks in the anti-apoptotic pathway of 5-HT
Under serum-free conditions, PD-098059 (50 µM), a specific inhibitor of mitogen-activated protein kinases MAPK (ERK1/ERK2), or MAPK down-regulation by transfection of MAPK antisense oligonucleotides resulted in about twice as many apoptotic cells than observed in cells treated with 5-HT alone. LY-294002 (10 µM), a specific inhibitor of phosphatidylinositol-3 kinase (PI3K), thus of Akt phosphorylation resulted in threefold more apoptotic cells than observed in cells treated with 5-HT alone (Fig. 1) . A dominant negative Akt (d3A-Akt) reversed 5-HT cytoprotective effect observed with LY-294002 with similar efficiency. 5-HT-mediated cytoprotective effect was completely reversed in the presence of PD-098059 and LY-294002.

These results show that the cytoprotective effects conferred by 5-HT against serum deprivation-induced apoptosis are dependent not only on ERK but also Akt pathways.

3. 5-HT activation NF-B is mediated by PI3K, but not ERK pathways
To investigate downstream regulators of ERK1/2 and PI3K/Akt kinase in cardiomyocytes, the effect of 5-HT on the activation of NF-B was assessed using the NF-B/luciferase-responsive element reporter gene. 5-HT stimulated NF-B (Rel A) -dependent reporter transcription in wild-type cardiomyocytes, whereas in 5-HT_2BR knockout cardiomyocytes LIF (but not 5-HT) induced NF-B activity. 5-HT phosphorylated IB- undergoes gradual degradation that was completely inhibited by the PI3K inhibitor LY-294002, but not by MAPK inhibitor PD-098059. These results show that in cardiomyocytes 5-HT via PI3K/Akt can induce IB- degradation and thereby NF-B nuclear translocation, which increases the transcription of NF-B-dependent genes.

4. 5-HT via 5-HT_2BR prevents cytoC redistribution and caspase cleavage in cardiomyocytes
We investigated the targets of 5-HT signaling for protecting cardiomyocytes from apoptosis. In wild-type cardiomyocytes, cytochrome C (CytoC) was localized in the mitochondrial fraction, and no cytoplasmic cytoC was observed (Fig. 2 A). However, the cytoC was substantially translocated from mitochondria to cytosol after serum deprivation. The mitochondrial release of cytoC into the cytoplasmic fraction was blocked after treatment of wild-type cardiomyocytes with 5-HT (1 µM) or LIF (5 nM). In 5-HT_2BR knockout cardiomyocytes, basal cytoplasmic cytoC was slightly increased; after serum deprivation, cytoplasmic cytoC reached the maximum observed in wild-type cardiomyocytes. Conversely, 5-HT did not prevent cytoC translocation from mitochondria to cytosol, whereas LIF totally prevented this translocation in the 5-HT_2BR knockout cardiomyocytes. Recent studies have demonstrated that release of cytoC from mitochondria leads to activation of caspase cascade in cardiomyopathic heart. We examined the effect of 5-HT on caspase cleavage as an indicator of caspase activity. A significant increase in cleaved caspase-3 and caspase-9 was observed after serum deprivation whereas the cleavage was negligible after the 5-HT treatment in wild-type cardiomyocytes. No 5-HT-dependent but LIF-dependent inhibition of caspase-3 and caspase-9 cleavage was observed in 5-HT_2BR knockout cardiomyocytes (Fig. 2B ). These data show that mitochondria are targets of 5-HT cytoprotective signaling.

View larger version (59K): [in this window] [in a new window]   Figure 2. Effects of 5-HT on mitochondria. A) 5-HT prevents translocation of cytoC from mitochondria to cytosol in wild-type cardiomyocytes. Wild-type and knockout cardiomyocytes were treated in serum (S) and serum-free (SF) medium with ±1 µM 5-HT or ± 5 nM LIF, then mitochondrial and cytosolic fractions were extracted and submitted to Western blot analyses to determine the relative quantities of cytoC. B) 5-HT prevents caspase activation in wild-type cardiomyocytes. Wild-type and knockout cardiomyocytes were treated in serum-free (SF) medium with ±1 µM 5-HT or ±5 nM LIF for 4 days, then extracted and submitted to Western blot analyses to determine the cleavage of procaspase-3 and procaspase-9 as an indicator of relative activities using anti caspase-3 and caspase-9 antibodies. Procaspase-3; CPP32F, cleaved caspase-3; CPP32C, IB: immunoblot.

5. 5-HT via 5-HT_2BR regulates Bax and ANT-1 expression
Next we investigated how 5-HT cytoprotective signaling prevents cytoC redistribution from mitochondria. In apoptotic conditions, expression of the proapoptotic factor Bax increased twofold, reduced in the presence of 5-HT. The effect of 5-HT was to down-regulate Bax expression, which was completely prevented by ERK1/2 inhibitor PD-098059. However, inhibition of PI3K by LY-294002 did not change 5-HT-mediated regulation of Bax expression. These data indicate that, in cardiomyocytes, ERK1/2 activation by 5-HT is involved in regulating Bax expression, whereas PI3K/Akt did not alter Bax levels. Since the adenine nucleotide translocator (ANT-1) is a component of mitochondrial membrane permeability transition pore and ANT-1 mutant mice exhibited severe cardiomyopathy, we investigated the possible regulation of ANT-1 in 5-HT cytoprotective signaling. In the apoptotic conditions, ANT-1 expression was increased in the wild-type cardiomyocytes which was reduced in the presence of 5-HT. Down-regulation of ANT-1 by 5-HT was completely inhibited by PI3K inhibitor LY-294002 or by inhibition of NF-B via overexpression of a superactive IB, but not by the ERK1/2 inhibitor PD-098059. In vivo immunodetection of Bax and ANT-1 analysis in the frozen sections of the hearts showed that the Bax and ANT-1 levels were increased by 59 ± 5 and 39 ± 4%, respectively, in the knockout mice heart. 5-HT cytoprotective signaling targets mitochondria by regulating Bax and ANT-1 expression, which are altered in the 5-HT_2BR knockout mice heart.

6. The 5-HT_2BR knockout mice heart demonstrates abnormal mitochondrial structure and functions
Electron microscopic analysis in neonatal 5-HT_2BR knockout mice heart revealed pronounced mitochondrial abnormalities such as interrupted inner membrane and swollen cristae. Although damage in mitochondria is a key step leading to programmed cell death, no ultrastructural nuclear fragmentation but myofibrillar breakdown was observed in the 5-HT_2BR knockout mice heart. These structural abnormalities are reflected in the in vivo functions of mitochondria, since enzymatic activities for cytoC oxidase and succinate dehydrogenase were reduced by 40 ± 5 and 55 ± 4%, respectively, in the 5-HT_2BR knockout mice heart.

CONCLUSIONS AND SIGNIFICANCE

In cultured cardiomyocytes and transgenic animal models, overexpression of Gq-coupled receptors or their signaling molecules Gq, PLC, or p38 MAPK triggers a hypertrophic response and/or extensive hypertrophy that leads to cardiomyocyte apoptosis. On the other hand, several lines of evidence showed that circulating or locally released catecholamine and adenosine via their Gq-coupled receptors contributes to adaptive responses against hemodynamic stress or myocardial injury. Adenosine A(3) receptor activation limits myocardial injury in the isolated rat heart and improves survival in isolated cardiomyocytes, possibly by anti-apoptotic and anti-necrotic mechanisms. Stimulation of -adrenergic receptors activates calcineurin, which protects against ischemia/reperfusion-induced cell death by regulating the expression of mitochondria-associated apoptosis regulatory genes and activating hypertrophic growth. Activation of -adrenergic receptors inhibits ß-adrenergic receptor induced apoptosis. These reports indicated that activation of Gq signaling is important for protecting heart against various stresses. However, the molecular mechanisms involved are not known.

Our data for the first time show that 5-HT binding to the Gq-coupled 5-HT_2BR activates both PI3K/Akt and ERK kinases to prevent cardiomyocytes from apoptosis. We describe the mechanism of 5-HT survival signaling. 5-HT prevents cytoC release and caspase activity after serum deprivation by inhibiting ANT-1 and Bax expression via cross-talks between PI3K/Akt and ERK1/2 signaling pathways, respectively. Regulation of ANT-1 expression results from activation of nuclear factor-B via phosphatidylinositol-3 kinase/Akt. Using 5-HT_2BR knockout mice as a model of dilated cardiomyopathy, we demonstrate that the Gq-coupled 5-HT_2BR signaling regulates mitochondrial structure and function, thereby controlling myofibrillar organization in the heart (Fig. 3 ).

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic presentation of 5-HT cytoprotective signaling pathways in cardiomyocytes. 5-HT binding to 5-HT2BR activates PI3K/Akt and ERK kinases. ERK activation inhibits serum deprivation-induced Bax expression that controls mitochondrial membrane permeability. Parallel to this pathway, upon Akt activation, phosphorylated IB- triggers the degradation of IB- to release NF-B-free. This activation of NF-B by 5-HT inhibits serum deprivation-induced ANT-1 expression to regulate mitochondrial permeability. A cross-talk between PI3K/Akt/ANT-1 and ERK/Bax pathways targets mitochondria to prevent cytoC release and caspase activation in the 5-HT2BR cytoprotective signaling.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1122fje; doi: 10.1096/fj.02-1122fje

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