Canan G Nebigil

Nelly Etienne

Nadia Messaddeq

Luc Maroteaux
email: lucm@igbmc.u-strasbg.fr

Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT 2Breceptor signaling

Keywords: PI3Kinase, Akt, serotonin, Gq, ERK, Bax

Identification of factors regulating cardiomyocyte survival and growth is important to understand the pathogenesis of congenital heart diseases. Little is known about the molecular mechanism of cardiac functions triggered by serotonin. The link between signaling circuitry of external stimuli and the mitochondrial apoptotic machinery is of wide interest in cardiac diseases. Utilizing cultured cardiomyocytes and 5-HT 2B -receptor knockout mice as an animal model of dilated cardiomyopathy, for the first time we show that serotonin via the Gq-coupled 5-HT 2B -receptor protect cardiomyocytes against serum deprivation-induced apoptosis as manifested by DNA fragmentation, nuclear chromatin condensation and TUNEL labeling. Serotonin prevents cytochrome c release and caspase-9 and -3 activation following serum deprivation via cross talks between phosphatidylinositol-3 kinase/Akt and ERK1/2 signaling pathways. Serotonin binding to 5-HT 2B -receptor activates ERK kinases to inhibit Bax expression induced by serum deprivation. Serotonin via phosphatidylinositol-3 kinase /Akt can activate NF-KB that is required for the regulation of the mitochondrial adenine nucleotide translocator (ANT-1). Parallel to these observations, ultrastructural analysis in the 5-HT 2Breceptor knockout mice heart revealed pronounced mitochondrial defects in addition to altered mitochondrial enzyme activities (cytochrome oxidase and succinate dehydrogenase), and ANT-1 and Bax expressions. These findings identify 5-HT as a novel survival factor targeting mitochondria in cardiomyocytes.

Introduction

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 (5hydroxytryptamine, 5-HT) is a neurohormone that regulates cardiovascular functions [START_REF] Villalon | Serotonin receptors as cardiovascular targets[END_REF]. 5-HT is secreted from enterochromaffin cells into the blood and stored in the platelets. Circulating 5-HT can also be taken up by sympathetic neurons and vascular endothelial cells and can be co-released [START_REF] Frishman | Serotonin and the heart[END_REF]. The various biological actions of 5-HT are mediated by numerous cognate receptors. It now appears that there are at least 15 receptor subtypes that belong to four classes: 5-HT 1/5 , 5-HT 2 , 5-HT 3 , and 5-HT 4/6/7 [START_REF] Hoyer | Molecular, pharmacological and functional diversity of 5-HT receptors[END_REF]. Binding of 5-HT to the Gq-coupled 5-HT 2A , 5-HT 2B or 5-HT 2C -receptors activates phospholipase C (PLC), which initiates a rapid release of inositol trisphosphate and increases intracellular calcium levels. 5-HT 2B -receptor (5-HT 2B R) is involved in 5-HT-induced mitogenesis in which c-Src is required for cell cycle progression via the mitogen activated protein kinase (MAPK) pathway [START_REF] Nebigil | 5-Hydroxytryptamine 2B receptor regulates cell-cycle progression: Cross talk with tyrosine kinase pathways[END_REF]. Stimulation of the 5-HT 2B R results in cross talk with the 5-HT 1B/1D receptor subtype via activation of phospholipase A 2 [START_REF] Tournois | Cross-talk between 5-hydroxytryptamine receptors in a serotonergic cell line. Involvement of arachidonic acid metabolism[END_REF]. The 5-HT 2B R also activates nitric oxide synthesis through a PDZ domain [START_REF] Manivet | PDZ-dependent activation of nitric-oxide synthases by the serotonin 2B receptor[END_REF]. We have recently shown that inactivation of the Gq-coupled 5-HT 2B R gene leads to partial embryonic and neonatal lethality due to the following defects in the heart: (i) 5-HT 2B R knockout embryos exhibit a lack of trabeculae defects leading to mid-gestation lethality. (ii) Newborn 5-HT 2B R knockout mice exhibit cardiac dilation resulting from contractility deficits and structural deficits at the intercellular junctions between cardiomyocytes. (iii) In adult 5-HT 2B R knockout mice, echocardiography and electrocardiography confirm the presence of dilated cardiomyopathy [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF].

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 [START_REF] Adams | G-proteins in growth and apoptosis: lessons from the heart[END_REF][START_REF] Molkentin | Cytoplasmic signaling pathways that regulate cardiac hypertrophy[END_REF]. On the other hand, several lines of evidence showed that circulating or locally released catecholamine and adenosine via their Gq-coupled receptors contribute 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 [START_REF] Maddock | Adenosine A(3) receptor activation protects the myocardium from reperfusion/reoxygenation injury[END_REF]. Stimulation of alpha-adrenergic receptors activates calcineurin leading to cardiac hypertrophy that protects against ischemia-reperfusion-induced cell death [START_REF] De Windt | Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: An apoptosis-independent model of dilated heart failure[END_REF]. Activation of alphaadrenergic receptors inhibits beta-adrenergic receptor induced apoptosis [START_REF] Iwai-Kanai | alpha-and beta-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes[END_REF]. Alpha-adrenergic receptor activation protects also cardiomyocytes against hypoxia and serum deprivation-induced apoptosis by regulating the expression of mitochondria-associated apoptosis regulatory genes, and activating hypertrophic growth [START_REF] Zhu | Phenylephrine protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis[END_REF]. These reports indicated that activation of Gq signaling is important for protecting heart against various stresses. However, the molecular mechanisms involved are not known.

Mitochondria are an important component of the apoptotic signaling [START_REF] Kroemer | Mitochondrial control of cell death[END_REF], especially in heart [START_REF] Gill | Losing heart: the role of apoptosis in heart disease--a novel therapeutic target?[END_REF]. In vitro apoptotic signals such as serum deprivation provoke release of cytochrome c (cytoC) from mitochondria to the cytoplasm. CytoC binds and activates Apaf-1, which in turn activates caspase-9 resulting in the culminating activation of caspase-3 [START_REF] Bratton | Apoptotic death sensor: an organelle's alter ego?[END_REF][START_REF] Green | Mitochondria and apoptosis[END_REF] that cleave key substrates during the apoptotic process [START_REF] Shi | A structural view of mitochondria-mediated apoptosis[END_REF]. Several complex signal transduction pathways have been implicated in the execution of cardiomyocyte apoptosis including Ras, Raf, MAPK, (30), phosphatidylinositol-3 kinase (PI3K), and protein kinase B/Akt [START_REF] Wang | The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB[END_REF]. Recently, the Bcl-2 gene family was shown as the central players of apoptosis regulation [START_REF] Adams | Life-or-death decisions by the Bcl-2 protein family[END_REF]. Some members such as Bcl-2 and Bcl-XL inhibit apoptosis while other such as Bax, Bad, and Bak, accelerate apoptosis by altering mitochondrial membrane permeability thereby inducing cytoC release. The link between signaling circuitry of external stimuli and the mitochondrial apoptotic machinery is of wide interest in cardiac diseases.

Although ablation of the Gq-coupled 5-HT 2B R in mice leads to dilated cardiomyopathy [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF], the functional role of 5-HT 2B R in heart remains undefined. In this study, for the first time, we identified a cytoprotective 5-HT 2B R signaling linking membrane to mitochondrial apoptotic machinery utilizing in vitro and in vivo models.

Materials and methods

Material. 5-HT, PD-09858 and SB-206553 were purchased from R & D Systems. SB-203580 and LY-294002 were purchased from Promega. Phospho-p44/p42 mitogen-activated protein kinase (MAPK), phospho-Akt, Akt, phospho-IKappaB and IKappaB (IKB) alpha antibodies were purchased from Cell Signaling Technology (Beverly, MA). Mouse MF-20 anti-myosin heavy chain antibody was purchased from Hybridoma Bank Laboratories. Anti-cytoC antibody was from BD Pharmingen. All other antibodies were purchased from Santa Cruz Biotechnology. Hoechst and Apoptag Tunel detection kit were from Boehringer Mannheim. All other reagents were purchased from Sigma.

Generation of 5-HT 2B R knockout mice. Targeted mutagenesis by homologous recombination has been previously described [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF]. All animal experimentation was performed in accordance with institutional guidelines, and the French Animal Care Committee in accordance with European regulations approved protocols.

Cardiomyocyte isolation and transfection. Ventricular cardiomyocytes from neonatal mice (3-5 days old) were isolated by Percoll gradient technique as previously described (32). More than 95% of cells exhibited specific myosin heavy chain (MHC)-positive cardiomyocytes staining. Cardiomyocytes were grown on plates precoated with fibronectin (Biocoat) in the medium (Dulbecco+ HamF-12) containing 10% FCS and 5% horse serum for overnight and cytosine arabinoside (10µM) was added to prevent proliferation of non-cardiomyocytes. The cells showed spontaneous contractility within 24h after plating. For TUNEL analysis and for immunocytofluorescence experiments, cardiomyocytes were plated at a density of 10 5 cells perLab-TekTM glass slide chamber that were fibronectin coated. For Western analyses, cardiomyocytes were plated at a density of 2 x 10 6 cells per 35-mm fibronectin coated plastic culture dish.

For MAPK downregulation, cells were treated with mouse p42 and p44 MAPK sense or antisense oligonucleotides (30 µM) during 48h, then the medium was replaced by serum free medium (Dulbecco) for 24h as described previously [START_REF] Nebigil | 5-Hydroxytryptamine 2B receptor regulates cell-cycle progression: Cross talk with tyrosine kinase pathways[END_REF]. Fluorescent-labeled synthetic phosphorothioate oligodeoxynucleotides that include the ATG initiation codon of mouse p42 and p44 MAPK mRNA, antisense (5'-GCCGCCGCCGCCGCCAT-3') or sense (5'-ATGGCGGCGGCGGCGGC-3') oligodeoxyribonucleotides were previously selected and tested for their efficiency [START_REF] Sale | Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis[END_REF]. The efficiency of transfection was verified by Western blot and fluorescent microscopic analysis revealing that p42 and p44 MAPK protein levels were reduced 80 % in antisense oligonucleotide treated cells when compared to control or the sense.

DNA laddering. Visualization of apoptotic DNA fragments was performed as described [START_REF] Huang | A quantitative assay for fragmented DNA in apoptotic cells[END_REF]. Briefly, after harvesting, cells were treated with lysis buffer (10 mM Tris-HCl, pH 8, 100 mM NaCl, 25 mM EDTA, 0.5% SDS and proteinase K 0,1 mg/ml) and incubated at 50 °C for 4h. After DNA extraction, the purified DNA fragments were labeled with ( 32 P) ATP and separated by electrophoresis using a 2% agarose gel. The dried gel was exposed to Kodak X-Omat film.

Apoptosis determination by TUNEL analysis. TUNEL analysis of fragmented DNA was performed according to manufacturer's protocol as described before [START_REF] Zechner | MKK6 activates myocardial cell NF-kappaB and inhibits apoptosis in a p38 mitogenactivated protein kinase-dependent manner[END_REF]. Cells were treated with either agonists or antagonist for 4-6 days in serum free conditions. After 6 day of serum deprivation, no induction of endogenous anti-apoptotic factors was observed in cardiomyocytes. Cells then were fixed in 4% formaldehyde and permeabilized. After washing, slides were incubated with TdT terminal transferase and fluorescein-dUTP. Slides were counterstained with anti-MHC antibody and Hoechst. Cells were scored for TUNEL positive nuclei corresponding to condensed Hoechst stained nucleus. The percentage of TUNEL-positive cells was evaluated by viewing each field at x 60 magnification. Generally, 10 different microscopic fields containing 10-15 cells each were recorded for each sample. Each experiment was repeated at least three times.

Cardiomyocyte virus infection.

The adenovirus construct encoding dominant negative Akt (d3A-Akt) with a K179A/T308A/S473A mutation has been described before [START_REF] Fujio | Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart[END_REF] and used on isolated cardiomyocytes. The adenovirus construct sIB encoding the IKB superactive form S32A/S36A was used as described before [START_REF] Condorelli | TNF-alpha signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF-alpha receptor[END_REF]. Cardiomyocytes were plated in medium containing 10% FCS and 5% horse serum overnight and then incubated with adenovirus vector at a multiplicity of infection of 35 in medium containing 2% FCS. After the overnight incubation, the virus was removed and cells were cultured in serum free medium. Infection efficiency was analyzed by GFP signaling using Adeno-GFP-infected cardiomyocytes, and is consistently > 80% by this method.

Extracellular Signal-Regulated Kinase (ERK) and Akt and IKB Activity Assay and Western Blot Analysis. MAPK activities were assayed by using phospho-p42/p44 MAPK (ERK1/2) antibodies [START_REF] Nebigil | 5-Hydroxytryptamine 2B receptor regulates cell-cycle progression: Cross talk with tyrosine kinase pathways[END_REF]. Stimulated cardiomyocytes were harvested in SDS sample buffer at various time points. Approximately 20 µg of protein was separated on 10% SDS/PAGE and blotted to nitrocellulose membranes. Two identical blots were incubated with antibody specific for the dually phosphorylated, activated forms of ERK1 and ERK2, and an antibody specific for ERK2 that is independent of its phosphorylation state. Similar Western blot analysis was performed using appropriate antibody for phospho-p38 or phospho-Akt or phospho-IKB alpha. Loading homogeneity was verified by stripping and reprobing the blots. Blots were stripped with 6.25 mM Tris pH 7.5, 2% SDS and 100 mM 2mercaptoethanol for 30 min at 45°C, washed 1h and reprobed with antisera specific for p38 or Akt or IKB antibodies.

Wild type and knockout cardiomyocytes were treated with serum free medium and ±1 µM 5-HT or ± 5 nM LIF for 4-6 days and then extracted and submitted to Western blot analyses to determine the cleavage of procaspase-3 and procaspase-9 as a indicator of relative activities using anti caspase-3 and caspase-9 antibodies. Cardiomyocytes were treated with serum free medium and ±1 µM 5-HT and ± 10 µM LY-294002, or 50 µM PD-098059 for the indicated times and then extracted and submitted to Western blot analyses to determine the relative quantities of Bax and adenine nucleotide translocator (ANT-1) expression. Antibody-antigen complexes were detected with ECL kit according to manufacturer instruction. Densitometric analysis was carried out using Molecular Dynamics Image Quant software.

NF-KB Luciferase assays. Cells were transfected with plasmid coding for a luciferase driven by a minimal TK promoter upstream NF-KB responsive element. 24 hours after plating, cardiomyocytes were transfected with a plasmid pGL3-NFKB-RE-tk-luc mock (empty vector) utilizing transferrin transfection in combination with Fugene or Lipofectamine according to manufacturer recommendation [START_REF] Nebigil | 5-Hydroxytryptamine 2B receptor regulates cell-cycle progression: Cross talk with tyrosine kinase pathways[END_REF]. With this protocol, transfection efficiency in cardiomyocytes was measured by cotransfection of a ß-Galactosidase-containing construct. Cells were stimulated with different concentration of 5-HT or LIF (5 nM) for 24h. After cell lysis and removal of cell debris by centrifugation 150 µl samples of cell lysate were combined with 50 µl of luciferase buffer (25 mM Tris pH 7.8, 1 mM DTT, 15 mM MgSO 4 , 4 mM EDTA, 45 mM KHP0 4 pH 7.8, 0.3 mM luciferin, 5% glycerol, 3 mM ATP and 270 µM Coenzyme A. An MGM Instruments Optolamp II luminometer was used to measure light emission of each sample for 5 sec [START_REF] Craig | The cytoprotective effects of the glycoprotein 130 receptor-coupled cytokine, cardiotrophin-1, require activation of NF-kappa B[END_REF].

Analysis of cytosolic and mitochondrial fraction of cytoC.

For cytosolic and mitochondrial fraction of cytoC, after cardiomyocyte treatment, cells were permeabilized, sampled on 12 % SDS gel and processed for immunoblotting as described [START_REF] Ekert | DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9[END_REF]. In brief, cardiomyocytes were plated then suspended in 200 µl of 0.025% digitonin (Calbiochem) in a lysis buffer (250 mM sucrose, 20 mM Hepes, pH 7.4, 5 mM MgCl 2 , 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin). After 10 min, the cells were centrifuged (2 min, 13 000 rpm) and the supernatant was removed (cytosolic fraction). The remaining pellet was resuspended in 200 µl mitochondrial lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% triton-X-100, 50 mM Tris-HCl, pH 8.0), lysis was allowed to proceed for a further 30 min, and centrifuged. 28µl of each fraction was sampled on 12% SDS-PAGE. Densitometric analysis was performed with image analyzer (Bio-rad, GS-700).

Immunostaining. Immunohistological detection was performed on isolated cardiomyocytes with antiserum against the anti-MHC antibody (MF-20), the cytoC, ANT-1, and Bax as previously described [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF][START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF]. Signal intensity was quantified on digitalized images and calculated as the product of averaged pixel intensity per area. Densitometric analysis was carried out using Molecular Dynamics Image Quant software.

Morphological analysis of mouse heart. Transmission electron microscopy and histological techniques were performed as previously described [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF][START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF]. For histochemistry, cryostat sections of heart (7 µM) were fixed and blocked. The standard techniques were utilized for the immunohistochemistry.

Mitochondrial enzyme activity assay. The histochemical enzymatic test for succinate dehydrogenase and cytoC oxidase activities were performed on cryostat sections of unfixed heart from wild type and knockout mice as described [START_REF] Wang | Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression[END_REF].

Data analysis and statistics. All values represent average of independent experiments ± S.E.M. (n = number of experiments as indicated in the text). Comparisons between groups were performed using an ANOVA followed by a Student's t test. Significance was set at p<0.05.

Results

5-HT via 5-HT 2B R inhibits apoptosis induced by serum-deprivation in isolated cardiomyocytes

To evaluate the effect of 5-HT on cardiomyocyte survival, cardiomyocytes from neonatal heart were isolated and apoptosis was induced by serum deprivation. Apoptosis was detected first by monitoring internucleosomal cleavage with DNA laddering. DNA fragmentation was observed after the day 2 of serum deprivation and persisted throughout day 6. Stimulation by 5-HT (1 µM) or neuregulin (NRG-1, 25 ng/ml) in the serum free condition protected wild type but not 5-HT 2B R knockout cardiomyocytes from apoptosis as manifested with DNA fragmentation (Fig. 1A). To further confirm the role of 5-HT 2B R in protecting cardiomyocyte from serum-deprivation induced apoptosis; we performed both TUNEL and Hoechst staining. As shown on figure 1B, apoptotic cardiomyocytes exhibited small-condensed nuclei detected by Hoechst staining (blue) corresponding to the TUNEL staining (green). A quantitative analysis revealed that cardiomyocytes subjected to serum free medium for 4-6 days displayed an approximate 38% increase in apoptosis (Fig. 1C). In the presence of 5-HT or NRG-1 (not shown) for 4-6 days, the number of TUNEL-positive cardiomyocytes declined to 8±5%. In the presence of specific 5-HT 2B R inhibitor, SB-206553 (1 µM), 5-HT was not able to inhibit apoptosis (Fig. 1C). Conversely, the 5-HT 2B R knockout cardiomyocytes exhibited an approximate 45% apoptotic cells 4-6 days after serum deprivation and still displayed 46±5 % apoptotic cells in the presence of 5-HT (Fig. 1C). These data indicate that 5-HT via 5-HT 2B R protects cardiomyocytes from apoptosis.

PI3K/Akt and ERK1/2 activities cross talks in the anti-apoptotic pathway of 5-HT.

To investigate the potential roles of the p38, Akt, or ERK pathways in 5-HT-mediated cytoprotection, cardiomyocytes were incubated with various cell-permeable inhibitors of these pathways in the presence and absence of 5-HT (1 µM). Under these conditions, PD-098059 (50 µM) a specific inhibitor of MEK1/2, and thus ERK [START_REF] Dudley | A synthetic inhibitor of the mitogen-activated protein kinase cascade[END_REF], compromised the ability of 5-HT to protect the cardiomyocytes, resulting in about two times more apoptotic cells than observed in cells treated with 5-HT alone (from 8% in presence of 5-HT to 16% in presence of 5-HT plus PD-098059). Moreover, MAPK downregulation by transfecting cells with MAPK (ERK1/ERK2) antisense oligonucleotides blocked 5-HT cytoprotective effect to nearly the same extent as seen in the PD-098059 treated cells. LY-294002 (10 µM), a specific inhibitor of PI3K, and thus Akt [START_REF] Moule | Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and betaadrenergic agonists in rat epididymal fat cells. Activation of protein kinase B by wortmanninsensitive and -insensitive mechanisms[END_REF], also reduced the cytoprotective effects of 5-HT, resulting in about three times more apoptotic cells than observed in cells treated with 5-HT alone (24% of apoptotic cells) (Fig. 2). Accordingly, a dominant negative d3A-Akt with K179A/T308A/S473A mutations reversed 5-HT-cytoprotective effect observed with LY-294002 at a similar efficiency. p38 inhibition by SB-203580 (10 µM) had no effect on the 5-HT 2B R-dependent cytoprotective effect of 5-HT in the wild type cardiomyocytes (data not shown). 5-HT-mediated cytoprotective effect was completely reversed in the presence of both PD-098059 and LY-294002. These results show that the cytoprotective effects conferred by 5-HT against serum deprivationinduced apoptosis are not only dependent upon ERK but also PI3K/Akt pathways.

Since inhibition of the PI3K/Akt and ERK pathways compromised the cytoprotective effects of 5-HT, the ability of the 5-HT to activate these pathways was evaluated using antibodies specific for each kinase at the residues that are phosphorylated upon activation. The relative level of phospho-ERK1/2 was time-dependent and reached a maximum value of about 2-fold over control following 10 min of exposure to 1 µM 5-HT without altering total ERK-2 level (Fig. 3A). Phosphorylation of ERK1/2 by 5-HT was blocked by 50 µM PD-098059 (Fig. 3A-C). In 5-HT 2B R knockout cardiomyocytes, 5-HT was not able to activate ERK1/2. However, leukemia inhibitory factor (LIF, 5 nM), a known survival factor still phosphorylates ERK1/2 in the 5-HT 2B R knockout cardiomyocytes. The levels of phospho-Akt (ser 473 and thr 308) were maximal following 15 min of exposure to 5-HT and amounted to about 3-fold over control (Fig 3B). Phosphorylation of Akt was blocked by 10 µM LY-294002 but not by 50 µM PD-098059 without altering total Akt expression (Fig. 3C). Both ERK and Akt were transiently activated by 5-HT or LIF. Moreover, no p38 phosphorylation in response to 5-HT could be evidenced (not shown), confirming that p38 activation is not involved in 5-HTdependent cytoprotection. These results are consistent with roles for ERK1/2 and Akt signaling pathways in 5-HT 2B R-mediated cytoprotection against serum deprivation-induced apoptosis.

5-HT via 5-HT 2B R activates the IKB-alpha/NF-KB signaling pathway

To investigate the downstream regulators of ERK1/2 and PI3K/Akt kinase in cardiomyocytes, the effect of 5-HT on the activation of NF-KB was assessed using NF-KB/luciferase responsive element reporter gene. 5-HT stimulated NF-KB (Rel A)-dependent reporter transcription in wild type cardiomyocytes, while in 5-HT 2B R knockout cardiomyocytes, LIF but not 5-HT induced NF-KB activity (Fig. 4A). NF-KB staining in cardiomyocytes, initially localized in the cytoplasm in a punctuate pattern, was strikingly increased in the nucleus after 15 min of 5-HT treatment (Fig. 4B). Subsequently, IKB-alpha phosphorylation in the presence of 5-HT was determined. 5-HT phosphorylated IKB-alpha reaching to maximum at 15 min (data not shown). Indeed, phosphorylated form of IKB-alpha undergoes gradual degradation at 15 min (Fig. 4C). The time for nuclear translocation of NF-KB by 5-HT is consistent with the IKB-alpha degradation (Fig 4B). This phosphorylation of IKB-alpha by 5-HT was completely inhibited by the PI3K inhibitor LY-294002 but not by MAPK inhibitor PD-098059 (Fig. 4C). These results show that in cardiomyocytes 5-HT via PI3K/Akt induces IKB-alpha degradation thereby NF-KB nuclear translocation, which activates NF-KB-dependent gene transcription.

5-HT via 5-HT 2B R prevents cytoC redistribution and caspase cleavage in cardiomyocytes

Next, we investigated the targets of 5-HT signaling for protecting cardiomyocytes from apoptosis. In wild type cardiomyocytes, cytoC was localized in the mitochondrial fraction, and no cytoplasmic cytoC was observed (Fig. 5A). 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) and LIF (5 nM). In 5-HT 2B R knockout cardiomyocytes, basal cytoplasmic cytoC was slightly increased and after serum deprivation, cytoplasmic cytoC reached to the maximum that was also observed in wild type cardiomyocytes. Conversely, 5-HT did not prevent cytoC translocation from mitochondria to cytosol, whereas LIF totally prevent this translocation in the 5-HT 2B R knockout cardiomyocytes (Fig. 5A). The total cytoC levels in wild type and 5-HT 2B R cardiomyocytes were similar. These data indicate that only 5-HT-mediated survival effects are impaired in 5-HT 2B R knockout cardiomyocytes.

Recent studies have demonstrated that release of cytoC from mitochondria leads to activation of caspase cascade in cardiomyopathic heart [START_REF] Adams | Cardiomyocyte apoptosis induced by Galphaq signaling is mediated by permeability transition pore formation and activation of the mitochondrial death pathway[END_REF][START_REF] Narula | Apoptosis and cardiomyopathy[END_REF]. 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 2B R knockout cardiomyocytes (Fig. 5B).

These data indicate that 5-HT via 5-HT 2B R prevents cytoC redistribution from mitochondria thereby inhibiting caspase activity to protect cardiomyocytes from serum deprivation induced apoptosis.

5-HT via 5-HT 2B R regulates Bax and ANT-1 expression

Next we investigated how 5-HT cytoprotective signaling prevents cytoC redistribution from mitochondria. The intrinsic mitochondria-dependent apoptotic pathway in cardiomyocytes is largely dependent on anti-apoptotic and pro-apoptotic members of the Bcl-2 family proteins. Bcl-2 inhibits apoptosis by blocking the release of cytoC from mitochondria during cellular stress whereas proapoptotic member Bax causes cytoC release [START_REF] Adams | The Bcl-2 protein family: arbiters of cell survival[END_REF]. We investigated whether 5-HT regulates Bax expression thereby controlling cytoC release in isolated cardiomyocytes. In the apoptotic conditions, Bax expression increased 2 fold that was reduced in the presence of 5-HT. The effect of 5-HT was to downregulate Bax expression that was completely prevented by ERK1/2 inhibitor, PD-098059 (Fig. 6A). When the same blot was revealed with anti-phospho-Bad antibody no significant difference was observed in the Bad level or phosphorylation stage. These effects of 5-HT were completely absent in 5-HT 2B R knockout cardiomyocytes. However, inhibition of PI3K by LY-294002 or inhibition of NF-KB by the adenovirus sIB encoding the IKB superactive form S32A/S36A did not change 5-HTmediated regulation of Bax expression (Fig. 5A-6A). These data indicate that, in the cardiomyocytes, ERK1/2 activation by 5-HT is involved in regulating Bax expression, whereas PI3K/Akt did not alter Bax levels.

Next, we asked if PI3K/NF-KB signaling regulates also the mitochondrial membrane permeability. ANT-1 is a component of mitochondrial membrane permeability transition pore [START_REF] Desagher | Mitochondria as the central control point of apoptosis[END_REF] and the only mitochondrial carrier for ADP and ATP. Since ANT-1 plays an important role in the disturbed cardiomyocyte metabolism in the dilated cardiomyopathy and ANT-1 mutant mice exhibited severe cardiomyopathy [START_REF] Graham | A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator[END_REF], we investigated possible regulation of ANT-1 in 5-HT cytoprotective signaling. In the apoptotic conditions, ANT-1 expression was increased in the wild type cardiomyocytes that was reduced in the presence of 5-HT. Downregulation of ANT-1 by 5-HT was completely inhibited by PI3K inhibitor LY-294002 but not by ERK1/2 inhibitor, PD-098059 (Fig. 6B). When NF-KB was blocked with the adenovirus sIB encoding the superactive IKB, the 5-HT effect on ANT-1 expression was completely inhibited (Fig. 6B).

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 38.9±4 % respectively in the knockout mice heart (Fig. 6B). These results were confirmed by RT-PCR analysis (not shown). Our in vivo and in vitro data indicate that 5-HT/5-HT 2B R cytoprotective signaling targets mitochondria by regulating Bax and ANT-1 expression and that 5-HT-cytoprotective signaling is impaired in the 5-HT 2B R knockout mice heart.

The 5-HT 2B R knockout mice heart demonstrates abnormal mitochondrial structure and functions

Next we investigated the mitochondrial structure in the 5-HT 2B R knockout mice heart. Electron microscopic analysis in neonatal (Fig. 7A) and 6 weeks old (Fig. 7B) 5-HT 2B R 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 2B R knockout mice heart. To investigate how these structural abnormalities are reflected in the in vivo functions of mitochondria, enzymatic histochemical staining for cytoC oxidase (COX-2) and succinate dehydrogenase (SDH) activity were performed. These staining revealed reduced activity of both SDH and COX-2 by 40±5 and 55±4% respectively in the 5-HT 2B R knockout mice heart (Fig. 7B). These data indicate that secondary to structural defect in mitochondria of 5-HT 2B R knockout mice heart, the electron chain transport and oxidative phosphorylation were disturbed as observed in human dilated cardiomyopathy [START_REF] Narula | Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy[END_REF][START_REF] Von Harsdorf | E2F-1 overexpression in cardiomyocytes induces downregulation of p21CIP1 and p27KIP1 and release of active cyclin-dependent kinases in the presence of insulin-like growth factor I[END_REF].

Discussion

Although a number of signaling pathways that lead to dilated cardiomyopathy and heart failure have been discovered, the factors that mediate distinct forms of cardiac hypertrophy, apoptosis and survival are not yet elucidated. Utilizing cultured cardiomyocytes, and 5-HT 2B R knockout mice as a model of dilated cardiomyopathy, we demonstrate that 5-HT 2B R signaling regulates mitochondrial structure and function thereby controlling apoptosis and myofibrillar organization in the heart.

Suppression of Apoptosis by 5-HT. Overexpression of Gq-coupled receptors or Gq protein itself in cardiomyocytes contributes to the development of hypertrophy and/or ultimate decompensation of cardiac hypertrophy leading to apoptosis [START_REF] Adams | G-proteins in growth and apoptosis: lessons from the heart[END_REF][START_REF] Dorn | Low-and high-level transgenic expression of beta2-adrenergic receptors differentially affect cardiac hypertrophy and function in Galphaq-overexpressing mice[END_REF][START_REF] Wettschureck | Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes[END_REF]. On the other hand, evidence suggests that hormones such as angiotensin II, endothelin 1, norepinephrine and prostaglandin F2 alpha via their cardiac Gq-coupled receptors contribute to adaptive responses after hemodynamic stress or myocardial injury [START_REF] Dorn | Gq signaling in cardiac adaptation and maladaptation[END_REF]. However, the mechanism by which Gq-coupled receptors mediate survival effects has not been clearly elucidated. For the first time, our data show that 5-HT can protect cardiomyocytes from apoptosis following serum deprivation. This protective effect is specifically mediated by the Gq-coupled 5-HT 2B R: the cytoprotective effect of 5-HT was completely blocked by a specific 5-HT 2B R inhibitor in wild type cardiomyocytes, and absent in the 5-HT 2B R knockout cardiomyocytes. Previously, neural crest cells have been shown to exhibit apoptosis following the treatment of embryos with a 5-HT 2B R antagonist [START_REF] Choi | 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells[END_REF], supporting the survival role of 5-HT.

Mechanisms of protective action of 5-HT. 5-HT protects cardiomyocytes from apoptosis by preventing of cytoC redistribution thereby inhibiting caspase-3 and -9 activation. Our data indicate that in the 5-HT anti-apoptotic signaling, ERK1/2 and PI3K/Akt crosstalk to regulate cardiomyocyte survival. In the present study, inhibitors of MAPK efficiently blocked the activation of ERK1/2 by 5-HT, but did not interfere with the function of PI3K/Akt indicating that these two signaling pathway are independent of each other. Our observations are consistent with previous findings showing that both MAPK and PI3K/Akt signaling pathways are essential for antiapoptotic action of both interleukin-5 and stem cell factor [START_REF] Huang | Mcl-1 is a common target of stem cell factor and interleukin-5 for apoptosis prevention activity via MEK/MAPK and PI-3K/Akt pathways[END_REF]. The downstream targets of MAPK and PI3K/Akt were unclear in cardiomyocytes. For the first time, we show that 5-HT mediated PI3K/Akt signaling is involved in IKB-alpha/NF-KB activation. Previously, it has been shown that PDGF activates Akt that is directly involved in IKB/NF-KB activation [START_REF] Romashkova | NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling[END_REF]. Akt is also involved in cardiotrophin activation of NF-KB (p65) and cytoprotection of cardiomyocytes. Our results indicate that Gq-coupled 5-HT 2B R activation leads to degradation of IKB-alpha and subsequent translocation of NF-KB (p50) to the nucleus where NF-KB can regulate anti-apoptotic gene expression.

Mitochondria are targets of 5-HT-mediated cytoprotection. CytoC release due to impaired mitochondria has been observed in several models of apoptosis in cardiomyocytes [START_REF] Narula | Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy[END_REF][START_REF] Von Harsdorf | Signaling pathways in reactive oxygen speciesinduced cardiomyocyte apoptosis[END_REF], however the importance of mitochondria as a direct target for factors protecting cardiomyocytes from apoptosis has not been examined yet. Our findings demonstrate that the 5-HT-induced cardiomyocyte cytoprotection involves cross talks between PI3K and ERK1/2 pathways that lead to the regulation of ANT-1 and Bax expression to protect of mitochondrial membrane permeability and cytoC release. In human pancreatic cancer cells, MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-XL, and Mcl-1 and promotes survival [START_REF] Boucher | MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells[END_REF]. While the Bcl-2 family of proteins, at least in part, controls the mitochondrial apoptosis, we demonstrate that only ERK1/2 inhibition can overturn 5-HT-induced down regulation of Bax in cardiomyocytes. Parallel to these in vitro findings, a robust increased expression of Bax was observed in the 5-HT 2B R knockout mice heart. Bax forms membrane pores that control mitochondrial permeability and release cytoC [START_REF] Saito | beta-Adrenergic pathway induces apoptosis through calcineurin activation in cardiac myocytes[END_REF]. Bcl-2 and Bcl-XL inhibit formation of these pores, however, no alteration in the expression of these genes could be detected by RT-PCR analysis of the 5-HT 2B R knockout mice heart mRNA (data not shown). We observed that Bax expression level was regulated by ERK1/2 activation in the 5-HT cytoprotective signaling, but the mechanism by which ERK1/2 activation controls Bax expression remains to be investigated.

We also observed that PI3K/Akt signaling maintains the integrity of mitochondrial permeability via a mechanism that is distinct from regulating Bcl-2 expression or Bad phosphorylation in cardiomyocytes [START_REF] Flusberg | Cooperative control of Akt phosphorylation, bcl-2 expression, and apoptosis by cytoskeletal microfilaments and microtubules in capillary endothelial cells[END_REF]. Moreover, our data show that PI3K/Akt/NF-KB controls mitochondrial membrane permeability by regulating the ANT-1 expression. ANT-1 is the only mitochondrial transport system for nucleotides, an important link for energy production and accumulation process. It plays an important role in the disturbed myocardial metabolism in the dilated cardiomyopathy. ANT-1 mutant mice exhibited severe cardiomyopathy [START_REF] Graham | A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator[END_REF]. Impaired ANT-1 function and increased ANT-1 levels were observed in heart tissue from patients with dilated cardiomyopathy [START_REF] Portman | Adenine nucleotide translocator in heart[END_REF] and point mutations in ANT-1 gene have been reported in humans to generate genetic mitochondrial disease [START_REF] Kaukonen | Role of adenine nucleotide translocator 1 in mtDNA maintenance[END_REF]. ANT-1 overexpression leads to the phenotypic alteration of the apoptosis, i.e. collapsed mitochondrial membrane potential, cytoC release, caspase activation and DNA degradation (5). In the 5-HT 2B R knockout mice heart, elevated expression level of ANT-1 is detected and we present evidence that ANT-1 is a main target of PI3K/Akt/NF-KB signaling that controls mitochondrial permeability in the cardiomyocytes. Moreover, transgenic mice overexpressing 5-HT 2B R in the heart exhibit decreased ANT-1 levels in the heart (unpublished observation). If Bax and ANT-1 interact in cardiomyocytes is currently unknown.

Evidence of mitochondrial involvement for 5-HT-mediated cytoprotection in vivo. Mitochondrial dysfunction has been reported in human cardiac diseases including ischemic and nonischemic heart failure, myocardial infarction, arrhythmia and myocarditis [START_REF] Cook | Cardiac myocyte apoptosis[END_REF]. We also observed mitochondrial structural defects in the 5-HT 2B R knockout mice heart by electron microscopy analysis. In the knockout mice heart, reduced SDH and COX-2 activities are indicative of altered functions of electron transport complexes II and IV, respectively. Increased lactate plasma levels in knockout mice confirmed this observation (not shown). Decreased oxidative phosphorylation and respiration that lead to lactate production have also been observed in mitochondrial myopathies of human and in other animal models for dilated cardiomyopathy [START_REF] Narula | Apoptosis and cardiomyopathy[END_REF] Although damage in mitochondria is a key step leading to programmed cell death, no typical apoptotic bodies were observed in the 5-HT 2B R knockout mice heart despite impaired myofibrillar structure [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF]. Knockout cardiomyocytes may be in the pre-apoptotic stage long before nuclear events became morphologically manifested in vivo. Activation of caspases by cytoC in the failing myocardium induces breakdown of contractile proteins, which constitute the basis of impaired systolic ventricular function without inducing nuclear apoptosis [START_REF] Communal | Functional consequences of caspase activation in cardiac myocytes[END_REF][START_REF] Moretti | Essential myosin light chain as a target for caspase-3 in failing myocardium[END_REF]. Increased troponin I plasma levels in knockout mice confirmed this observation [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF]. Accordingly, evidence of cytoC redistribution from mitochondria to cytoplasm and caspase activation without nuclear morphology of apoptosis has also been observed in idiopathic dilated cardiomyopathic in human heart [START_REF] Reed | Postmitochondrial regulation of apoptosis during heart failure[END_REF]. Our in vivo and in vitro data clearly show that 5-HT/5-HT 2B R signaling targets mitochondria. Recently, we observed that transgenic mice overexpressing 5-HT 2B R in the heart exhibit mitochondrial proliferation and hypertrophy in the ventricular wall (unpublished observations).

Summary. Our data for the first time shows that 5-HT binding to 5-HT 2B R activates both PI3K/Akt and ERK kinases in cardiomyocytes to protect mitochondrial damage thereby preventing apoptosis (Fig. 8). 5-HT prevents cytoC release and caspase-9 and -3 activation following serum deprivation by inhibiting ANT-1 and Bax expression via cross talks between PI3K/Akt and ERK1/2 signaling pathways, respectively. The regulation of ANT-1 expression results from activation of NF-KB via PI3K /Akt. Utilizing 5-HT 2B R knockout mice as a model of dilated cardiomyopathy, we demonstrate that the Gq-coupled 5-HT 2B R signaling regulates mitochondrial structure and function (Fig. 8). or antisense (MAPK-AS) fluorescent-labeled synthetic phosphorothioate oligodeoxynucleotides (30 µM) during 48 hours, then in serum free medium for 24 hours, then with ±1 µM 5-HT for 4 days. Expression levels of each protein were approximately equal, as determined by Western blot analysis (not shown). To inhibit Akt activity, similar experiment was performed with ±1 µM 5-HT and ±10 µM LY-294002 (LY). To downregulate Akt, cardiomyocytes were plated in medium containing 10% FCS and 5% horse serum overnight and then incubated with adenovirus construct encoding a dominant negative Akt (d3A-AKT) with a K179A/T308A/S473A mutation in medium containing 2% FCS. After the overnight incubation, the virus was removed and cells were cultured in serum free medium (SF). Infection efficiency was evaluated by counting GFP positive cells using Adeno-GFP-infected cardiomyocytes. Each inhibitor trial was paired with its own control, 6 days after cells were fixed for TUNEL analysis. The number of TUNEL-positive cells in each microscopic field was determined, as described in methods and then normalized to the total number of cells in that field to obtain the numbers of TUNEL-positive cells as a percent of the total. Bars: 500 µm. Each value represents the mean of 10 separate fields (~150 cells/field) ± S.E.M. These experiments are representative of three similar experiments. M indicates mock transfection with the virus.

Legends to figures

Figure 1 . 5 -

 15 Figure 1. 5-HT via 5-HT 2B R acts as a survival factor in cardiomyocytes A) 5-HT prevents DNA laddering induced by serum deprivation in isolated wild type cardiomyocytes. DNA laddering was observed from 2, 4, to 6 days of serum deprivation (SF). The effect of 5-HT (1 µM) or neuregulin (NRG-1, 25 ng/ml, neu) was studied by DNA laddering at 4 days of serum deprivation in wild type (+/+) or knockout cardiomyocytes (-/-). The DNA size marker is in base pairs (bp). B) 5-HT prevents apoptosis induced by serum deprivation in wild type cardiomyocytes. Myocardial cells plated 4-6 days in serum-free (SF) media, ±1 µM 5-HT or ±1 µM SB-206553 (SB), a specific 5-HT 2B R receptor antagonist, were then fixed for TUNEL (green) analysis and Hoechst (blue) staining. MHC staining (red) shows the pure population of cultured cardiomyocytes. Illustrations show cardiomyocytes that are representative of the cell population observed following the treatments indicated above. White arrows indicate double stained, Hoechst and TUNEL positive cells with small and condensed nuclei. C) Quantitative analysis of TUNEL staining. The number of TUNEL-positive cells in each microscopic field was determined, as described in methods, and then normalized to the total number of cells in that field to obtain the numbers of TUNEL-positive cells as a percent of the total. Each value represents the mean of 10 separate fields (~15 cells/field) ± S.E.M. (P<0.05, star). These experiments are representative of three similar experiments.

Figure 2 .

 2 Figure 2. Effects of cell-permeable inhibitors and dominant negative signaling proteins on 5-HTmediated inhibition of apoptosis. Illustrations show wild type cardiomyocytes that are representative of the cell population following treatments inhibiting either MAPK or PI3K pathways. For MAPK inhibition, cardiomyocytes were treated in serum free (SF) conditions with ±1 µM 5-HT and ± 50 µM PD-098568 (PD). For MAPK downregulation, cells were treated with mouse p42 and p44 MAPK sense (MAPKS)or antisense (MAPK-AS) fluorescent-labeled synthetic phosphorothioate oligodeoxynucleotides (30 µM) during 48 hours, then in serum free medium for 24 hours, then with ±1 µM 5-HT for 4 days. Expression levels of each protein were approximately equal, as determined by Western blot analysis (not shown). To inhibit Akt activity, similar experiment was performed with ±1 µM 5-HT and ±10 µM LY-294002 (LY). To downregulate Akt, cardiomyocytes were plated in medium containing 10% FCS and 5% horse serum overnight and then incubated with adenovirus construct encoding a dominant negative Akt (d3A-AKT) with a K179A/T308A/S473A mutation in medium containing 2% FCS. After the overnight incubation, the virus was removed and cells were cultured in serum free medium (SF). Infection efficiency was evaluated by counting GFP positive cells using Adeno-GFP-infected cardiomyocytes. Each inhibitor trial was paired with its own control, 6 days after cells were fixed for TUNEL analysis. The number of TUNEL-positive cells in each microscopic field was determined, as described in methods and then normalized to the total number of cells in that field to obtain the numbers of TUNEL-positive cells as a percent of the total. Bars: 500 µm. Each value represents the mean of 10 separate fields (~150 cells/field) ± S.E.M. These experiments are representative of three similar experiments. M indicates mock transfection with the virus.

Figure 3 . 5 -

 35 Figure 3. 5-HT activates ERK1/2 and Akt kinases.Cardiomyocytes were treated ± 1 µM 5-HT and 10 µM LY-294002 (LY), or 50 µM PD-098059 (PD) for the indicated times, then extracted and submitted to Western blot analysis to determine the relative quantities of phosphorylated and total, Akt, or ERK, as described in methods. A) 5-HT increases ERK1/2 phosphorylation in a time-dependent manner. As revealed using anti-phospho-MAPK antibody (anti-pMAPK thr 202; tyr 204) (pTEpYMAPK), the ERK1/2 phosphorylation peaks at 10 min in wild type cardiomyocytes (+/+) but not in knockout cardiomyocytes (-/-). Two identical blots were incubated with antibody specific for the dually phosphorylated, activated forms of ERK1 and ERK2, and an antibody specific for ERK2 that is independent of its phosphorylation state to verify samples homogeneity. ERK activation was evaluated in presence of 5-HT ± PD-098059 or ± LY-294002 and in presence of LIF in both wild type and knockout cardiomyocytes. B) 5-HT enhances Akt phosphorylation in a time dependent manner. As revealed with anti phospho-Akt antibodies (anti-pAkt ser 473 or anti-pAkt thr 308) Akt phosphorylation peaks at 15 min in wild type cardiomyocytes (+/+). 5-HT does not modify the Akt expression as revealed by anti Akt antibody (anti-Akt) after blot restripping. C) Akt activation by 5-HT is completely blocked by LY-294002, but not by PD-098059. Akt activation by 5-HT was evaluated in presence of inhibitors LY-294002, or PD-098059. Quantification of these experiments presented in the graphs was determined using densitometry and Molecular Dynamics ImageQuant software and shown as average S.E.M.. IP: immunoprecipitation, IB: immunoblot. Blots are representative of 3 separate experiments.

Figure 4 .

 4 Figure 4. Effects of 5-HT on IKB degradation, NF-KB-mediated transcription and cellular localization. A) 5-HT activates NF-KB-mediated transcription. Wild type and knockout cardiomyocytes were co-transfected with an (pGL3-NF-KB-RE-tk-luc,12 µg) and ß -galactosidase reporter (20 µg).Following 24 hours in serum-free medium (SF), the cells were treated for another 24 hours with serumfree medium ± 5-HT or LIF at the concentrations indicated, then extracted and assayed for luciferase and ß-galactosidase activities as described in methods. This experiment is representative of three similar experiments. B) 5-HT induces p50/NF-KB nuclear translocation. Cardiomyocytes were incubated in serum-free media for 24 hours, treated ±1 µM 5-HT for the indicated times and stained for p50/NF-KB (NFKappaB). Primary antibody localization was visualized using CY3-conjugated secondary antibody to show the localization of NF-KB after 5-HT treatment. Shown are single myocytes that are representative of the cell population following the treatments indicated. This experiment is representative of three similar experiments. Bars: 5 µm. C) 5-HT-mediated IKB phosphorylation is inhibited by PI3K inhibitor. Cardiomyocytes were treated in serum free medium with ±1 µM 5-HT and ± 10 µM LY-294002 (LY), or ± 50 µM PD-098059 (PD) for 4 days, then extracted and submitted to Western blot analyses to determine the relative quantities of phosphorylated and total IKB after blot restripping. This experiment is representative of three similar experiments.

Figure 5 .

 5 Figure 5. Effects of 5-HT on cytoC cellular localization. A) 5-HT prevents translocation of cytoC from mitochondria to cytosol in wild type cardiomyocytes. Wild type and in 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, as shown and as described in methods. IP: immunoprecipitation; IB: immunoblot. Blots are representative of 3 separate experiments. The values presented in the graphs were determined using densitometry and Molecular Dynamics ImageQuant software and shown as average S.E.M. 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 a indicator of relative activities using anti caspase-3 and caspase-9 antibodies. Procaspase-3; CPP32F, cleaved caspase-3; CPP32C, IB: immunoblot. This experiment is representative of three similar experiments.

Figure 6 .

 6 Figure 6. Effects of 5-HT on Bax and ANT-1 levels. A) Reduction of Bax expression by 5-HT. Wild type cardiomyocytes were treated in serum free (SF) medium with ±1 µM 5-HT and ± 10 µM LY-294002 (LY) or 50 µM PD-098059 (PD) for 4 days, then extracted and submitted to Western blot analyses to determine the relative quantities of Bax, as described in methods. Phospho-Bad (anti-pBad ser112, ser 155) expression was assessed after restripping of the same blot. The values determined by densitometry using Molecular Dynamics ImageQuant software are shown as a graph ± S.E.M.. B) Reduction of ANT-1 expression by 5-HT. Cardiomyocytes were treated ±1 µM 5-HT and ± 10 µM LY-294002 or 50 µM PD-098059 or ± Adenovirus sIB (sIB) or ± Adenovirus GFP (M), then extracted and submitted to Western blot analyses to determine the relative quantities of ANT-1, as described in methods. Actin expression was assessed after restripping of the same blot. The values determined by densitometry using Molecular Dynamics ImageQuant software are shown as a graph. C) Bax and ANT-1 expression are increased in knockout mice heart. Bax and ANT-1 expression were evaluated by immunohistochemical staining of cryosections of wild and knockout mice heart. Bars: 500 µm. The values of staining intensity determined using Molecular Dynamics ImageQuant software are shown as a graph ± S.E.M.. This experiment is representative of three similar experiments.

Figure 7 .

 7 Figure 7. In vivo mitochondrial defects in 5-HT 2B R knockout mice heart. A) Mitochondrial ultrastructural defects in 5-HT 2B R knockout newborn mice heart. The analysis of the compact layer of newborn mice heart was performed by transmission electronic microscopy on ultra-thin sections. Mitochondria (m) with apparent intact morphology in wild type heart (+/+) and mitochondria with defective membrane (arrow) are indicated in knockout mice heart (-/-). Bars: 0.5 µm. Inserts show higher magnification of the mitochondrial defects. Bars: 0.2 µm. N: nucleus, Z: z-stripe; B) Mitochondrial ultrastructural defects in 5-HT 2B R knockout adult mice heart. Analysis by transmission electronic microscopy of ultra-thin sections of 6 weeks old mice heart knockout (-/-) and wild type (+/+) in the compact layer was performed. Fused mitochondria (m) with apparent intact outer membrane (arrow) and defective inner membrane are indicated. N: nucleus, Z: z-stripe; Bars: 0.5 µm. C) Decreased mitochondrial enzyme activities in the knockout mice heart. Enzymatic histochemical staining for cytoC oxidase (COX-2, brown) and succinate dehydrogenase (SDH, blue) activity were determined in sections of the heart of wild type (+/+) and knockout newborn mice (-/-). Bars: 500 µm. SDH and COX-2 activities in the mice heart are shown as percent of wild type values. The values of staining intensity were determined using Molecular Dynamics ImageQuant software and shown as a graph ± S.E.M.. Illustrations are representative of at least 3 separate experiments.

Figure 8 .

 8 Figure 8. Schematic presentation of 5-HT cytoprotective signaling pathways in cardiomyocytes. 5-HT binding to 5-HT 2B R activates both 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 IKB-alpha triggers the degradation of IKB-alpha to release NF-KB free. This activation of NF-KB by 5-HT inhibits serum deprivation-induced ANT-1 expression to regulate also mitochondrial permeability. A cross talk between PI3K/Akt/ANT-1 and ERK/ Bax pathways targets mitochondria to prevent cytoC release and caspases activation in the 5-HT 2B R cytoprotective signaling. In the 5-HT 2B R knockout mice as a model of dilated cardiomyopathy, we demonstrate that the Gq-coupled 5-HT 2B R signaling regulates mitochondrial structure and function thereby controlling myofibrillar organization in the heart.

Acknowledgements

We thank Dr. K. Niederreither for critical reading of the manuscript and for helpful discussions. We wish to acknowledge Dr. K. Walsh for the dominant negative Akt adenovirus, Drs. M.V.G. Latronico and G. Condorelli for the adenovirus expressing the mutant form of IKB and Dr. H. Gronenmeyer for GL3-NF-KB-Luc. We thank P. Hickel for excellent technical assistance. This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Hôpital Universitaire de Strasbourg, the Université Louis Pasteur, and by grants from the Fondation de France, and the Association pour la Recherche contre le Cancer # 9503, 7389. C. G. N. is supported by a fellowship from the Fondation pour la Recherche Médicale.


Jana Dlouhá
email: dlouha@lmgc.univ-montp2.fr

Joseph Gril

Bruno Clair

Tancrède Alméras

Evidence and modelling of physical aging in green wood

Keywords: Physical aging, Creep, Green wood

Physical aging typical of semi-crystalline polymers was evidenced in green (never dried) wood from three tropical hardwood species. The assumption of uniform aging rate was verified by the construction of master curves from series of tensile creep tests in the fibre direction, performed at increasing time elapsed after a quench following heating above the glassy transition. The rheological response during periods of creep small enough to neglect the progress of aging was described by a model made of a spring in series with a parabolic dash-pot where only the characteristic time depends on the aging time. The model was able to describe results obtained by a previous author on softwood loaded transversally to the fibres. The possible role of a transient adsorption process consecutive to the quench is discussed.

Introduction

As a natural hygroscopic polymer, wood exhibits a complex viscoelastic behaviour depending both on temperature and moisture content. In the living stem, however, the cell cavities contain an excess of free water that keeps the cell walls above moisture saturation. Thus, the rheological behaviour of the building material of a tree, "green" or never dried wood, can be approached by considering essentially the action of temperature, although small variations of bound water content are likely to accompany temperature changes [START_REF] Skaar | Thermodynamics of the swelling of wood[END_REF].

On the other hand, the process of wood formation involves the production of considerable pre-stressing. Newly formed wood generally contracts after deposition at tree periphery, producing the forces required to control the shape of the stems and enhance their stability and resulting in a well-known field of residual stresses at the macroscopic (stem) level [START_REF] Archer | On the distribution of tree growth stresses. Part 2: stresses due to asymmetric growth strains[END_REF][START_REF] Wilson | Tree design: some biological solutions to mechanical problems[END_REF]. This pre-stressing process, however, needs to be considered also at the cellular level. Wood cell wall is a multilayered composite where each layer contains cellulosic reinforcements (microfibrils) oriented at different angles relative to the cell axis and deposited concentrically during cell wall formation [START_REF] Kollmann | Principles of wood science and technology[END_REF]. Cell maturation and death, occurring within few weeks of its life, induce anisotropic deformations of each layer, incompatible to a great extent, so that at the end of the process the cell wall is pre-stressed [START_REF] Archer | On the origin of growth stresses in trees. Part 1: micro mechanics of the developing cambial cell wall[END_REF]. The external stress supported by the wood is modified in the later years when new wood rings have been added to the stem, but a large proportion of these residual stresses and strains remain locked in the cell walls all through the tree life and even after the wood has been cut and processed [START_REF] Kubler | Growth stresses in trees and related wood properties[END_REF][START_REF] Gril | Tree mechanics and wood mechanics: relating hygrothermal recovery of green wood to the maturation process[END_REF].

To produce biomechanical models valid over the whole life, the time-dependent behaviour of green wood needs to be predicted. The use of series of creep or relaxation tests at increasing temperature levels is the obvious way to identify such long-term rheological models, but several obstacles might produce difficulties. First, the complex polymeric composition might produce overlapping of phenomena so that the observed mechanical response is not described easily by simple equivalence between time and temperature [START_REF] Bardet | Modelling the transverse viscoelasticity of green wood using a combination of two parabolic elements[END_REF]. Second, the recovery of locked-in strain due to the pre-stressing during wood formation at stem periphery is likely to occur during the tests at increasing temperature levels and produce non-negligible perturbation of the viscoelastic data [START_REF] Gril | Tree mechanics and wood mechanics: relating hygrothermal recovery of green wood to the maturation process[END_REF].

Residual strains can be removed or at least significantly recovered by heating above the glassy transition (T g ), typically 65 • C for the in situ saturated lignins of a hardwood [START_REF] Kelley | Relaxation behaviour of the amorphous components of wood[END_REF]. However, when the wood is cooled below T g after the heating, physical aging is likely to occur: Upon cooling below T g , due to the low chain mobility, the polymer cannot shrink fast enough to keep equilibrium volume, and the material properties undergo slow and gradual changes towards its new equilibrium state [START_REF] Struik | Physical aging of amorphous polymers and other materials[END_REF]. Consequently, the tests performed too shortly after the heating and subsequent cooling may produce data poorly connected with the real behaviour of a never-heated green wood.

Physical aging has been already observed in wood. A number of arguments for its occurrence were given for wood in the hygroscopic range [START_REF] Hunt | Evidence of a physical ageing phenomenon in wood[END_REF]. The effects of a quench (rapid cooling) on wet wood properties was studied by Nakano [START_REF] Nakano | Effects of quenching on relaxation properties of wet wood[END_REF]) who reported a temporary change in viscoelastic properties with a new equilibrium state definitely reached at the end of 10 h. Free volume creation has been ascribed to the freezing of molecular chains of wood components, most likely lignin, during the quench. On the other hand, [START_REF] Ishimaru | Mechanical properties of wood in unstable states caused by changes in temperature and/or swelling[END_REF] has investigated the effect of quenching rate on wood creep properties and observed that the effect of cooling had not completely disappeared after 40 days, but they have not examined the aging phenomenon in more details. In these studies, the wood was usually tested transversally to the fibres, in the radial (R) or tangential (T) direction, whereas for biomechanical consideration, the behaviour in the longitudinal direction (L) is the most important.

The objective of this work was to verify the occurrence of physical aging in green wood, and, if possible, quantify it based on tensile creep tests in L direction. In his pioneer study on physical aging of polymers, Struik proposed an experimental protocol to investigate the effect of aging time on small-strain creep properties after a quench based on the "snapshot" assumption-testing time is very short relative to the aging time. Assuming that aging changes by a negligible amount during each creep test, the creep response can be separated from the aging effect. Struik has also shown that in the case of simple polymers, creep curves for different aging times can be superimposed into a single master curve, and he proposed a simple mathematical description of the log time shift. We adopted Struik procedure, intending to verify the applicability of his descriptive model to green wood.

Experimental

Wood material and creep equipment Wood samples of three tropical species, Oxandra asbeckii (Pulle) R.E. Fries, Licania alba (Bernoulli) Cuatrec and Virola michelii Heckel, exhibiting contrasting mechanical properties and various anatomical features were used to investigate the diversity of the aging phenomenon in green wood. Plant material coming from tilted trees was included in the study because trees in reorientation process produce particular wood tissues with high levels of pre-stresses and specific cell wall structure [START_REF] Scurfield | Reaction wood: its structure and function[END_REF], called "tension wood" in hardwoods, unlike normal wood produced a priori by straight growing tree. Sample names are built as the initials of the specie's name with an index specifying the wood type (e.g. Oa N and Oa T for Oxandra asbeckii normal and tension wood, respectively). Samples L=150 × T=15 × R=2mm 3 were kept in the wet conditions during all test procedure and storage.

Tensile creep tests were performed on the experimental device used previously by Hunt and Darlington (1978) newly equipped by a water chamber to enable testing of green wood samples (Dlouhá et al., submitted). Water bath was controlled in temperature by a thermo regulator with an accuracy of ±0.1 • C. Constant stress was prescribed by assigning an initial displacement of around 500 με corresponding to the generally observed level of pre-stresses in wood tissues. Axial strain in the central part of the sample was measured by a couple of strain gages glued on both side of the sample. Readings were taken every 0.1 s during the first 1,000 s and every second afterwards. We used three-wire gages to avoid the influence of temperature variations on the resistance of the wires and silicon coating to reduce influence of water flow in the bath and risk of gage detachment.

Experimental procedure

After being heated at 80 • C for 30 min to release all macroscopic residual strains and establish the thermodynamic equilibrium, the sample was quenched to 30 • C and kept at 30 ± 0.1 • C. The quench was done by replacing the sample from the bath at 80 • C to the water chamber of the testing device regulated at 30 • C, the operation taking about 60 s. After that, a set of short-term creep tests was performed on the same specimen at various aging times t a elapsed after the quench as shown in Fig. 1a. It is important to distinguish aging time t a , counted from the moment when the specimen was moved from the hot to the cold bath, and t c , the creep time counted from the moment of each loading. It is always quite delicate to decide which point is to be considered as a starting point of the loading corresponding to t c = 0 s. In this case, the point corresponding to the half load was considered (Fig. 2a). Points corresponding to t c < 3 s were not included in further analysis because of the important dispersion of deformation values attributed to vibrations resulting from the manual weight application (Fig. 2b).

To separate the creep response from the aging effect, testing time t c has to remain much shorter than t a at the onset of the test. This is called the snapshot assumption because a test performed during such a short time

(t c < 0.3 × t a )
will exhibit negligible effects of aging. Strain was measured during both creep and recovery periods. By extrapolation of the recovery curve in logarithmic timescale into the next creep period, the strain built up during each individual creep test could be singled out (Fig. 1b). To obtain a reliable extrapolation, testing time has to be short relative to the previous (3 × t c ) recovery period. We also need to have t c short relative to the following recovery period (10 × t c ). This condition takes into account the time necessary to complete the recovery of previous loading so that at the end strain signal is quite stable and easy to extrapolate. In fact, corrections due to the extrapolation of recovery tests revealed to be negligible (∼0.002% for the longest creep test) so that the correction was not applied systematically. The total duration of the test procedure is 3.5 days.

Results and discussion

Suitability of experimental procedure and evidence of aging phenomenon

The experimental procedure was first tested omitting the step of preliminary heating above T g . In this case, curves corresponding to creep tests performed at different elapsed times after the onset of the test were superimposed into one single curve (Fig. 3a). This shows that the effect of loading history of the sample on a given creep test is negligible and we can use the proposed procedure to investigate the effect of aging time. Figure 3b represents a set of creep tests performed on the same specimen after the quench. We can notice that higher creep strains were developed during the tests following the quench. The relative creep for the creep test performed at t a5 (Fig. 3a) amounted to ∼7.5% against ∼12.2% for the creep test (t a5 ) in Fig. 3b. The shape of the creep curve corresponding to the first aging time (t a1 ) was quite different from others. This effect was ascribed to the non-achieved thermal equilibration of the specimen with the surroundings: In later tests, we have always applied the first loading at log t a1 > 2, or 100 s after the quench.

The gradual decrease of instantaneous compliance with increasing aging time as visible in Fig. 3b clearly confirms the occurrence of physical aging in green wood. The drop between successive creep curves does not reduce with increasing aging time. This suggests that the logarithmic aging rate remains approximately constant during the test; even at the end of the test period, 3.3 days after the quench, the specimen is far from a new equilibrium state. It definitely shows that the stabilisation period of our samples is much longer that expected considering Nakano's results transversally to the fibres. Figure 4a shows a master curve obtained by shifting the curve for each aging time along the log time axis toward a reference curve corresponding to t a8 . The smoothness of the resulting master curve suggests that the aging dependence of the creep response is rather uniform and the material could be considered as rheologically simple. A commonly used method to describe the aging behaviour is to display the shift factor a versus the aging time t a on double logarithmic scale. The slope yields the aging shift rate μ (Fig. 4b):

μ = -d(log a)/d(log t a ) 1 .
(1)

Identification of a rheological model

To improve the reliability of aging quantification, a procedure based on rheological modelling was tried. As a starting point for the model identification, we have used the representation of experimental data in the approximated complex plane (ACP) as proposed by [START_REF] Huet | Représentation des modules et complaisances complexes dans les plans complexes arithmétique et logarithmique[END_REF]) for analysis of viscoelastic data obtained by static tests. This method allows checking the presumed validity of creep time-aging time equivalence and offers the possibility to deduce a descriptive model of the phenomenon from the graphical representation 1 In this paper, decimal logarithms are used. of the data without any presumed hypothesis about its mathematical form. To represent data from static tests, we have used [START_REF] Alfrey | Mechanical behaviour of high polymers[END_REF] approximation to obtain storage J and delayed J components of the complex compliance:

J ≈ J (t c ) J ≈ π 2 × dJ (t c ) d ln t c . ( 2
)
The ACP can be understood as a kind of phase diagram showing, for each J(log t c ) curve, the slope against the value. Alfrey's approximation is valid for small J /J ratio and broad viscoelastic spectra, which is usually the case in wood and can be verified a posteriori. In the case of a simple solid rheological behaviour described by a spring in series with a Kelvin block, the representation of the compliance in the ACP deviates significantly from the half circle in the complex plane (CP) used to represent a dynamic response. However, when the dashpot compliance becomes proportional to t k with k < 1, the model is described as "parabolic". For k << 1, which corresponds to a broad viscoelastic spectrum, ACP and CP coincide. The applicability of parabolic models to wood was suggested by [START_REF] Huet | Accurate measurement of creep of nylon-6,6 at constant temperature and humidity[END_REF] and verified for air-dry wood creep in longitudinal direction [START_REF] Gril | Using wood creep data to discuss the contribution of cell-wall reinforcing material[END_REF]) and green wood relaxation transversally to the fibres [START_REF] Bardet | Modelling the transverse viscoelasticity of green wood using a combination of two parabolic elements[END_REF]. The value of k was in the range 0.1-0.5 in these cases.

In complex plot, the creep curves measured at various aging times by form into one single straight line (Fig. 5). This indicates that the aging phenomenon in green wood can be described by a "parabolic Maxwell" model [START_REF] Huet | Représentation des modules et complaisances complexes dans les plans complexes arithmétique et logarithmique[END_REF]) with an aging-dependent dashpot element. Creep behaviour of such a model obeys to the power law:

J (t c ) = J 0 1 + t c τ k , ( 3
)
where J(t) is the creep compliance, J 0 the instantaneous compliance, τ the doubling time of the creep response and k the power parameter. The use of a power law has been often proposed to describe the creep response of wood (Le [START_REF] Govic | Mise en évidence d'une équivalence temps-température et modélisation du fluage du bois 1 Documents généraux-Thèmes 1 et 2. Actes du 2e colloque Sciences et Industries du Bois[END_REF]. This behaviour can be considered as the beginning of a three-element parabolic response as described above for durations considerably smaller than the characteristic time of the dashpot, so that the spring in parallel to the dashpot is subject to a negligible stress. Estimates of J 0 and k can be obtained graphically as shown in Fig. 5 and have the same value for all aging times. Only τ depends on t a , so that the creep compliance for any aging time can be written as:

J (t c ; t a ) = J 0 1 + t τ (t a ) k . ( 4
)
In this equation, the relation τ (t a ) is not imposed. Assuming, in addition, the validity of aging rate uniformity claimed by Struik (Eq. 1), we obtain:

μ = -d (log τ ) d (log t a ) = -d (log a) d (log t a ) , ( 5
)
where μ is the aging shift rate where parameter a represents the horizontal shift used in Struik's procedure

log τ (t a ) = log [τ 0 ] -μ log (t a ) -log (t a0 ) (6)
where t a0 is the reference aging time andτ 0 the doubling time at t a0 . The combination of Eqs. 4 and 6 allows predicting a whole data set like that of Fig. 3b using four parameters only: J 0 , k, μ and τ 0 . As a reminder, Eq. 4 is only valid for t c values verifying the snapshot condition (t c << t a ), so that the age of the specimen did not change significantly since it was loaded.

Optimisation of model parameters

Model parameters were adjusted by least-square optimisation method using smoothed experimental data with regular step in log timescale (δlog t c = 0.1). First, we have tested the suitability of the proposed description of aging behaviour deduced from the complex plot (Eq. 4). Parameters J 0 , k and τ i were adjusted omitting any presumption about the dependency of τ i on aging times. This optimisation method will be called "fitting 1". An example of a master curve and adequacy between experimental and modelled creep curves is displayed in Fig. 6. As output of fitting 1, we have obtained a set of doubling times τ i corresponding to different aging times t ai at which were performed individual creep tests. Plotting the change in τ i versus aging times t ai in double logarithmic scale (Fig. 6), we can assess the validity of the presumed aging rate uniformity. Excepting the sample Oa T , the assumption of linearity is well satisfied. Next, we have proceeded to the optimisation of model parameters J 0 , k, τ 0 and μ taking into account both Eqs. 4 and 6, referred to as "fitting 2". An example of master curve using shift factors obtained by fitting 2 is shown in Fig. 8a. We can notice some discrepancies compared to the smooth master curve resulting from fitting 1 represented in Fig. 7a; however, agreement between experimental and modelled creep curves based on parameters issued from fitting 2 remains very satisfactory (Fig. 8b).

Table 1 summarises parameters of the agingdependent model for all tested specimens adjusted by different methods and/or corresponding to different hypothesis about the doubling times dependency on aging times. Parameter values resulting from fitting methods 1 and 2 are very close. This indicates that the assumption of uniformity of aging rate (Eq. 4) is valid, and so, we can reduce the number of model parameters without introducing an important error to the prediction of aging behaviour. It is also interesting to notice that the accuracy of visual fitting of the master curve is quite good (see values of μ by visual fitting and fitting 1). This study proved that complex plots are very useful in deciding about the mathematical form of the behaviour law; however, the accuracy of value of parameter k deduced from the graphical representation is not sufficient.

Concerning the variability of instantaneous compliances between samples, they can be easily explained by the diversity of densities and Young moduli of our sampling. On the other hand, parameters μ, k and τ 0 were very similar for all samples (excluding aging shift rate μ measured for the sample Oa T ), suggesting the Values of aging rate are consistent with bibliographic data. Struik has observed aging rate μ close to unity for amorphous polymers and μ ∼ 0.75 for rigid chain cellulose acetate butyrate ester. Considering partially crystalline and partially amorphous nature of wood, lower aging rate can be explained by the slowing effect of rigid chains as proposed by [START_REF] Govic | Mise en évidence d'une équivalence temps-température et modélisation du fluage du bois 1 Documents généraux-Thèmes 1 et 2. Actes du 2e colloque Sciences et Industries du Bois[END_REF]. Application to the analysis of Nakano's data Nakano studied Picea jezoensis (Carrhas), a Japanese softwood, loaded in bending in the R direction. Although the wood was studied in the water saturated state, it had been previously kiln-dried so cannot be considered as green wood. The experimental procedure has been different from that suggested by Struik. To examine in more details Nakano's data, we have applied the optimisation method 1-fitting without presumed linear logarithmic dependency of doubling times on aging times. As creep tests were not performed on the same sample, we have allowed J 0 to vary accounting for possible differences in instantaneous and delayed behaviour between samples. Only data fulfilling the snapshot condition t c > t a 3 were used for the fitting. Agreement between experimental and modelled creep curves is shown in Fig. 9a. Relation between shift factors and aging times is quite linear, yielding aging rate equal to 0.73 (Fig. 9b) that is very close to our values for tropical hardwoods loaded in longitudinal tension. It suggests that the dynamics of aging phenomenon is not dependant on the wood type or the loading direction.

Discussion on the physical processes involved

According to [START_REF] Skaar | Thermodynamics of the swelling of wood[END_REF], the fibre saturation point (FSP), representing the maximum content of bound water, increases by 0.1% per 1 • C decrease. This estimate, deduced from sorption isotherms, is representative of phenomena occurring in the hygroscopic domain. However, even in wet conditions, with the cellular cavities (lumens) filled with free water and the cell walls saturated with bound water, small moisture movements are likely to occur through the cell wall as a result of temperature changes.

The phenomenon stated by Stamm is qualitatively confirmed by the negative reversible thermal expansion observed in green wood and attributed to a moisture 9 Model fitting based on Eq. 3 applied on Nakano's data: a Agreement between experimental and modelled creep curves for fitting 1. Solid lines represent the prediction for values of t c < t a /3; dashed lines correspond to modelled values not satisfying the snapshot condition t c > t a /3. b Relationship between shift factors a and aging times t a in double logarithmic scale uptake during cooling [START_REF] Yokota | Changes in dimension on heating green wood[END_REF]. But the intensity of the phenomenon seems to be smaller. An expansion ratio of -7.10 -6 / • C in the tangential direction can be evaluated for green wood from a previous study [START_REF] Gril | Recouvrance hygrothermique du bois vert. I. Influence de la température. Cas du jujubier (Ziziphys lotus (L) Lam)[END_REF]. Taking +33.10 -6 / • C as the expansion ratio for dry wood [START_REF] Kollmann | Principles of wood science and technology[END_REF]), the expansion resulting from the moisture uptake can be calculated as -40.10 -6 / • C. Assuming a typical expansion ratio of 0.3% per percent moisture content, this would correspond to a moisture content increase of 0.7% for a 50 • C temperature decrease to be compared to the 5% predicted from FSP estimates.

Even by that small amount, the moisture uptake resulting from quenching is likely to influence the mechanical response. In the hygroscopic range, humidity variations, both adsorption and desorption, considerably accelerate the viscoelastic response, a phenomenon known as mechanosorptive effect [START_REF] Grossman | Requirements for a model that exhibits mechano-sorptive behaviour[END_REF]) and described as a transient increase of molecular mobility [START_REF] Back | Transient effects of moisture sorption on the strength properties of paper and wood-based materials[END_REF] of the hygroscopic matrix made of lignin, hemicelluloses and non-crystalline cellulose. However, at high humidity levels, the contributions of mechanosorptive and stable-state creep are difficult to separate. Our data do not allow distinguishing that "hygrothermomechanical" process from the progressive return to equilibrium of temporary frozen molecular segments. The quenching provoked, for a number of reasons, an unstable molecular configuration of the matrix that is progressively recovered according to the identified kinetics.

The fact that Struik's formalism seemed to be relevant to describe the physical aging of green wood suggests that the pertinent level of description is that of a semi-crystalline polymer where the heating above lignin glassy transition followed by a quench had induced a delayed return to a stable molecular configuration. The transient instability induced by the small moisture uptake does not necessarily contradict this interpretation as long as it only adds a reason for higher molecular mobility. However, if the variation in moisture content was the main cause for the change in behaviour with t a , the remarkable superimposition of the creep curves in Fig. 5, resulting in a model where only τ varies with t a , may not have been observed.

Higher levels of structural organisation should be also evoked to complement this molecular interpretation. In the longitudinal direction, because of the axial orientation and tubular shape of most cells, the cell wall level is most appropriate. When a green wood specimen is isolated and heated, stress resulting from the cell maturation process is easily recovered as soon as the temperature exceeds T g , which was the case at 80 • C. However, the condition of structural integrity limits the extent of recovery; a part of the locked-in strains and pre-stresses remain present locally, as their complete relaxation would require the destruction of the cell wall structure. The local swelling anisotropy, and its incompatibility between layers, might interact with this initial pre-stressing resulting from cell wall formation and be only partially released by the heating.

Conclusion

The effect of quench on creep persists for a long time in green wood and could be described as a physical aging process. The analysis of aging behaviour was performed in three steps. First, rough assessment of the validity of the aging rate uniformity was made by visual shifting of individual curves into a master curve. Second, complex plots were used to choose a rheological model and identify its parameters for each aging time t a without any presumption about the influence of t a . The model used, a parabolic Maxwell, describes the delayed compliance by a simple power law. Only the "doubling time" theoretically required to double the instantaneous response was affected by t a (fitting 1). Based on results of this fitting, the validity of the assumption of the uniform aging rate was verified and introduced in the model in a third stage (fitting 2). The same approach was successfully applied on experimental data provided by Nakano. Model based on the linear dependency between doubling and aging times was considered satisfying for all studied samples suggesting common feature of the aging phenomena in wood. The transient adsorption process consecutive to the quench was considered as one of the possible causes of the higher molecular mobility observed.

Fig. 1 Fig. 2 a

 12 Fig. 1 Experimental procedure for investigating the aging time effect on small strain creep properties. a Loading schedule: dashed line, temperature; solid line, load. t a aging time, t c creep time. b Accounting for the correction of the creep signal. Dashed line, extrapolation of the recovery signal to the creep test duration

Fig. 4

 4 Fig. 4 Relationship between retardation and ageing time based on visual assessment (sample Vm N ). a Master curve obtained by visual shifting of individual creep curves from Fig. 3b. b Double logarithmic plot of shift factors a versus the aging time t a

Fig. 6 Fig. 7

 67 Fig. 6 Model fitting based on Eq. 4. Relationship between shift factors a and aging times t a in double logarithmic scale for different samples

Fig. 8

 8 Fig. 8 Example of model fitting based on Eqs. 4 and 5 for sample Oa N : a Master curve produced by shifts resulting from optimisation accounting for linear logarithmic dependency between doubling time and aging time. b Example of agreement between experimental and modelled creep curves issued from fitting 2. t a aging time

  Fig. 9Model fitting based on Eq. 3 applied on Nakano's data: a Agreement between experimental and modelled creep curves for fitting 1. Solid lines represent the prediction for values of t c < t a /3; dashed lines correspond to modelled values not satisfying the snapshot condition t c > t a /3. b Relationship between shift factors a and aging times t a in double logarithmic scale

AGING TIME(s) TEMPERATURE & LOADING

	QUENCH
		3 t c	t c	10 t c
	t a1	t a2	t a3

Table 1

 1 Adjusted parameters of aging-time-dependant model Last line corresponds to results obtained for Nakano's data presented in the second part of the paper, k power exponent, J 0 instantaneous compliance (GPa -1 ), log τ 0 doubling time of the reference element, μ the aging rate a Value of μ for fitting 1 is deduced from the plot represented in Fig.6

		Vis. fitting	Complex plot	Fitting 1				Fitting 2
		μ	k	J 0	μ a	k	J 0	logτ 0	μ	k	J 0	logτ 0
	La N	0.70	0.21	0.035	0.71	0.17	0.041	11.7	0.72	0.18	0.041	11.4
	La T	0.79	0.12	0.027	0.79	0.14	0.033	12.4	0.80	0.14	0.033	12.4
	Oa N	0.77	0.21	0.042	0.73	0.19	0.048	10.8	0.75	0.19	0.048	10.7
	Oa T	0.98	0.14	0.037	0.97	0.13	0.033	14.2	0.97	0.13	0.033	13.8
	Vm N	0.77	0.14	0.087	0.80	0.14	0.086	9.5	0.80	0.14	0.086	9.3
	Nakano				0.73	0.27		5.2	0.61	0.27		5.2
	Whilst in our case aging times at which individual creep
	tests are performed are equidistant in logarithmic scale
	(step = 0.5), in Nakano's procedure, the aging time step
	is not constant and tends to decrease for longer aging
	times (log t an+1 -log t a n : 0.78, 0.48, 0.30, 0.37, 0.22,
	0.27, 0.19 ). Hence, it is difficult to deduce from the
	graphical representation of creep curves with respect
	of testing time if the material is close to its equilibrium
	state or not.

Acknowledgements

The authors would like to thank Professor Takato Nakano for allowing the use of his experimental data as well as for his useful comments. The research reported in this paper was carried out as a part of the project "Woodiversity" ANR-05-BDIV-012-04 supported by the French National Research Agency.