Damien Campello

Nicolas Tardif
email: nicolas.tardif@insa-lyon.fr

Jean Desquines

Marie-Christine Baietto

Michel Coret

Anne Maynadier

Philippe Chaudet

D Campello A

J Desquines B

M.-C Baietto A

A Maynadier D

Validation of a multimodal set-up for the study of zirconium alloys claddings' behaviour under simulated LOCA conditions

Keywords: Digital Image Correlation, Near Infra-Red Thermography, uncertainties, measurement error

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1� INTRODUCTION

During its operating life, the water in the primary loop of a Pressurized Water Reactor (PWR) is pressurized at 155 bar and heated by fuel pellet stacks up to 320 °C. The fuel pellets are inserted in claddings assembled in a 27x27 bundle. They are made of zirconium alloys.

The claddings are designed to be the first safety barrier in French PWR. In operating lifecycle, filling gas and fission gas release also internally pressurizes the fuel claddings. A Loss of Coolant Accident scenario (LOCA) postulates a breach in the primary loop system. The water is depressurized limiting the coolant efficiency. Both the water and the fuel rods are then heated and the claddings are loaded with internal pressure. It can lead to rod ballooning and, potentially, to rod bursting. Re-flooding the nuclear core reactor interrupts this accidental sequence. The fuel rod ballooning can impede the core cooling capacity by flow blockage.

The PERFROI project, detailed in [1], aims to study this complex scenario.

In [2], the authors presented the global picture of the coupled experimental/numerical approach to identify the steady-state creep behavior of as-fabricated Zircaloy-4 claddings under simulated LOCA conditions. The study targeted thermal-mechanical conditions corresponding to the lower bound of phase transformation from α towards α + β where several creep mechanisms are potentially activated (i.e. temperatures between 750 and 850 °C, stress between 7 and 45 MPa). Fig. 1 summarizes the methodology. The experimental setup enabled the internal pressurization of an unirradiated Zircaloy-4 cladding by stage. An induction heating system induced an axisymmetric but axially heterogeneous thermal loading. Thermal and kinematic surface fields were monitored by optical full-field measurement methods during the ballooning. Hence, several thermal-mechanical conditions were studied during each experiment. The parameters of local Norton power laws were identified by Finite Element Model Updating by minimizing the difference between experimentally measured and calculated secondary creep strain rates. The approach enables a substantial reduction of the test matrix and associated discrepancies compared to other traditional setup [3][4][5][6][7][8][9].

Since the nineties, non-contact techniques in both kinematic and thermal fields measurements have been improved. Digital Image Correlation [10,11] and edge detection [12] are frequently used for strain measurements in experimental mechanics. Recent improvements in algorithms (global approaches with finite element basis [13], multi-grid solver [14], , FEMU [16]...) allow for material behavior identification as proposed by Réthoré et al. in [17].

Infra-Red (IR) cameras are now commonly used in the field of experimental mechanics for computing thermal fields as demonstrated in [18] with Infra-Red Image Correlation or in [19] to explore the energy balance associated with high-cycle fatigue. The IR cameras remain very expensive compared to standard cameras, and do not allow for high spatial resolution. In order to avoid these drawbacks the Near Infra-Red (NIR) measurement methods have also been developed [20,21,22]. Their use (for the steel industry [23] for example, or a hot specimen in an aggressive environment [24]) has been greatly improved in the recent years. The homemade experimental setup is depicted in Fig. 2. The setup enabled a combined biaxial compression/internal pressurization loading of the tubular Zircaloy-4 sample. A local thermal loading was applied by induction heating. The specimen was surrounded by an enclosure to maintain an inert environment during the test. Windows were distributed over the circumference in order to enable the thermal and kinematic optical full-field measurements.

Details on the setup are given in the following.

2.1� MATERIAL

The material was Stress Relieved Annealed (SRA) Zr-4 manufactured by CEZUS. The claddings in service have a 9.5 mm outer diameter, a 0.57 mm thickness and are 4 m long.

The claddings were cut by an electro discharge machining into 90 mm long samples.

2.2� SPECIMEN PREPARATION

High temperature image correlation measurement requires meticulous preparation. A black undercoating (ULFALUX thermo-coating 1200 °C) was sprayed on the outer specimen surface. A white speckle was then applied using BND painting. The speckle size ranged from 10 to 120 µm (usual pixel size of the optical measurement was 9µm). A degreaser was used for removing the painting along the z direction at two circumferential positions separated by a 180 °C angle. Three type K thermocouples (⑭) were spot-welded in each of the painting free azimuth. The two wires of each thermocouple were separated using a single hole round ceramic insulator (SH-1-24). The diameter of the wires (CAB KX 04) was 0.078 mm to lower the intrusive effect of the thermocouples.

2.3� SETUP

The test specimen was connected (⑮ in Fig. 1) onto a Schenck 10kN servo-hydraulic tensile machine (①, ②) using custom grips (③) designed by Tardif et al. [24]. An enclosure (⑤) was fitted to the machine for working in an inert environment. Argon flushing (⑥, ⑦) was kept during the entire test with a constant flow. Two 12MPx CMOS cameras (VC-12MC-M65E0-FM) performed the 2D-DIC on the tubular specimen surface at temperatures ranging from 750 to 850°C. At such temperatures, the cladding surface emits radiation in the Near Infra-Red (NIR) spectral range. To get rid of incandescence, blue LED rings (CCS LDR2-70-BL2, 470nm, ⑪) were set up above and below the induction coils. The LED rings were pulsing blue light. Band-pass filters (MIDOPT FIL BP470/62) centered on 470 nm (useful range: 425-495nm) were fitted to the 12 MegaPixels cameras used for DIC as proposed by Pan et al. in [25,26].

Additionally, two 16MPx CCD cameras (Prosilica GE4900) shot images without any filtering when the blue lights were put off for NIRT. This way, only the light emitted by the specimen surface radiation was captured.

3� DEDICATED TEST PROCEDURES FOR ERRORS AND UNCERTAINTIES ASSESSMENT OF THE FULL FIELD MEASUREMENTS

3.1� TEST PROCEDURE TEST A FOR NIRT ASSESSMENT

A thermal cycling test was performed on a cladding at three different temperature levels. The thermal loading history is plotted versus time in Fig. 3. The cladding was first heated then three thermal cycles were successively performed and had actual both heating and cooling rates of 2.2°. . Argon flushing was kept during the whole test. The force was also set to zero. Note that the dedicated test conditions were aggravating compared to those in [2] (isothermal creep). Radiometric models were calibrated for each cycle during its own heating part. A Prosilica GE4900 camera shot pictures at a 1 frequency, with a /11 aperture.

The test procedure will be referred as Test A in the following.

3.2� TEST PROCEDURE TEST B FOR 2D DIC ASSESSMENT

The same test procedure as in [2] was chosen to assess the 2D-DIC. The test procedure is shown in Fig. 4. At the beginning of the test, the enclosure was filled with argon gas and flushing was kept on during the whole test. A controlled axial force of 0 N was set during the 3° • thermal ramp up to a first plateau. The first plateau enabled the evacuation of the smoke induced by the heating of the paint. Then the thermal ramp was started again up to the set point (795 °C). Then, the mechanical loading was applied by the combination of the compressive force and the internal pressure. Three mechanical plateaus were set so that the J2 equivalent strain reaches at least 4% at each plateau.

The cameras configuration is shown in Fig. 5a. Images shot by Cam 1 and Cam 2 (see 5b and 5c) were respectively dedicated to 2D-DIC and edge detection. This additional digital image analysis provided the out-of-plane displacement due to the tube ballooning.

The test procedure will be referred as Test B in the following.

4� POST-PROCESSING METHODS

4.1� NEAR INFRA-RED THERMOGRAPHY

The design of the setup was chosen to apply a 20°C heterogeneous thermal axial distribution in the ROI. This temperature distribution had a strong impact on the creep strain rates as can be seen in Fig. 1. Hence Full field thermal measurement must be preferred to local measurements such as type K thermocouples. Near-Infrared thermography is well suitable to the thermal measurement of metal at temperature higher than 750°C as it can be set up with a cheap CCD or CMOS camera. These cameras types have a higher spatial resolution than usual infrared cameras. The calibration of the radiometric model requires a reference temperature measurement provided by type K thermocouples. Any surface emits radiation (with a wavelength ) at a given temperature . The surface is characterized by an emissivity (0 ≤ ε (λ) ≤ 1) depending on the radiation wavelength. The total emittance , of this surface is estimated using its emissivity and the Planck law according to equation (1). The Planck law provides the total emittance of a blackbody , in a hemisphere using the Planck constant ℎ, the speed of light and the Boltzman constant . Note that the emissivity of a black-body is equal to 1. , ,-, = , + ,

4.1.1� PRINCIPLE OF RADIOMETRIC MEASUREMENTS

, = !. , = !. 2ℎ. " . # $%& ' ℎ. . . ( -1 (
*+ = !. / , ! + 01 -!1. *2 (2)
, *+ comes from the flux , *2 that the surface receives from the surroundings. A single detector (gain 3 and exposure time 4 5 ) of a digital radiometer is now considered. Its digital level output 6 is written in equation (3) using the spectral response of the optical system 7 ! and the total radiant flux of the target , ,-, . Finaly, the radiometric model gives the relation between the Temperature and the digital level recorded by the camera. The spectral response 7 ! of the optical image acquisition chain combined the argon's, the sapphire's and the objectives' transmittances (respectively : ;* ! , : <;= ! , : ->? !), and the detector's quantum efficiency @ A, !, as written in equation (4).

6 = 4 5 . 8 7 !. , ,-, 9 (3)
7 ! = : ;* !. : <;= !. : ->? !. @ A, ! (4)
A review of several calibration methods can be found in Rotrou et al. [28]. The radiometric model was calibrated using the thermocouples local measurements during the heating phase of each test. This method is particularly well adapted since the spectral response 7 ! is not known. Note that the thermocouples and the NIRT acquisition were triggered at exactly the same time.

Fig. 6 shows five pictures shot during the end of the heating. The thermocouples are located on the left side of the digital images. Three digital thermocouples were numerically built, close to actual Tc. They are depicted by the blue, red and green boxes in Fig. 6. An averaged gray level intensity 6 B was calculated into these boxes to be correlated to the associated thermocouple measurements.

Inside the digital thermocouples, the emissivity of the surface is heterogeneous because of the speckle applied onto the specimen. In order to achieve a good sensitivity to the radiation emitted by the surface, only the brightest 30% pixel intensities were averaged for computing 6 B .

As detailed by Rotrou in [29], the concept of equivalent wavelength B is useful to analytically assess a measured temperature C< based on a digital level intensity 6 B . The chosen radiometric model is detailed in equation ( 5). The parameters 3 and 3 " of equation ( 6)

were calibrated using the correlation between digital and real thermocouple measurements.

C< = 3 DE F 3 " 6 B + 1G (5) 3 = ℎ. . 3 " = 2. !. . H . 7 !. 4 5 (6)
The calibration was performed using a least squares minimization between temperature C< and averaged grey level intensity 6 B data. The ROI (white box in Fig. 5b) was discretized by isotropic linear Q4 finite elements. Their size was 30 x 30 px 2 . The ill-defined problem of the optical flow conservation was solved using a non linear least squares method relying on a finite element basis. Axial and hoop displacements were extracted from 2D-DIC results at the mesh nodes. From the gradient tensors, logarithmic strains were computed. True Von Mises equivalent creep strain was calculated over the sample surface assuming material incompressibility (equation ( 7)) during high temperature creep.

II J= + KK J= + ** J= = 0 B J= = L 2 3 M II " + KK " + ** " NO " (7)
In Test B, a coupled edge detection / 2D-DIC approach was used to quantify the error The coupled edge detection / 2D-DIC approach needs two steps. The first step allows for calibrating the intrinsic parameters of the optical system used for 2D-DIC. Then the displacements and strain measured by 2D-DIC are corrected by the out-of-plane displacements measured by the edge detection measurement.

4.2.1� CALIBRATION OF THE OPTICAL MODEL

Several authors [31,32] reported the effect of out-of-plane motion on in-plane displacements. These displacements can be corrected as proposed by using the dedicated experiment detailed in Fig. 7. The test aimed to calibrate the initial distance between the object and the optical system d , and the position of the intersection between the optical axis OA! and the detector plane O -9 5 , % 2 , S 2 !. Cam 1 (i.e. Fig. 5a) was mounted onto a sliding caliper and was translated in order to induce in-plane displacements T * . When sliding the caliper, the 2D-DIC measures the virtual displacement of the measured point M in the detector plane ( % " -% !% U + S " -S !S U). This virtual displacement depends on three parameters d , % 2 and S 2 . Considering a linearized optical system model, the virtual displacement can be deduced from system equations (8).

9 . % -% 2 ! = 9 -T * !. % " -% 2 ! 9 . S -S 2 ! = 9 -T * !. S " -S 2 ! (8)

The optical system was translated up to a maximum amplitude T * = 1 then moved back to the initial position with 0.1 mm displacement steps. Parameters are determined using a least squares minimization between the displacements calculated with the optical model and the 2D-DIC. Resulting parameters d , % 2 and S 2 were respectively identified as 613 mm, -122 px and -62 px.

The virtual strain associated to the virtual displacement is depicted as a function of the normalized in-plane displacement T * in Fig. 8. The linearization of the optical system model is validated at least for an in-plane displacement T * of 1mm. Note that the optical system used in these experiments is not strongly affected by the out-of-plane displacement.

4.2.2� CORRECTION OF THE CLADDING BALLOONING

Fig. 9 illustrates the description of the combined edge detection / 2D-DIC approach.

2D-DIC was performed on the Region of Interest (white box in Fig. 5b) of the pictures shot during Test B. Displacements and strains in both % and S directions of the detector plane were extracted along the yellow line.

The edge detection provided the radial displacement T * of every point of the deformed edge (Fig. 10) in the eulerian coordinate system (Fig 5c). The resulting point V′ in the detector plane corresponding to point X′ can be calculated using equation ( 9) where X = 9 5 9 Y is the magnification of the optical system. The initial position V of the deformed point V′ was determined in the detector plane using the 2D-DIC results as follows:

S Z -S 2 = [.A \ A \ ] ^. _ -_ 2 !. (9)
S -S 2 = S Z -S 2 -T ` V′!, (10)
with T ` V′!, the displacement of Q measured by the 2D-DIC in the y direction of the detector plane.

The initial axial position _ -_ 2 of the deformed point X′ was finally calculated using equation ( 11):

_ -_ 2 = S -S 2 X ( 11
)
The axial and radial displacements of each point of the yellow line during the transformation from the initial to the deformed configurations were thus determined. The logarithmic strains were calculated using deformed curvilinear positions and their associated initial positions. The uncertainties induced by the BEP304 take into account the internal cold junction measurement. It has the highest influence on the temperature measurement uncertainties. This manufacturer data accounts for the heating time of the cold junction. Performing measurement once the converter behavior is stabilized can reduce the uncertainties.

A calibrator (Fluke 725 Multifunction Process Calibrator) was placed before the compensation wire for assessing errors due to the compensation wire combined with the converter. It simulated the temperature of a K-type thermocouple. Five tracks of the converter were tested. The measurements were performed after 1.5 operating hour of the converter.

Temperature were imposed in a range of 720 to 880 °C temperature steps. The maximal error was 0.4% of the setting temperature, and the averaged error was 0.18%. The calibration of the NIRT relied on the thermocouple measurements. The errors related to the thermocouples acquisition chain were thus passed on the final temperature estimation.

5.1.2� TEST A : NIRT UNCERTAINTIES

The temperatures calculated using the calibrated radiometric model were compared to the thermocouples measurements during Test A (Fig. 3). .

The parameters determined using the calibration process and the silicon sensor time exposure 4 5 are reported in Tab. 4 for each cycle.

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The effective wavelength was calculated using equation ( 6). Note that is decreasing with increasing temperature level as expected. The Digital Thermocouple (DT)

and thermocouples (TC) measurements are plotted for the second temperature level cycle in Fig. 11a. The comparison between the NIRT and the thermocouple measurements are reported in Table 3 for each cycle. The maximal difference on the temperature prediction compared to thermocouples measurements remained lower than 0.7% when considering the entire test data.

Finally, the uncertainties estimation of the NIRT method was inherent to the thermocouples measurements and remains lower than 0.7% of the true temperature and the averaged error was 0.32% of the true temperature.

���� TEST B : 2D-DIC ASSESSMENT� Several aspects of the 2D-DIC measurements will be discussed in the following. First, the influence of the setup on the 2D-DIC noise will be studied at room temperature and at high temperature (respectively at markers t A and t B in Fig. 4). Then, the error induced by the out-of-plane displacement will be regarded at different ballooning levels (markers t 1 to t 4 in Fig. 4) by using the combined 2D-DIC/edge detection approach.

5.2.1� ANALYSIS OF THE INFLUENCE OF NOISE IN 2D DIC MEASURE

At markers t A and t B , (respectively at room and high temperature), Cam 1 shot 30 pictures, under a constant thermal mechanical loading, for a noise analysis purpose. A stationary spatial thermal distribution was reached at t B .

Fig. 12a shows the Grey level histogram of the ROI (red box, 1851x301 px 2 ). The histogram is plotted considering all the data (i.e. the pixels grey level of all pictures associated to each marker). The first pictures of the sets t A and t B are also shown as an example of the effect of the heating on the speckle pattern. The heating results in a decrease of the contrast 4. Note that the histograms include the DIC noise but also the physical displacements induced by the thermal mechanical loading noise (see Fig. 4). The increase of noise between room and high temperature can be explained by several reasons.

The dynamic of the pictures are narrower at high temperature. Little residual smoke induced by the heating of the paint could affect the measurement. However, no mirage effect was observed presumably because of the combined effect of the induction volume heating and the

��������������������������������������������������

Hence, the strain uncertainty at high temperature is less than 1.0 10 -4 . The strain noise is less than 1.5 10 -3 . The identification process shown in Fig. 1 used the stationary strain rates profile along the axial position of the ROI at each mechanical loading plateau. These stationary strain rates are calculated over a Von Mises equivalent strain range that is typically higher than 0.01 and over more than 20 pictures. The strain range is far higher than the strain uncertainty. The 20 pictures are enough to smooth the noise effect. The error of the 2D DIC measurements induced by the out-of-plane displacement is quantified in the following at different ballooning levels (markers t 1 to t 4 in Fig. 4) by using the combined 2D-DIC / edge detection approach. The marker 4 H is picked because of its extremely large ballooning state

5.2.2� CORRECTION OF THE CLADDING BALLOONING

The axial strain, calculated by 2D-DIC, and the curvilinear strain, calculated by the combined edge detection / 2D DIC approach, are plotted in Fig. 13a.

The correction effect is negligible for times 4 → 4 q but it becomes significant for time 4 H when the balloon is pronounced. After correction, the axial deformation is lowered in the middle of the specimen because of the out-of-plane displacement combined to a low curvature.

The opposite phenomenon is observed at the sample regions that were no longer perpendicular to the optical axis. The in-plane projection induced an artefacted contribution to ``.

The hoop strain levels were higher than those of axial strains. The tube curvature was not accounted for considering the low chord error induced by the Finite Element discretization of the circumference of the tube at the generator location. The hoop strains calculated using 2D-DIC were thus only corrected by subtracting the virtual strain related to the out-of-plane displacement (see Fig. 8). The 2D-DIC hoop strain and the corrected one are plotted versus the position _ in Fig. 13b. The correction is not significant in the hoop direction.

In [2], the equivalent Von Mises plastic strain was used for the FEMU based identification. The equivalent true strains were computed with and without the out-of-plane displacement correction. The error $r defined in equation ( 12) is plotted in Fig. 13c at the 4 markers.

$r " = M B "s -B 2-* N " M B 2-* N " (12)
The error associated to the 2D-DIC was lower than 1% of the equivalent strains accounting for the out-of-plane displacement even when the balloon was pronounced.

6� CONCLUDING REMARKS

The combined experimental/numerical approach detailed in [2] for identifying the creep behavior of Fresh zirconium alloys under simulated LOCA conditions was assessed. The paper focused on uncertainties and error quantification of the two key optical full field measurement method: NIRT and 2D-DIC.

The NIRT uncertainties mainly results from the thermocouples acquisition chain used for the calibration of the radiometric model. The physically based radiometric model tends to lower these uncertainties during the calibration by correcting the discrepancies between

thermocouples. An uncertainty of 0.7% of the actual temperature was quantified.

2D-DIC measured the ballooning of the tube. A correction method is proposed to take into account the out-of plane displacement of the tube during the ballooning. The method relies on a combined 2D-DIC / edge detection approach.

Applied to the used optical system, the correction is lower than 1% of the equivalent plastic strain even for a 20% increase of radius during the ballooning, value far higher than the ones used in [2] for the identification process. However, in the test configuration, axial strains are unaccurately estimated at this strain level and must be corrected from a radius increase of 10%. The proposed approach would have been particularly necessary for anisotropy identification in multiple biaxial stress states as enables the setup.

The combined 2D-DIC / edge detection approach can be used with a single camera for axisymmetric ballooning as during the above discussed testing conditions. It simplifies the setup and test procedure compared to 3D DIC.

7� ACKNOWLEDGMENT

The study was performed in the framework of the PERFROI ANR project (n • ANR-11-RSNR-0017-01). The authors would like to acknowledge all the participants of the project:

EDF, IRSN-SEREX, LEMTA, Ecole Centrale of Paris. The authors would also like to thank J.

Réthoré for the open access to the Ufreckles software and his caring help, and S. Comas-Cardona for his kind assistance in the improvement of this paper.

  This paper focuses on the validation of the experimental setup detailed in [2] and more specifically on uncertainties and error quantification of the 2 key optical full-field measurement methods applied in the test configuration: NIR Thermography (NIRT) and 2D Digital Image Correlation (2D-DIC).First, the experimental setup is recalled. The Post-processing of NIRT and 2D-DIC are detailed. The NIRT protocol is then assessed. Finally, error made by the use of 2D-DIC to analyze the ballooning of the tubular specimen is then quantified by the comparison with a more accurate combined edge detection / 2D-DIC method.

  induction generator (CELES) control the tube heating. The regulation was enclosed in a K-type thermocouple (⑭) measurement loop. The coils (⑨, ⑫) let a visual access to the warmest part of the specimen. The height of the region of interest (ROI, ⑬) is = 20 . A proportional pressure regulator (IMF) linked to an argon cylinder controlled the pressurization (⑧) of the specimen. Two pressure gages (P8AP from HBM, ④) mounted onto the upper and lower grips measured the internal pressure. The pressure induced bottomend effect was mechanically compensated by an axial compressive force inducing a uniaxial hoop loading. Five sapphire glasses were distributed over the enclosure to enable optical monitoring of the ROI by 4 digital cameras (⑩) equipped with 200 mm macro optics (NIK AF MICRO-NIKKOR 200MM F/4 D IF-ED).

  1) Meriaudeau et al. detailed the concept of a radiation thermometer (RT) in [20]. A radiometer senses the radiant flux of a target. This flux is the sum of the emitted flux , and a reflected flux , *+ as detailed in equation (2).

4. 1 .

 1 2� CALIBRATION OF THE RADIOMETRIC MODEL DURING THE HEATING STEPIn the proposed setup, induction heating induces negligible environmental heating (enclosure, argon,...). There was no other hot object in the surroundings. Moreover, the tubular geometry cannot lead to the reflection of the specimen itself. The calculation of , ,-, was simplified to the emitted flux assuming that , *2 = 0.

  KINEMATIC FULL FIELD MEASUREMENTS In [2], 2D-DIC measured the creep rate distribution along the yellow line in front of the cameras as depicted in Fig 5b.

  made by the 2D-DIC during the ballooning of the specimen. The edge detection method is based on the analysis of the gray level gradient of an image. Recent improvements allow for a sub-pixel definition of a curvilinear shape position and low CPU time calculations [12]. The reference line (���� edge before loading) was defined between -8mm and +8mm of the eulerian variable Z (see Fig 5c). It was discretized by 30 elements associated to a B-spline shape functions basis. The software used to perform 2D-DIC [30] and Edge detection [12] is called Ufreckles. It has been developed by Réthoré.

  1� UNCERTAINTIES RELATED TO THE THERMOCOUPLES MEASUREMENT Five components were involved in the acquisition chain: a thermocouple wire, a LEMO connector, a compensation wire, a converter TEPI (BEP304 model) and a FlexTest SE controller. Each of the components is a source of error affecting the temperature uncertainties.

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  Temperatures were calculated using the three radiometric models and grey level intensities ranging from 120 to 200. Results are plotted in Fig.11bversus the grey levels normalized by their respective time exposure. The radiometric model continuity from one calibration to another is highlighted. It was expected because of the linearity between the pixel intensity and the time exposure.

  white speckles. The white speckles Grey levels diminished and were smoothed. It is worth mentioning that the test constraints induce a poor speckle pattern quality either at high or room temperature as the highest quality being a wide and uniform histogram.2D-DIC was performed with these data sets. Fig.12bshows the displacement histogram over the whole data sets t A and t B (i.e. the displacement of all the nodes of each pictures). Fig 12c and Fig. 12d depict the histograms of the strains respectively at room temperature and at high temperature. The mean values and the standard deviations of each histogram are summarized in Table

FIG. 1 :

 1 FIG. 1: Summary of the identification process detailed in [2]. FIG. 2: Experimental setup FIG. 3: Procedure Test A : Near Infrared Thermography assessment. FIG. 4: Procedure Test B : 2D -DIC assessment. The dashed black lines are the setting point. The plain lines are the measurements. FIG. 5: Procedure Test B. a) Top view of the experimental setup -position of the cameras. b) Picture of CAM1, 2D-DIC performed inside the white box, data extracted along the yellow line. c) Picture of CAM2, Edge detection performed along the white line, edge in the initial configuration. FIG. 6: Procedure Test A. Pictures shot during the end of a heating with a 850°C setting point. FIG. 7: Procedure Test B. Scheme of the experiment used to calibrate the optical system parameters: 9 , % 2 , S 2 . FIG. 8: Procedure Test B. Virtual in-plane strain versus the normalized out-of-plane displacement T * r Y FIG. 9: Procedure Test B. Scheme of the combined edge detection / 2D-DIC approach. FIG. 10: Procedure Test B. Normalized out-of-plane displacement T * r Y of each marker versus the normalized axial coordinate _ _ Y FIG. 11: Procedure Test A a) Comparison NIRT -Thermocouples measurements for the second cycle of Test A. b) Radiometric model identification -one model per thermal cycle. FIG. 12: Procedure Test B. a) Grey level histograms of the sets of pictures associated to markers t A and t B , respectively at room and high temperature (See Fig. 4). b) Displacement histograms of the sets of pictures associated to markers t A and t B . c) Axial (ε yy ) and hoop Strains (ε xx ) histograms related to marker t A . d) Axial (ε yy ) and hoop Strains (ε xx ) histograms related to marker t B . FIG. 13: Comparison between the 2D-DIC and the combined edge detection / 2D-DIC approach, (a) axial strain, (b) hoop strain, (c) J2 equivalent strain.

  FIG. 12: Procedure Test B. a) Grey level histograms of the sets of pictures associated to markers tA and tB, respectively at room and high temperature (See Fig. 4). b) Displacement histograms of the sets of pictures associated to markers tA and tB. c) Axial (εyy) and hoop Strains (εxx) histograms related to marker tA. d) Axial (εyy) and hoop Strains (εxx) histograms related to marker tB. 381x317mm (300 x 300 DPI)

Table 1

 1

	summarizes the manufacturers data.
	TC wire	Compensation	BEP304	MTS FlexTest
				recorder
	0.004||	±2.5°	±6°	< 3. 10 # ||
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P Atten

J Raisin

J.-L Reboud G2elab

Field Induced Disruption of a Planar Water-Oil Interface Influenced by a Close Metallic Sphere

The problem of instability of a horizontal interface between water and an insulating fluid, electrically influenced by a metallic sphere located just above it, is considered here with its relevance to the basic electrocoalescence phenomenon of close water droplets suspended in an insulating medium. Results are presented of preliminary experiments performed using visualisation and shadowscopy techniques. The evolution of the interface shape is characterised for various applied step voltages and for different values of the initial spacing s 0 between sphere and interface. An order of magnitude analysis leads to good estimates of the characteristic parameters and time scales.

I. INTRODUCTION

In the electrocoalescence phenomenon, the mutual attraction between two close drops first induces a quasi-static deformation of the facing interfaces [START_REF] Atten | Critical conditions for electrically induced coalescence of two very close water droplets in oil[END_REF]; in the critical conditions characterised by a finite spacing between interfaces, we guess that there is an instability leading to a fast deformation and eventually to the contact between the liquid of the two drops. Observations on droplets of medium size (~ 300 µm) using a fast camera suggest that this instability process occurs in a rather short time < 100 µs [START_REF] Lundgaard | [END_REF]. The analogy with the exploding instability of a planar interface subjected to a uniform field [START_REF] De Surgy | Emission of liquid metal in vacuum[END_REF] and with some observations in the electro wetting phenomenon [Mugele] suggests that there is a tendency to formation of a kind of spike.

In order to gain some insight on the fast deformation and disruption of the interfaces subjected to electric fields, we consider here a model experiment of a horizontal water layer influenced by a metallic sphere located at some distance above the interface. The high enough sphere-interface spacing (~ 1 mm) makes it possible to observe the interface shape. Preliminary results are presented.

II. EXPERIMENTAL SETUP

The test cell is a rectangular tank with four vertical Plexiglas walls to allow easy visualisation. Deionised water fills a cylindrical hole (φ = 8 cm) and we can add a synthetic polybutene oil as dielectric fluid. A grounded metallic sphere (φ = 8 mm) welded on a thin rod is hanging at a controllable level above the water surface [ICDL05]. High step voltage is applied to the water (a rectangular signal of duration = 40 ms delivered by a low frequency generator is amplified by a 20 kV high voltage amplifier -TREK Model 20/20C).

The cell is illuminated by a parallel light through a flash given by a LED and the magnified image is formed on photocathode of a CCD camera; the signal is digitized and recorded on a PC. The light flash is triggered by the sudden drop of the voltage applied to the water electrode either by the prescribed voltage pulse drop or by the water-sphere contact inducing a short-circuit.

A second optical arrangement was also used with a steady illumination; the magnified image is then formed on a plane with a narrow slit; the total light passing through the slit is measured by a photodiode. When the slit is located along the symmetry axis of the system, we recover information on the vertical rise of the water interface (the water absorbs lightsee Fig. 1).

III. DEFORMATION OF WATER-AIR INTERFACE

The problem which would correspond to the coalescence stage under the action of the field should start from a static deformation of the interface in conditions very close to the critical ones. This is not easy as a marked deformation is very sensitive to perturbations and its control is difficult. In this preliminary study, we worked in conditions where the voltage is suddenly applied and we examine how the interface evolves starting from the initial planar shape. We first considered the case of air-water interface.

Fig. 1, relative to the initial spacing s 0 = 0.6 mm, illustrates the fact that applying a voltage V appl during 40 ms induces a limited deformation for V appl < 800 V. For V appl > 900 V the interface shape becomes very sensitive to the voltage value 800 V 900 V 940 V 950 V Fig. 1. Photographs (taken at t = 40 ms) of the deformed water-air interface for various applied voltages (s 0 = 0.6 mm).

and its curvature at the axis is more and more important as V appl is raised. The marked change in shape for very small voltage increase is induced by the rather fast uprising of the interface when the instantaneous spacing s between the water surface and the metallic sphere becomes small. This is due to the high field strength (~ V appl /s) and the subsequent high electrostatic pressure at the apex of the deformed interface.

The fast final rise of the interface is clearly characterized by using the shadowscopy technique. In the oscillograms of Fig. 2, the upper curves correspond to the applied voltage (the marked drop at t < 40 ms is due to the sphere-water contact) and the lower curves can be viewed as giving the time variation of the level of the interface on the system axis (the apparent stay of this level during the first 5 ms is due to imperfections in the optical arrangement). We see that, up to about 0.6 s 0 , the rising interface velocity is close to 1 cm/s; the final stage is much faster and the velocity is ~ 20 cm/s. An extra phenomenon when working with air as insulating fluid is the occurrence of discharges in the gas in appropriate voltage and spacing conditions. Such discharges which play but a minor role for small initial spacing s 0 (for instance s 0 = 0.6 mm) induce serious disturbances for larger s 0 values as can be seen on Fig. 3 (s 0 = 1 mm). The discharge which occurs before the water reaches the sphere suppresses the electrostatic pressure and the subsequent evolution of the interface shape is mainly controlled by capillary waves (see the photographs for V appl = 1560 and 1600 V in Fig. 3). The picture taken at t = 25 ms for V appl = 1800 V (Fig. 3) does not exhibit such a clear disturbance because the instantaneous spacing is higher (V appl is higher) and the interface deformation is at a earlier stage (note in Fig. 4 that the last stage of interface uprising for this voltage is slower than for V appl = 1540 V). 1540 V (10 ms/cm) 1800 V (5 ms/cm) Fig. 4. Oscillograms given by the shadowscopy technique : applied voltage (upper curve) and approximate rise (lower curve) of the apex of the water-air interface (s 0 = 1 mm).

IV. CASE OF WATER-OIL INTERFACE

When working with oil as insulating medium, the electrical discharges are eliminated. For small gaps s 0 , the deformation of the water-oil interface is qualitatively analogous to the one of water-air interface (see Figs. 5 and1). Increasing the initial spacing s 0 results in a more and more marked bending of the interface (Figs. 6 and7). We also observed that the deformation around the axis is sharper and sharper (see the example of Fig. 6 for V appl = 722 V). For an applied voltage high enough, the interface impacts the metallic sphere at a time lower than the predetermined duration (40 ms) of the voltage pulse. Photographs taken shortly after the water-sphere contact reveal an interface shape to a first approximation independent of the applied voltage (Figs. 6 and7). This suggests that the voltage amplitude affects the dynamics of the interface deformation and of the associated motions of water and oil but not the shape at the successive stages of interface uprising (at least to a first approximation). By closely examining the zones near the water-sphere "contact", it appeared that the short ligament has a diameter increasing rapidly with time because of the flux of uprising water. it can then be inferred that the last stage of interface deformation is a transition toward a jet presumably having a high velocity. After numerous trials we obtained the -magnified -picture of Fig. 8 unambiguously showing a very thin jet emitted by an elongating cone-like zone. Attempts to visualise such a jet in the case s 0 = 1 mm were unsuccessful, very likely because the reproducibility is not very good (after each voltage pulse the sphere is removed, cleaned and repositioned). In any case the question arises concerning the occurrence of such a jet-like disruption of the interface for small values of the initial spacing s 0 .

V. ORDER OF MAGNITUDE ANALYSIS

A very rough model consists in following the rise of a certain amount of water (and oil) under the action of the electric forces. The time evolution is governed by the basic equation :

cap g es F F F dt dw M - - = ( 1
)
where M stands for the mass of liquid, w for the interface velocity on the axis and F es , F g and F cap are the magnitudes of electrostatic, gravitational and capillary forces. The electrostatic pressure applies on an effective area decreasing as the water level rises; it has been shown that the pertinent scale then is R eff = (R 0 s) 1/2 [START_REF] Atten | A simplified model of electrocoalescence of two close water droplets in oil[END_REF] where R 0 is the sphere radius and s = s 0z int the minimum distance between water and sphere (z int denotes the height of the interface apex and w = dz int /dt). With this scale we deduce F es ~ (π/2) R eff 2 ε V appl 2 /s 2 (ε : fluid permittivity). The extra pressure due to the interface rise is ∆ρ g z int where ∆ρ is the density difference between the two fluids; the area concerned by the interface rise does not significantly change during the interface rising and we take F g ~ π R 0 s 0 ∆ρ g z int . At the axis the capillary pressure due to the interface curvature is 2T/R where T is the surface tension and R the radius of curvature; as the zone of strong curvature is closely related to the zone of high electrostatic pressure, we have F cap ~ 2π R eff 2 T/R. We can link R with z int by stating that, in the vicinity of the axis, the interface has a parabolic shape with the parabola intersecting the plane z = 0 at a distance x 0 from the axis ; this leads to 1/R = z int /x 0 2 .

Taking x 0 = α 1/2 R eff (α : numerical constant) finally gives F cap ~ (2π/α) T z int .

The deformation and rise of the interface implies the motion of a volume of liquid that we assume to be again characterised by the length scale R eff so that M = β ρ water R eff 3 , β being a constant. By introducing the relative height f 0 of the interface (f 0 = z int /s 0 ), equation ( 1) can be rewritten as follows:

( ) ( )

1 2 1 1 1 3/2 0 0 5/2 0 2 2 0 2               + - = f - f Bo f - Be dt f d α τ (2)
The non dimensional Bond number Bo and electrical Bond number Be have the expressions [START_REF] Atten | Field-induced deformation and disruption of a planar water-oil interface under the influence of a conducting sphere[END_REF] :

2 1 2 0 0 2 0 0 s T R V Be T s R g Bo appl ε ρ = ∆ = (3)
and the time constant τ defined by :

2 / 1 2 / 3 0 3/2 0 π         = T s R water ρ β τ (4)
is the capillary time built with the length scale R eff . Its typical value in the case R 0 = 4 mm and s 0 = 1 mm is on the order of 10 -2 s. Of course the time scale of the problem also depends on the applied voltage through the parameter Be but, as the Be values typically range from 0.2 to 0.6 in the conditions of interest here, this does not change the order of magnitude of the effective time scale.

Static solutions for the interface deformation correspond to the right hand side of (2) being equal to zero and can be obtained only for small enough Be values. The maximum of Be corresponds to (f 0 ) crit = 0.5, which leads to Be crit ≅ 1/(2α) + Bo/4, an estimate in fair agreement with theoretical and experimental results [START_REF] Atten | Field-induced deformation and disruption of a planar water-oil interface under the influence of a conducting sphere[END_REF] when taking α ≅ 2. Considering the case of water-air interface for an initial spacing s 0 = 1 mm, integration of (2) with the constants α = 2 and β = 5 gives results as shown in Fig. 9 (in this model the critical voltage is defined by Be crit = 0.39, a value close to the measured ones [START_REF] Atten | Field-induced deformation and disruption of a planar water-oil interface under the influence of a conducting sphere[END_REF]). The two curves shown in Fig. 9 are qualitatively similar to the oscillograms of Figs. 2 and4. The mean rising velocity (~ 2 cm/s) for the two curves of Fig. 9 is a fairly good estimate for the first stage (f 0 up to about 0.6) of interface deformation as shown by Fig. 4. However, this model does not predict the fast final uprising. This is not surprising because for an interface which sharpens and tends to emit a jet, the very rough estimations of the various forces and of M are not valid.

VI. DISCUSSION AND CONCLUSIONS

These preliminary experiments have shown that for a large enough spacing s 0 the water-oil interface raises and deforms tending to a more or less conical shape; in the last stage of the apex rising, due to the very strong electrostatic pressure there is a strong acceleration of the motion associated with the transition towards a jet. Although this has not been visualised unambiguously, we guess that a similar phenomenon of interface disruption occurs for small gaps. A refined technique of visualisation is under development to investigate the last stage of interface disruption and to characterise the length scale of jet-like formation.

Fig. 2 .

 2 Fig. 2. Oscillograms given by the shadowscopy technique : applied voltage (upper curve) and approximate rise (lower curve) of the apex of the water-air interface (s 0 = 0.6 mm).

Fig. 5 .Fig. 6 .

 56 Fig. 5. Photographs (t = 40 ms) of the deformed water-oil interface for various applied voltages (s 0 = 0.4 mm).

Fig. 8 .

 8 Fig. 7. Photographs of the deformed water-oil interface for various applied voltages in the case of a rather large gap s 0 = 2 mm.

Fig. 9 .

 9 Fig. 9. Variation with time of the normalized level of the apex of water-air interface (solution of (2) in the case α = 2, Bo = 0.56 and τ ≅ 13 ms).

ACKNOWLEDGMENT

This work is developed in the framework of a collaborative research between the French CNRS and the Consortium working on the project "Electrocoalescence -Criteria for an efficient process in real crude oil systems"; co-ordinated by SINTEF Energy Research. Contact person is L.E. Lundgaard. The project is supported by The Research Council of Norway, under the contract no: 169466/S30, and by the following industrial partners: Vetco Aibel AS, Aker Kvaerner Process Systems AS, StatoilHydro ASA, BP Exploration Operating Company Ltd, Shell Technology Norway AS, Petrobras.


PharmD-PhD Canan G Nebigil

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

Functional consequence of serotonin/5-HT 2B receptor signaling in heart: A role of mitochondria for transition between hypertrophy and heart failure?

Keywords: Cardiomyopathy, genetics, hypertrophy, receptors, signal transduction

Functional consequence of serotonin/5-HT2B receptor signaling in heart: A role of mitochondria for transition between hypertrophy and heart failure?

The role of serotonin in heart Serotonin (5-hydroxytryptamine, 5-HT) was first isolated from blood and found to function as a vasoconstrictor. 5-HT is found in three main areas of the body: the intestinal wall (where it causes increased gastrointestinal motility), blood vessels (where it causes constriction of large vessels) and the central nervous system. Several lines of evidence suggest that 5-HT regulates cardiovascular function during embryogenesis and adulthood. 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. The effects of 5-HT in the cardiovascular system are complex: for example, 5-HT has been associated with bradycardia or tachycardia, hypotension or hypertension and vasodilatation or vasoconstriction. Recent pharmacological studies suggested that compounds acting on 5-HT receptors could be of therapeutic use in treatment of migraine, hypertension, heart and vascular diseases [START_REF] Villalon | Serotonin receptors as cardiovascular targets[END_REF][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 of receptors: 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/11-coupled, 5-HT 2 (A, B, C) receptors activates phospholipase C (PLC), which initiates a rapid release of inositol trisphosphate and increases intracellular calcium levels. The 5-HT 2B receptor (5-HT 2B R) is expressed in the adult cardiovascular system, as well as in the gut and brain. 5-HT 2B R mediates 5-HT-induced mitogenesis in transfected fibroblasts by recruiting c-Src for cell cycle progression via the Mitogen Activated Protein Kinase (MAPK ERK1/2) 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 R subtype via activation of PLA 2 and nitric oxide synthesis via a PDZ recognition motif [START_REF] Nebigil | Developmentally regulated serotonin 5-HT2B receptors[END_REF] .

In clinical disorders associated with increased 5-HT plasma levels such as carcinoid tumors, valvular heart disease has been commonly observed. This valvulopathy is characterized by hyperplasic valvular and endocardial lesions with increased extracellular matrix. The 5-HT mitogenic signaling has been proposed to trigger the valvular hyperplasia observed in carcinoid patients [START_REF] Robiolio | Carcinoid heart disease. Correlation of high serotonin levels with valvular abnormalities detected by cardiac catheterization and echocardiography[END_REF] . Antidepressant drugs that selectively inhibit the membrane 5-HT transporter (increasing the availability of 5-HT) have also been shown to produce arrhythmia including atrial fibrillation, bradycardia and heart block [START_REF] Sheline | How safe are serotonin reuptake inhibitors for depression in patients with coronary heart disease?[END_REF] . Antidepressants might contribute to cardiac damage in patients with coronary heart disease by amplifying the platelet aggregation, thus continuing vasoconstriction through increasing 5-HT levels. The prevalence of major depression in individuals with coronary artery disease is estimated to range from 15% to 23% [START_REF] Musselman | Depression really does hurt your heart: stress, depression, and cardiovascular disease[END_REF] . Another drug that causes the efflux of 5-HT, 3,4methylenedioxymethamphetamine (MDMA) can produce significant cardiovascular toxicity [START_REF] Badon | Changes in cardiovascular responsiveness and cardiotoxicity elicited during binge administration of Ecstasy[END_REF] . Some patients taking appetite-suppressants such as fenfluramine or ergot derivatives also experience valvular hyperplasia [START_REF] Rothman | Therapeutic and adverse actions of serotonin transporter substrates[END_REF] . Fenfluramine is a potent 5-HT releaser acting at the 5-HT transporter. However, its main metabolite, norfenfluramine has preferentially high affinity for human 5-HT 2 Rs acting as an agonist [START_REF] Rothman | Therapeutic and adverse actions of serotonin transporter substrates[END_REF] .

Norfenfluramine -mediated valvular disease was recently reported to be mediated predominantly by 5-HT 2A Rs via ERK1/2 activation trough both a protein kinase C and a Src stimulation [START_REF] Xu | Serotonin Mechanisms in Heart Valve Disease II: The 5-HT(2) Receptor and Its Signaling Pathway in Aortic Valve Interstitial Cells[END_REF] . In addition, use of fenfluramine resulted in a recent outbreak of pulmonary hypertension in humans. It has also been reported that activation by 5-HT or fenfluramine of the 5-HT 2B R is a limiting step that controls lung vascular proliferation and remodeling including the elastase activity and TGF-beta release associated with pulmonary hypertension [START_REF] Launay | Role of serotonin 5-HT2B receptor in pulmonary hypertension[END_REF] .

The diversity of 5-HT receptors and the lack of 5-HT receptor isotype specific pharmacological agents have complicated attempts to identify the specific receptors involved in 5-HT-induced cardiac functions. As an alternative to classical pharmacological approaches, mice lacking or overexpressing 5-HT receptors are useful tools for defining the specific 5-HT receptor subtypes involved in 5-HT responses.

5-HT 2B R is required for myocardial trabeculation during embryogenesis.

We have created 5-HT 2B R gene knockout (KO) mice to address the roles of 5-HT 2B R during development and in the adult. Genetic ablation of 5-HT 2B R induces trabeculation defects in the developing heart, causing partial lethality at mid-gestation [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF] . KO mice undergo two periods of lethality, one before embryonic day embryonic day 11.5 and the other shortly after birth. Histological analysis of hearts from 5-HT 2B R KO embryos revealed defects of the subepicardial layer and lack of trabeculation of the ventricular myocardium. The myocardial abnormalities of the 5-HT 2B R embryo mice correlate with 5-HT 2B R expression in the compact zone and in the myocardial trabeculae. Since the Gq-coupled 5-HT 2B R transduces mitogenic signals in fibroblasts [START_REF] Nebigil | 5-Hydroxytryptamine 2B receptor regulates cell-cycle progression: Cross talk with tyrosine kinase pathways[END_REF] , impaired differentiation and proliferation in the heart of 5-HT 2B R KO embryos probably accounts for the mid-gestation lethality. Interestingly, analysis of the mutant mice for Gq/G11 protein demonstrated a strict dependence on Gq/G11 dosage. Mice lacking both Gproteins Gq -/-/ G11 -/-died at embryonic day 11 due to ventricular hypoplasia. In addition, Gq -/-/ G11 +/-newborn mutants exhibit craniofacial defects. Thus, two active Gq and G11 alleles are necessary for postnatal life and at least one intact Gq or G11 allele is required to bring the embryo to term [START_REF] Offermanns | Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice[END_REF] . G11 seems to be preferentially required for 5-HT 2B R-dependent heart developmental events, since the skeletal abnormalities observed in Gq -/-/G11 +/-embryos could not be detected in the 5-HT 2B R KO mice

5-HT 2B R mutations alter cardiomyocyte function and structure.

Newborn 5-HT 2B R KO mice developed cardiac dilation at 100% penetrancy. The thinning of the ventricular wall and the reduction in ventricular mass observed in the 5-HT 2B R KO mice result from both a reduction in the number of myocardial cells and a decrease in cell size. The decrease in cardiomyocyte size could be due to impaired growth during postnatal development [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] . Cardiomyocytes from KO mice also exhibit impaired contractility and beating rates in response to dobutamine (a b-adrenergic receptor agonist) in the absence of sympathetic innervation, indicating cell autonomous defects. The impaired contractility and myofibrillar degeneration could be related to the altered intercalated disc structures observed ultrastructurally in 5-HT 2B R KO hearts [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] . Myofibril loss is the most obvious structural change in human cardiomyopathy and sarcomeric disarray is characteristic of failing hearts [START_REF] Schaper | Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy[END_REF] . Interestingly, most mutations leading to dilated cardiomyopathy in humans affect structural proteins involved in cytoskeleton-extracellular matrix interactions at the Zstripe [START_REF] Beltrami | The cellular basis of dilated cardiomyopathy in humans[END_REF] . Z line-associated structures are responsible for the lateral alignment of myofibrils, and their anchorage to N-cadherin-and vinculin-containing costameres along the cell membrane. N-cadherin plays an important role in maintaining myofibril integrity, in cardiomyocyte interaction and in myofibrilogenesis [START_REF] Radice | Developmental defects in mouse embryos lacking N-cadherin[END_REF] . The 5-HT 2B R KO mice exhibit decreased N-cadherin levels [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] . Downregulation of N-cadherin and disruption of intercellular adhesion have also been reported in failing guinea pig hearts [START_REF] Wang | Chronic pressure overload cardiac hypertrophy and failure in guinea pigs: III. Intercalated disc remodeling[END_REF] . Myofibrillar organization and intercellular junction structures are impaired in the absence of 5-HT 2B R, suggesting that its activation plays a crucial and specific role in the organization and expression of cytoskeletal structures in cardiomyocytes [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] .

The 5-HT 2B R KO heart phenotype is reminiscent of the natural history of patients with dilated cardiomyopathy. All 5-HT 2B R KO mice that survived until the first postnatal week reached adulthood and exhibited dilated cardiomyopathy-like symptoms. Dilated cardiomyopathy in human is diagnosed with two typical features, left ventricular (LV) dilatation and depressed LV systolic performance. Both features were observed in adult 5-HT 2B R KO mice hearts. Echographic analysis showed that the LV end-diastolic diameter and the LV endsystolic diameter were significantly increased in 5-HT 2B R KO male mice [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] . Moreover, serum biochemical indicators of myocardial infarction (creatine kinase-MB mass, troponin I-C) are increased in the 5-HT 2B R KO mice. 5-HT 2B R KO mice therefore, provide a novel model of mouse cardiomyopathy, characterized by a phenotype of dilation with a "failed" hypertrophic response (figure 1).

Heart-specific overexpression of 5-HT 2B R leads to cardiac hypertrophy.

Overexpression of 5-HT 2B R in the heart using a MHC promoter containing transgene also leads to specific defects. Transgenic (TG) mice overexpressing the Gq-coupled 5-HT 2B R exhibit hypertrophy that is due to increased number and growth of cardiomyocytes. The overexpression of 5-HT 2B R raises the expression of hypertrophic genes such as ANF and b-MHC as an evidence of hypertrophic response in the TG heart. This hypertrophic response is a direct effect of overexpression of 5-HT 2B R in the myocardium, since hypertrophy was observed without evidence of hemodynamic overload. Cardiac fibrosis was not observed in TG hearts, indicating that cardiomyocyte-specific overexpression of 5-HT 2B R does not alter remodeling in nonmyocyte cells through paracrine mechanisms [START_REF] Nebigil | Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy[END_REF] .

Several factors might be responsible for the development of compensated hypertrophy after overexpression of 5-HT 2B R. A first possibility to explain the development of hypertrophy is the change in signaling pathways and Gq protein repertoire coupling. Overexpression of Gq-downstream signaling molecules has been associated with the induction of a cardiomyopathy characterized by a ventricular hypertrophy: cardiac-specific overexpression of constitutively active d-PKC and e-PKC isoforms caused identical, non-pathological hypertrophic phenotypes [START_REF] Takeishi | Transgenic overexpression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy[END_REF][START_REF] Bowman | Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates[END_REF] ; TG expressing an activated MEK1 cDNA in the heart also demonstrated concentric hypertrophy without signs of cardiomyopathy [START_REF] Bueno | The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice[END_REF] .

Another possible cause of hypertrophy after overexpression of 5-HT 2B R could be secondary to compensation for the early apoptosis and increased proliferation of cardiomyocytes. 5-HT 2B R KO newborn mice exhibit ventricular hypoplasia due to impaired cardiomyocyte proliferation [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF] . It is possible that overexpression of 5-HT 2B R in the heart activates mitogenic pathway(s) before birth and then inhibits apoptosis in terminally differentiated and non-proliferative cardiomyocytes after the birth.

The involvement of Gq-coupled receptors in regulating cardiomyocyte growth has not yet been fully understood despite the use of both loss-of-function and gain-offunction experiments to a given receptor in vivo. For example, TG mice overexpressing the angiotensin AT1 receptor in the myocardium develop cardiac hypertrophy [START_REF] Paradis | Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling[END_REF] , but pressure overload and stretch-induced hypertrophy still occur in AT1 KO mice [START_REF] Harada | Angiotensin II type 1A receptor knockout mice display less left ventricular remodeling and improved survival after myocardial infarction[END_REF] ; cardiac specific overexpression of constitutively active alpha-1B adrenergic receptor in TG results in mild cardiac hypertrophy [START_REF] Milano | Myocardial expression of a constitutively active alpha 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy[END_REF] but no hypertrophy developed in TG mice overexpressing the wild type alpha-1B adrenergic receptor [START_REF] Akhter | Transgenic mice with cardiac overexpression of alpha1B-adrenergic receptors. In vivo alpha1-adrenergic receptor-mediated regulation of beta-adrenergic signaling[END_REF] . Mice with selective Gq and G11 ablation in cardiomyocytes showed no detectable ventricular hypertrophy in response to pressure-overload induced by aortic constriction [START_REF] Wettschureck | Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes[END_REF] .

Mitochondrial defects are observed in the 5-HT 2B R KO and transgenic hearts.

Mitochondrial dysfunction has been reported in human cardiac diseases including ischemic and non-ischemic heart failure, myocardial infarction, arrhythmia, myocarditis and dilated cardiomyopathy [START_REF] Towbin | The failing heart[END_REF][START_REF] Chien | Converging pathways and principles in heart development and disease: CV@CSH[END_REF] . Although damage in mitochondria is a key step leading to programmed cell death, no typical apoptotic bodies were observed in the heart of 5-HT 2B R KO [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] . Altered mitochondrial membrane integrity was observed, however, in the cardiomyocytes of 5-HT 2B R KO mice that presented dilated cardiomyopathy (figure 2). Furthermore, reduced succinate dehydrogenase (SHD) and cytochrome C (cytoC) oxidase (COX) indicated altered functions of electron transport complexes II and IV, respectively [START_REF] Nebigil | Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a direct of 5-HT2B-receptor signaling[END_REF] . Increased lactate plasma levels in KO mice confirmed these observations [START_REF] Nebigil | Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a direct of 5-HT2B-receptor signaling[END_REF] . Decreased oxidative phosphorylation and respiration that lead to lactate production have also been observed in human mitochondrial myopathies and animal models for dilated cardiomyopathy [START_REF] Narula | Apoptosis and cardiomyopathy[END_REF] . 5-HT 2B R KO cardiomyocytes may be in the pre-apoptotic stage long before nuclear events became morphologically manifested in vivo. 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] . These data raise the issue how cardiomyocytes seemingly tolerate some cytosolic cytoC. The balance between nuclear fragmentation and protein cleavage is variable in cardiomyocytes [START_REF] Communal | Functional consequences of caspase activation in cardiac myocytes[END_REF] . Other factors might explain the presence of processed caspase-3 in cells that are not undergoing apoptosis.

Overexpression of 5-HT 2B R in the mouse heart leads to abnormally high mitochondrial numbers as observed by transmission electron microscopic analysis and red ragged fibers staining. Moreover, mitochondrial enzyme activities such as COX and SDH are increased in TG mice heart. This increase in oxidative phosphorylation activity is also suggestive of an accumulation of mitochondria in the TG heart [START_REF] Nebigil | Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy[END_REF] .

Mitochondria are targets in 5-HT 2B R survival signals in isolated cardiomyocytes.

Mitochondria represent an essential component of many apoptotic pathways by controlling the release of cytoC into the cytosol [START_REF] Reed | Postmitochondrial regulation of apoptosis during heart failure[END_REF][START_REF] Zamzami | The mitochondrion in apoptosis: how Pandora's box opens[END_REF] . Released cytoC from mitochondria forms an activation complex with apoptotic protein-activating factor (APAF)-1 and caspase-9, which activates downstream caspases to execute final morphological and biochemical alterations [START_REF] Li | Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade[END_REF] . This pathway is tightly regulated by a group of antiapoptotic proteins, such as Bcl-2, and proapoptotic proteins, such as Bax [START_REF] Green | Mitochondria and apoptosis[END_REF] . Further downstream regulation occurs through various inhibitors of caspases [START_REF] Deveraux | IAP family proteins--suppressors of apoptosis[END_REF] . Cardiomyocytes also use a mitochondria-dependent apoptotic pathway [START_REF] Suleiman | Mitochondria: a target for myocardial protection[END_REF] . In isolated cardiomyocytes, serum withdrawal is a classical inducer of apoptosis. Our work has shown that 5-HT via the 5-HT 2B R acts as a survival factor, by inhibiting serum withdrawal-induced apoptosis as manifested by DNA fragmentation, nuclear chromatin condensation and TUNEL labeling [START_REF] Nebigil | Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a direct of 5-HT2B-receptor signaling[END_REF] . 5-HT prevents cytoC release and caspase-9 and -3 activation following serum deprivation via cross talks between phosphatidylinositol-3 kinase (PI3K) / Akt and ERK1/2 signaling pathways. Upon activation of Akt by 5-HT, phosphorylation of IKappaB-alpha triggers its degradation to release NF-KappaB. This activation of NF-KB inhibits serum deprivation-induced the adenine nucleotide translocator (ANT) expression to maintain mitochondrial permeability [START_REF] Nebigil | Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a direct of 5-HT2B-receptor signaling[END_REF] . Parallel to this pathway, ERK activation by 5-HT inhibits elevation of Bax expression induced by serum deprivation. Defects in survival pathway in the 5-HT 2B R KO heart are not secondary to the cardiomyopathy, since defective cardiomyocyte survival can be reverse by transfection of 5-HT 2B R cDNA and LIF survival signaling is intact. In addition, 5-HT did not induce hypertrophy of cultured cardiomyocytes (unpublished observation).

5-HT via 5-HT 2B R regulates the expression of ANT-1 in the heart.

Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT-1 emerges as a major player in the regulation of cell death [START_REF] Vieira | Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator[END_REF] . The ANT-1 combines mitochondrial energy-producing and cytosolic energy-consuming processes. Impaired ANT-1 functions have been observed in heart tissue from patients with dilated cardiomyopathy [START_REF] Portman | The adenine nucleotide translocator: regulation and function during myocardial development and hypertrophy[END_REF] . A shift in the ANT isoform transcription profile was found in heart tissue from patients with dilated cardiomyopathy, but not in that from such patients with ischemic or valvular cardiomyopathy. The shift is characterized by an increase in ANT-1, a decrease in ANT-2 and an unchanged ANT-3 proportion. This alteration in the ANT isoform pattern is not a general phenomenon of end-stage heart failure, and clearly occurs already before the heart is terminally damaged. Therefore, an altered ANT isoform expression appears to be a feature of a dilated cardiomyopathy-specific gene program [START_REF] Dorner | The myocardial expression of the adenine nucleotide translocator isoforms is specifically altered in dilated cardiomyopathy[END_REF] . ANT-1 KO 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] and point mutations in the ANT-1 gene have been reported in cases of human mitochondrial diseases [START_REF] Kaukonen | Role of adenine nucleotide translocator 1 in mtDNA maintenance[END_REF] . ANT-1 overexpression leads to phenotypic alterations typical of apoptosis, i.e. collapsed mitochondrial membrane potential, cytoC release, caspase activation and DNA degradation [START_REF] Bauer | Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis[END_REF] . In the 5-HT 2B R KO hearts, elevated expression level of ANT-1 was detected. In cardiomyocytes, ANT-1 is a main target of PI3K / Akt signaling that control mitochondrial permeability [START_REF] Nebigil | Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a direct of 5-HT2B-receptor signaling[END_REF] . Moreover, ANT-1 downregulation was observed in 5-HT 2B R overexpressing TG hearts [START_REF] Nebigil | Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy[END_REF] (figure 3). Interestingly, histological and ultrastructural examination of muscle from ANT-1 null KO mice revealed red ragged fibers, COX and SDH activation, cardiac hypertrophy with mitochondrial proliferation similar to 5-HT 2B R TG mice [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] .

5-HT via 5-HT2B R regulates Bax expression in the heart.

The proto-oncogenes Bcl-2 -an inhibitor of apoptosis and Bax -an inducer of apoptosis play critical roles in the molecular circuit controlling apoptosis in cardiac muscle. The ratio of Bax to Bcl-2 determines survival or death after an apoptotic stimulus. Patients with mild heart failure show significantly higher Bax/Bcl-2 ratios than patients with advanced heart failure. The susceptibility of myocytes to apoptosis is significantly increased in the early phase of heart failure, but decreases with worsening of the disease due to depressed expression of the Bax onco-protein. This increased susceptibility to apoptosis may have a role in the transition from mild to severe heart failure in patients with idiopathic dilated cardiomyopathy [START_REF] Akyurek | Association between the severity of heart failure and the susceptibility of myocytes to apoptosis in patients with idiopathic dilated cardiomyopathy[END_REF] .

In cardiomyocyte culture, the effect of 5-HT via 5-HT 2B R was to downregulate Bax expression that was completely inhibited by the ERK1/2 inhibitor PD-09858. In neonatal cardiomyocytes, Bax is necessary, but not sufficient, to control cytoC release; cytoC diffusion is controlled by both ERK1/2 and PI3K/Akt. While the Bcl-2 family of proteins, at least in part, controls the mitochondrial apoptosis, only ERK1/2 inhibition can overturn 5-HT-induced down regulation of Bax in cardiomyocytes [START_REF] Nebigil | Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a direct of 5-HT2B-receptor signaling[END_REF] . Bax forms membrane pores that control mitochondrial permeability and release cytoC. Bcl-2 and Bcl-XL inhibit formation of these pores, however, no alteration in the expression of these genes could be detected in 5-HT 2B R KO heart mRNA. In vivo, analysis in KO mice heart showed that the Bax expression level was increased. Our data indicate that 5-HT/5-HT 2B R cytoprotective signaling targets mitochondria by regulating Bax and ANT-1 expression in heart.

A proposed model for transition from hypertrophic to dilated cardiomyopathy.

Hypertrophy is viewed as compensatory and the transition to heart failure occurs via the constitutive activation of proapoptotic signals due to extensive hypertrophic stimulus. In cardiac tissue, apoptosis is a form of programmed cell death distinct from necrotic cell death. It has been proposed that ventricular dilatation and neurohormonal activation during heart failure lead to up-regulation of transcriptional factors induced during cardiomyocyte hypertrophy that prepared the cells for entry in the cell cycle [START_REF] Chien | Genomic circuits and the integrative biology of cardiac diseases[END_REF] . However, terminally differentiated cardiomyocytes cannot divide and, instead, they undergo apoptosis. Initiation of apoptosis is associated with activation of upstream cascades, including the release of cytoC from mitochondria to cytoplasm and processing of proteolytic caspases [START_REF] Wallace | Mitochondrial defects in cardiomyopathy and neuromuscular disease[END_REF] . Persistent aerobic conditioning induces a compensated hypertrophy that is thought to be beneficial to the heart. These functional changes may increase the mitochondria proliferation rate. Failing mitochondrial functions may lead to decompensation due to induction of apoptotic signaling. Intrinsic determinants of mitochondrial apoptosis have recently been elucidated that modify or abort decompensation: Cardiac hypertrophy induced by the Gq pathway upregulates Nix expression whereas, a generalized increase in apoptotic mediators did not occur [START_REF] Yussman | Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy[END_REF] . Recent data on 5-HT 2B R signaling pathway indicate that 5-HT can control expression of the nuclear genes Bax and ANT-1, which regulate mitochondrial function. It is becoming clear that 5-HT signaling via 5-HT 2B R converges onto mitochondria, major cellular compartments, which can modify cardiac hypertrophy or decompensation (figure 4). These findings promise to have important implications for the understanding of congenital heart disease and the development of potential therapeutic interventions for cardiovascular disease.

Figure 1 .

 1 Figure 1. Ablation of 5-HT 2B R in mice leads dilated cardiomyopathy without compensatory hypertrophic response. A representative sagittal section from adult hearts (12 weeks old) show thinning of the left venticle (lv) for in 5-HT 2B R KO (-/-) mice compared to wild type (+/+) . rv: right ventricle; Bar: 200 µm. Adapted from 15 .

Figure 2 .

 2 Figure 2. 5-HT 2B R mutant mice display alterations of cardiomyocyte cytoarchitecture. Transmission electronic microscopy on ultra-thin sections of the compact layer from left ventricles of 5-HT 2B R wild type (+/+) and KO (-/-) newborn mice shows mitochondrial defects (m, arrow). Z-bands (Z). Bars: 1 µm.

Figure 3 .

 3 Figure 3. Histological analysis of 5-HT 2B TG mouse heart. The analysis of the compact layer of adult mice heart was performed on semi-thin sections (A) and by transmission electronic microscopy on ultra-thin sections (B) of wild type (NTG) and transgenic (TG) mice. Lipid (l), mitochondria (m, arrow), and T-tubules (t) alterations. Z-bands (Z). Bars: A 40 µm, and B 1 µm. As a mitochondrial marker, the ANT-1 protein expression (C) was evaluated by immuno-histochemical staining. ANT-1 protein expression levels evaluated by densitometric analysis of western blot are expressed as percent of NTG. Bars=120 µm. Adapted from 20 .

Figure 4 .

 4 Figure 4. The role of mitochondria for the transition between hypertrophy and dilated cardiomyopathy. 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 that inhibits ANT-1 and Bax expression, respectively to control mitochondrial membrane permeability (middle). In the 5-HT 2B R knockout mice as a model of dilated cardiomyopathy, the Gq-coupled 5-HT 2B R signaling regulates mitochondrial structure and function thereby controlling myofibrillar organization in the heart (left). In the 5-HT 2B R overexpressing mice, the Gq-coupled 5-HT 2B R signaling regulates ANT-1 expression and mitochondria number thereby giving rise to hypertrophic heart (right).

Acknowledgements

The excellent drawings of Dr. Laurent Désaubry are greatly appreciated. We thank Dr P. Dollé for critical reading of the manuscript and for helpful discussions. 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, the Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer. CGN is supported by a fellowship from the Lefoulon-Delalande Fondation.