James Braun

Cédric Sauder

Jacques Lamon

Fanny Balbaud-Célérier

Influence of an original manufacturing process on the properties and microstructure of SiC/SiC tubular composites

Keywords: A. Ceramic-matrix composites (CMCs), B. Mechanical properties, C. Acoustic emission, D. . icrostructural analysis

SiC/SiC composites reinforced with Hi-Nicalon S 3 rd generation SiC fibers are promising candidates for high temperature aeronautical applications and fuel cladding in nuclear reactors. An original patented process was used to produce SiC/SiC tubular samples for dimensional and tensile mechanical characterizations. The process route is described in detail to further understanding of specific features.

SiC/SiC samples show a low porosity and excellent geometrical tolerance. Highly reproducible tensile mechanical behavior is observed with good mechanical failure characteristics. Some specific features were highlighted during mechanical characterizations with loading/unloading sequences. For instance, an original mechanical behavior was observed before the onset of matrix multicracking (deviation from linearity). To our knowledge, it is the first time that this kind of behavior has been detected on SiC/SiC composites, due to the processing route leading to the microstructure of specific samples. 1. Introduction Among the Ceramic Matrix Composites (CMC), SiC/SiC composites and affiliates have aroused interest for challenging high-temperature applications such as aerospace [1], aeronautics [2], defense [3] or nuclear applications [4] for their excellent mechanical properties [5] and oxidation resistance [6] at

2 elevated temperature. Initially considered for fusion application (breeding blankets) [START_REF] Giancarli | R&D issues for SiCf/SiC composites structural material in fusion power reactor blankets[END_REF] [START_REF] Katoh | Current status and critical issues for development of SiC composites for fusion applications[END_REF], SiC/SiC can also improve performance and safety of current nuclear reactors in the framework of EATF [START_REF] Terrani | Accident tolerant fuel cladding development: Promise, status, and challenges[END_REF] but is also one of the few options for generation IV nuclear reactor core materials. For instance, Gas-cooled Fast Reactors (GFR) are designed to operate at high temperatures even in nominal conditions, so the fuel cladding has to sustain temperatures between 450 and 1000°C for several years. Moreover, the fuel cladding will have to maintain its dimensions and integrity in extreme situation (up to 1600°C) [START_REF] Gasparini | Considerations in the development of safety requirements for innovative reactors: application to modular high temperature gas cooled reactors[END_REF]. In this aim, the SiC/SiC tubular composites reinforced with 3 rd generation Hi-Nicalon S fibers are potential candidates that can meet the GFR criteria and are, currently, the more advanced cladding solution [START_REF] Katoh | Current status and recent research achievements in SiC/SiC composites[END_REF].

The Chemical Vapor Infiltration (CVI), enabling also the pyrocarbon interphase deposition, was applied for densification. This process was chosen because the SiC matrix deposited by this technique leads to better properties under irradiation [START_REF] Snead | Radiation Effects in SiC and SiC-SiC[END_REF]. A three-layered tubular fibrous structure was chosen for the texture, made of filament winding and/or 2D braiding [START_REF] Sauder | Ceramic Matrix Composites: Nuclear Applications[END_REF], which achieves a good level of surface roughness (R a < 3 µm), with a final cladding thickness below 1 mm (Fig 1). Tubular shaped CMCs were not studied extensively, especially on such fine structure, due to the lack of application outside of the nuclear industry.

For high temperature heat exchangers, if thick tubes are needed (over 1 mm and up to 10 mm) [START_REF] Wimpory | Residual Strain Measurement of C/C-SiC Tubes at High Temperature[END_REF], the structure of the material is commonly made of woven tissues of carbon or silicon carbide fibers, rolled on a mandrel and embedded in a resin. Its pyrolysis combined with Melt Infiltration (MI) lead to the sample densification [START_REF] Wimpory | Residual Strain Measurement of C/C-SiC Tubes at High Temperature[END_REF] [START_REF] Ortona | Fabrication of cylindrical SiCf/Si/SiC-based composite by electrophoretic deposition and liquid silicon infiltration[END_REF]. However, this technique is not suitable for thin layer materials considered for fuel cladding (< 1 mm), which is around the thickness of the 2.5D woven fabrics made of Hi-Nicalon S fibers, even though 80% dense materials can be manufactured [START_REF] Deck | Fabrication of SiC-SiC composites for fuel cladding in advanced reactor designs[END_REF]. The MI is also not compatible with nuclear applications due to swelling mismatch between SiC and Si under irradiation [START_REF] Katoh | Ceramic matrix composites in fission and fusion energy applications[END_REF]. Some cylindrical structures were also developed for aerospace applications, such as turbine vanes and blades, out of Sylramic fibers by alternate methods (2D and 3D braiding, filament winding, pin-weave, orthogonal-weave, …) with a matrix made of a combination of CVI and melt infiltration. It was noted that this tube structure induces addition constituent and process-related issues that are not experienced 3 for flat panels: fiber bending, limited fiber fraction in key directions and limited interphase and matrix formation caused by one-sided infiltration from the mandrel [START_REF] Yun | Issues on Fabrication and Evaluation of SiC/SiC Tubes With Various Fiber Architectures[END_REF]. Hot pressing may be employed for matrix densification [START_REF] Yu | Fabrication of the tube-shaped SiCf/SiC by hot pressing[END_REF] but the necessity of high temperature may diminish the fibers mechanical properties [START_REF] Sha | Effect of heat treatment on the tensile strength and creep resistance of advanced SiC fibers[END_REF]. This issue can also occur for NITE-based materials [START_REF] Nakazato | Sic/Sic Fuel Cladding by Nite Process for Innovative LWR-Cladding Forming Process Development[END_REF], which leads to composites with low level of porosities.

In previous studies, the laboratory has manufactured 3D Interlock tubes, while the material reaches high damage tolerance, this technique leads to very important level of porosities [START_REF] Rohmer | Mechanical properties of SiC/SiC braided tubes for fuel cladding[END_REF].

In the present work, specific SiC/SiC cladding sections were produced out of filament winding and 2D braiding along with CVI densification. Both techniques can produce down to 300 µm thick composites.

These methods, along with the use of a specific mandrel, allow the production of materials that reach excellent dimension tolerance.

A lack of data on manufactured CMC tubes is noted. Mainly, studies are focused on the hoop [START_REF] Feinroth | Mechanical strength of CTP Triplex SiC fuel clad tubes after irradiation in MIT research reactor under PWR coolant conditions[END_REF] [23] or the flexural [START_REF] Deck | Fabrication of SiC-SiC composites for fuel cladding in advanced reactor designs[END_REF] strength. Thus, tensile mechanical behavior of samples (produced with an original manufacturing process) and their reproducibility were assessed. Loading/unloading cycled specific tensile tests were performed to achieve data regarding damage evolution and the friction mechanism as a function of the strain/stress levels, which were not evaluated in previous multiaxial testing [START_REF] Bernachy-Barbe | Anisotropic damage behavior of SiC/SiC composite tubes: Multiaxial testing and damage characterization[END_REF]. After being damaged at several levels and up to ultimate failure, the crack density of the composites was measured and compared to the cumulated acoustic emission signal (number of events, representative of the number of cracks generated during mechanical solicitation) [START_REF] Morscher | Modal acoustic emission of damage accumulation in a woven SiC/SiC composite[END_REF]. Finally, the thermal residual stresses, due to thermomechanical mismatch between the matrix and the fibers, were determined both experimentally and by modelling [START_REF] Michaux | Young's modulus, thermal expansion coefficient and fracture behavior of selected Si-B-C based carbides in the 20-1200°C temperature range as derived from the behavior of carbon fiber reinforced microcomposites[END_REF]. All these characterizations were used to discuss the specificity of tensile mechanical behavior of these SiC/SiC cladding specimens.

Materials and method

2.1. SiC/SiC composites elaboration 2.1.1. Fibrous reinforcement processing Third generation Hi-Nicalon S (NGS supplier) SiC fibers were chosen as SiC/SiC CMC (Ceramic Matrix Composite) reinforcement. The manufactured SiC/SiC composite material has a 3 successive layer structure. The inner layer is made of ± 45° filament wound structure (usually modelled as laminated structure) and the two others of ± 45° 2D braiding structure (modelled as twill fabric). In this aim, a 32 carriers 2D braiding machine was used. All the layers have similar thicknesses (≈ 300 µm). The filament winding process (interlaced plies) leads to a very good internal circular shape for tubular structure in contrast with the 2D braiding (cross plies) that leads to more irregular internal and external surfaces [START_REF] Sauder | Ceramic Matrix Composites: Nuclear Applications[END_REF].

Parameters (speed, fiber tension, etc…) have to be chosen wisely to avoid any tow fiber fracture during manufacturing. To obtain similar characteristics under tensile and swelling (internal pressure) mechanical solicitations, the fibers orientation was fixed to ± 45° [START_REF] Bernachy-Barbe | Anisotropic damage behavior of SiC/SiC composite tubes: Multiaxial testing and damage characterization[END_REF]. The fiber structure is processed on a mandrel that has to match several criteria. At first, it has to be chemically inert at high temperature (800 to 1100°C) to withstand the CVI densification process (MTS, H 2 , HCl, …). Secondly, its removal has to be simple, without damaging the composite. At last, this mandrel must have a sufficient stiffness and be easy to grind to reach a high tolerance diameter dimension along its length, directly influencing the dimension tolerance of the composite. In addition, the silica glass mandrel was retained because it can be easily removed by hydrofluoric acid chemical etching [START_REF] Sauder | Patent: Method for producing a composite including a ceramic matrix[END_REF]. Finally, the fibrous reinforcement has respectively an external diameter, a length and a fiber volume fraction of 9.4 mm, 200 mm and ≈ 40 to 45%.

Densification / Machining

Chemical Vapor Infiltration (CVI) was used for the densification of fibrous reinforcement. First, a 30 -50 nm pyrocarbon interphase and a few micrometers of SiC were successively deposited to give, respectively, the composites expected mechanical properties [START_REF] Buet | Influence of surface fibre properties and textural organization of a pyrocarbon interphase on the interfacial shear stress of SiC/SiC minicomposites reinforced with Hi-Nicalon S and Tyranno SA3 fibres[END_REF] and the material a sufficient rigidity for mandrel withdrawal [START_REF] Sauder | Patent: Method for producing a composite including a ceramic matrix[END_REF]. The SiC matrix volume fraction is around 5 -20 % after this step (consolidation step), so that the mandrel can be removed by a HF chemical etching, dissolving the silica.

In the case that the mandrel would have not been withdrawn, the densification would be incomplete on the inner side. To complete the densification, a second CVI densification step is repeated. At the end of this second step, matrix volume fraction is between 30 and 40 %. Centerless grinding is then employed to reach desired external dimensions. An ultimate CVI densification is then executed for seal coat deposition. After this last step, the matrix volume fraction reaches 40 to 55 %. The internal surface is then grinded (running-in process) to reach the final internal dimension with ± 0.01 mm tolerance. The final porosity volume fraction of the composites is below 10 %. The fibers and matrix volume fractions cannot be measured precisely because of the grinding steps. If the hypothesis that no fibers remained undamaged and that only the SiC matrix is removed during the grinding steps, the mean values (and SD) of the fiber, matrix and porosities volume fractions would be 42.26 (0.53)%, 50.05 (0.94)% and 7.70 (0.72)%, respectively. However, those values can only be considered as qualitative values. The tube cross-section was observed by X-ray tomography (Fig. 2). Cylindrical alumina standards and an optical profilometer (SCAN 52 TESA Technology) were used to determine respectively the internal (7.88 mm) and the external (9.58 mm) average diameters along the length of the tubes. Geometrical characteristics of the manufactured composites clads are presented in Table 1. (± 1 mm course) sensor. These extensometers were calibrated with a high-resolution numeric calibrator (0.1 µm). An acoustic emission sensor coupled with a signal conditioner and data acquisition system (resp. VS700-D and ASCO-DAQ2 by Vallen Systeme) (90 -295 kHz), fixed on the upper end collar, was systematically used to detect the matrix multicracking and the possible microstructural coupling. The measured signal is presented as a cumulated signal and corresponds to events. To avoid noise influence, only the acoustic emission over 50 dB were counted, corresponding to the background plus 10 dB. and are the number of events for a given level of stress reached and the number of events at the sample fracture, respectively.

Displacement speeds were controlled through the displaceable cross-rail, ranging from 0.05 to 0.2 mm/min. No effect on the mechanical behavior of those materials in this strain rate range has never been observed. 7 unloading -reloading cycles were done for some tensile tests at different levels of longitudinal strains before unloading (0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5 %). The speed level was increased along with the strain, to diminish the test duration, while still having a good precision at the lowest strains.

Analysis of the unloading -reloading cycles

The analysis of the unloading -reloading cycles give some information on damage evolution (matrix multi-cracking) in the composites microstructure during uniaxial mechanical loading [START_REF] Camus | Development of damage in a 2D woven C/SiC composite under mechanical loading: I. Mechanical characterization[END_REF] [31] (Fig. 4).

The consequence is a progressive decrease of the longitudinal elastic modulus ( ), along with the increase of the anelastic residual strains after unloading ( ), the full width at half maximum ( ) and the area of the cycles ( ), corresponding to the energy dissipated in the material. The evolution of those parameters are characteristic of the damaging and allows a better understanding of the microstructural changes in the material during mechanical loading.

Before matrix cracking, the composite exhibit a linear elastic mechanical behavior, allowing the measurement of the initial Young's modulus ( ). Therefore, cycles below this domain are closed.

Beyond this domain, matrix multicracking takes place. Cycles are open and can take the appearance of an ellipse with or without stiffening at the end of the unloading sequence. This possible stiffening, schematized on Fig. 4, is the consequence of the early reclosing of the matrix cracks and/or is due to the fibers roughness. In all the case, and always increase along with the mechanical loading and constitute a relevant parameter to follow the material damage progress during mechanical tensile test [32] [33]. The area of the cycles is characteristic of the dissipated energy in the material, hence the hysteresis loop. It increases as a consequence of the dissipative frictional mechanisms occurring at the interfaces. The shape of the cycles can also evolve during a test as a function of the reached longitudinal strain. Fig. 5 shows the method applied to determine both the initial Young's modulus ( ) and the elasticity modulus ( ) during the unloading -reloading tensile tests. The latter is determined by measuring the slope of the line passing through the summit of the cycle ( , ) and the median point of the cycle for a stress [START_REF] Domergue | Relationships between Hysteresis Measurements and the Constituent Properties of Ceramic Matrix Composites: II, Experimental Studies on Unidirectional Materials[END_REF]. The evolution of the elastic modulus is generally given thanks to the evolution of the reduced modulus, , as a function of the applied stress or strain, which is pertinent to follow the matrix multicracking evolution of the composite. For some specific composites structure (mechanical solicitation in one of the reinforcement axes and a good damage tolerance), when the longitudinal strain is close to failure (saturation of matrix multicracking), only the fibers orientated in the loading axis ensure the mechanical loading. Then, the reduced modulus reaches an asymptotic value, corresponding to the fiber fraction Young's modulus aligned in the mechanical solicitation direction. This value is given by Eq. ( 1) for a 2D plate composite solicited at a angle towards the tows direction [START_REF] Lynch | Effects of Off-Axis Loading on the Tensile Behavior of a Ceramic-Matrix Composite[END_REF]. and are the tow Young's modulus and the fiber volume fraction, respectively.

However, it has never been highlighted that this formula is also suitable for a ± 45° angle tubular structure.

(

) 1
The evolution of the reduced Poisson's ratio ( ) as a function of the applied stress usually follows the same trend that the longitudinal Young's modulus. However, a decrease to zero reveals the reduction of the lateral interactions, attributed to debonding at the fiber/matrix interface [START_REF] Bertrand | Hi-Nicalon/SiC minicomposites with (Pyrocarbon/SiC) n nanoscale multilayered interphases[END_REF]. The residual strains ( ) correspond to the longitudinal strains observed after complete unloading, over the composite elastic domain (Fig. 4), which take their origin from several phenomena:

-The release of the residual axial stresses (parallel to the loading direction) coming from the CVI densification (temperature going up to 1100°C). The residual thermal strains ( ) can be determined from the total residual strains [36] [37];

-friction mechanisms at different energies, which generate partial irreversible sliding [START_REF] Bouquet | Toughness assessment of ceramic matrix composites[END_REF];

-mechanical obstruction to the complete reclosing of the cracks due to the fibers roughness and/or fiber and matrix debris whose formation is enhanced by load cycling, leading to an increase in the sliding resistance at the end of the unloading sequence [START_REF] Bouquet | Toughness assessment of ceramic matrix composites[END_REF].

Results

Tensile behavior

The typical envelope of the strain/stress response of the manufactured SiC/SiC tubes, extracted from the cycled tensile tests, is presented along with the normalized acoustic emission ( ) as function of the longitudinal strain and the fracture surface after ultimate failure of the sample in Fig. 6.

The signal corresponds to the whole events occurring, even during the unloading sequences, that is why is not strictly growing as a function of the longitudinal strain. Different regimes are observed. At the beginning, the material exhibits a linear elastic response, whose slope is the material initial Young's modulus ( ). The deviation from linearity quickly appears ( and = 34 MPa) (Table 2), at point (A). However, the latter is not accompanied by any acoustic emission signal and slowly deviates from linearity. This phenomenon has never been observed on 2D SiC/SiC plates, even for offaxis solicitations [START_REF] Aubard | Model of the nonlinear mechanical behavior of 2D SiC-SiC chemical vapor infiltration composites[END_REF]. It can be concluded that it is not the consequence of the beginning of matrix multicracking. Indeed, the first acoustic signal ( and MPa), corresponding to the appearance of the first matrix crack, induces a clear and distinct change in the slope of the longitudinal strain/stress curve (Fig. 6) (B). Around 200 MPa (C), an increase in the stress response is again observed, evidencing a strengthening mechanism following the load displacement on the fibers, until ultimate failure (D). Afterwards, the AE follows an almost linear evolution as a function of the applied strain. The cumulative AE count is proportional to the number of cracks generated in the composite [START_REF] Morscher | Fiber effects on minicomposite mechanical properties for several silicon carbide fiber-chemically vapor-infiltrated silicon carbide matrix systems[END_REF]. It should be noted that the acquisition device only allows a simple counting over a predefined threshold. The analysis of the AE energy signal (rather than AE cumulative count) for similar tests (analogous materials and mounting) exhibit a saturation above a stress of 200 MPa and a 0.5 % longitudinal strain [START_REF] Bernachy-Barbe | Anisotropic damage behavior of SiC/SiC composite tubes: Multiaxial testing and damage characterization[END_REF]. The same Identical regimes are observed on diametric strain/stress curve.

The fracture surface gives a qualitative evaluation of the interfacial strength. On the internal side of the tube (filament winding structure), the debonding length ( ), i.e. extracted from the matrix sheath, is shorter from the one observed on the external side (2D braiding structure). These debonding lengths (several dozens of micrometers) are distinguishing features of a moderate bonding.

To verify the homogeneity and reproducibility of the manufacturing process, 8 different tubes (REF 1 to REF 8) were tested following the same procedure. Fig. 7 shows an assessment of the longitudinal strain/stress envelopes under uniaxial tensile loading. Table 2 summarizes the tensile characteristics of all the tested SiC/SiC tubular specimens. A very good reproducibility of the mechanical behavior is observed. A 279 (±13) MPa stress at fracture was determined. Given the fact that some of the samples are issued from different lots of fibers and that there were not manufactured on the same mandrel, it can be concluded that the employed process lead to homogeneous tubes. The noticeable differences appear mainly for high strain and stress levels and are mainly due to discrepancies in the defect levels in the composites, slighting changing the Weibull moduli of the composites and, as a consequence, the material ultimate tensile strength [START_REF] Curtin | Theory of Mechanicla Properties of Ceramic-Matrix Composites[END_REF].

Cycle analysis

A representative tensile stress-strain curve of a specimen (with unloading -loading sequences) is presented in Fig. 8. The extended hysteresis loops are characteristic of sliding with high fiber/matrix interface friction. The impeded crack closing at the end of the unloading step is present, inducing an apparent stiffening [START_REF] Bouquet | Toughness assessment of ceramic matrix composites[END_REF]. It appears that over a given threshold (longitudinal strain of 0.2 %), during the reloading, the stress does not reach the exact value of the maximal stress obtained before the previous unloading sequence for a similar strain. This phenomenon tends to reveal that, over a given damage level, additional damages take place during further tensile mechanical solicitations, even for lower stresses than the maximum stress previously reached. This may be related to the opening of the preexisting microcracks and/or to the abrasion of the different interfaces due to friction. This result is confirmed by the AE signal analysis (Fig. 8), where the count increase below the maximum strain and stress reached during the previous loading cycle.

The evolution of reduced elastic modulus ( ) as a function of the maximum stress applied before unloading sequences is presented Fig. 9. The whole tests show a good homogeneity of the processed specimens. The regimes highlighted on Fig. 6 can also be observed through the reduced modulus. Its value is fixed to the unity until the beginning of the loss of linearity of the tensile strain/stress curve [START_REF] Bertrand | Hi-Nicalon/SiC minicomposites with (Pyrocarbon/SiC) n nanoscale multilayered interphases[END_REF] (A). The reduced modulus at about 100 MPa of longitudinal stress correspond to the one measured during the unloading of the 0.05 % cycle, i.e. at the onset of AE, highlighting the beginning of the matrix multi-cracking (B). Therefore, a noticeable decrease (20 %) appears before the beginning of the matrix multicracking ( , which is not linked to any damaging of the material upon mechanical sollicitation.

Moreover, the main decrease of the composites reduced modulus occurs just above this domain in a small stress range [100 -150 MPa]. The measurements were stopped at a longitudinal strain of 0.5%, corresponding to an applied stress of about 200 MPa. At this level, the reduced modulus seems to converge towards an asymptote, following the load displacement on the fibers and a saturation of the matrix multicracking (C). As mentioned earlier, the evolution of AE energy signal is characterized by a saturation at high stress and strain [START_REF] Bernachy-Barbe | Anisotropic damage behavior of SiC/SiC composite tubes: Multiaxial testing and damage characterization[END_REF] whereas the AE event signal evolves linearly. Therefore, over those level of deformation, the contribution of the strongest events decrease a lot, in favor of low energy ones, such as interfacial debonding or interfacial shear cracking [START_REF] Moevus | Analysis of damage mechanisms and associated acoustic emission in two SiCf/[Si-B-C] composites exhibiting different tensile behaviours. Part II: Unsupervised acoustic emission data clustering[END_REF]. A combination between AE energy and events signal would lead to a better understanding of the material properties under mechanical loading.

The limit value of the reduced modulus is given on a 2D plate composite by relation [START_REF] Cavalier | Composites in aerospace industry[END_REF].

The tow Young's modulus (319 GPa) [START_REF] Buet | Influence of chemical and physical properties of the last generation of silicon carbide fibres on the mechanical behaviour of SiC/SiC composite[END_REF] was taken into account instead of the fiber one (372 GPa) [START_REF] Sauder | Influence of interface characteristics on the mechanical properties of Hi-Nicalon type-S or Tyranno-SA3 fiber-reinforced SiC/SiC minicomposites: influence of interface characteristics on the mechanical properties[END_REF].

Given a fiber volume fraction between 40 and 45% and a 45° angle, the ultimate reduced Young's modulus is comprised between 0.114 and 0.129. The later asymptotes are plotted in Fig. 9. Therefore, this relation seems to be also available for 2D tubular SiC/SiC composites, up to the composites ultimate failure (D). However, mechanical testing cannot be conducted at such high levels of damage because the material become sensitive to fatigue testing over 70% of the ultimate tensile strength [START_REF] Kostopoulos | Fatigue behaviour of 3-d SiC/SiC Composites[END_REF].

Fig. 8.

Fig. 9.

The longitudinal residual strains are anelastic strains ( ), which appears during the composite damaging under mechanical loading (Fig. 4). Two different contributions can be extracted. Residual thermal strains are generated in the composite as a consequence of the CVI process (during cooling down to room temperature) due to the mismatch of thermomechanical properties of its constituents (fiber, interphase and matrix). During a uniaxial tensile test, those axial stresses are released consecutively to the matrix multicracking (debonding of fiber and matrix) and induced a contribution in . The other component, , is the partial irreversible sliding and/or the mechanical hindrance of the cracks reclosing. The whole is linked by Eq. ( 2):

(2) corresponds to the intersection of the asymptotes of the curves during the reloading step at the end of the cycle and the abscissa axis for all cycles (Fig. 10 (a)). All the curves cross at the same point at about 35 MPa ± 5 MPa. This point corresponds to the axial thermal residual strains in the fibrous reinforcement after manufacture. The evolution of , and as a function of the maximum stress reached before unloading sequences are presented in Fig. 10 (b). A saturation of the thermal longitudinal residual strains appears at high stress (over 200 MPa), which may again confirm the saturation of the matrix multicracking. In the same way, the irreversible sliding strains ( ) also seems to tend to an asymptotic value over 200 MPa. On the contrary, the anelastic residual strains increase quite linearly with the loading stress level.

The evolution of the area and the full width at half maximum of the cycles are representative of the energy dissipated in the material and the interfacial shear strength ( ). The experimental results validate the reproducibility of the fabrication process given the homogeneity of materials (Fig. 11). The full width at half maximum and the area of the cycles follow a comparable evolution, even though the full width at half maximum saturates for high loading levels. On the contrary, the areas evolution is linear over 0.1% strain.

Crack density

The crack density, i.e. the inverse of the mean distance between cracks, was measured, either on the tubes surfaces (internal or external) or in its core. The matrix multicracking is directly related to the type and to the density of defects in the materials and follows a Weibull's distribution. Discrepancies in the crack density between the core (low matrix volume) and the surface (high matrix volume) are observed.

Fig. 12 presents a microscopic observation of the crack density for a tested specimen after ultimate failure. The crack density was also observed on samples that were damaged at several levels of longitudinal strain (0.1%, 0.2%, 0.3%, 0.4% and 0.5%). However, in this case, the matrix multicracking is hardly observable in the sample core because of the important cracking closure phenomena, even after chemical etching (Murakami's reagent). This explains why only the crack density at the surface of the tested specimen was retained. In Fig. 13, the evolution of crack density as a function of the applied stress, either on the internal (filament winding) or on the external (2D braiding) sides, are plotted. No obvious differences were noted in the crack density between both sides. The first cracks appear over a 110 Mpa applied stress. The comparison between the normalized crack density (i) and the normalized AE (ii) as a function of the applied stress is also plotted in Fig. 13 . The same evolution is observed, which confirms that the counting of the AE events is a good method to evaluate damage kinetics [START_REF] Rohmer | Mechanical properties of SiC/SiC braided tubes for fuel cladding[END_REF].

Therefore, this result highlights that no matrix multicracking saturation appear until the material failure, even though the reduced modulus and the full width at half maximum may evidence this saturation. It seems that cracks generated above 200 MPa or 0.5 % strain do not have any influence on the evolution of reduced modulus or full width at half maximum during cycled tensile tests. However, it could explain the continuous evolution of the area of cycles and the anelastic residual strains up to complete failure of specimens. This results is consistent with results previously obtained [START_REF] Moevus | Analysis of damage mechanisms and associated acoustic emission in two SiCf/[Si-B-C] composites exhibiting different tensile behaviours. Part II: Unsupervised acoustic emission data clustering[END_REF][START_REF] Momon | Unsupervised and supervised classification of AE data collected during fatigue test on CMC at high temperature[END_REF].

Discussion

The saturation of AE energy [START_REF] Bernachy-Barbe | Anisotropic damage behavior of SiC/SiC composite tubes: Multiaxial testing and damage characterization[END_REF] can explain the saturation of reduced modulus, full width at half maximum, irreversible sliding and anelastic residual strains. Nevertheless, the cumulative AE signal do not saturate at high stress or strain (< 200 MPa) and is a sign of a specific damage mechanism that still occur, up to complete failure of specimen (Fig. 8). This could explain the absence of saturation of the anelastic residual strains and the area of cycles signals at high stress (> 200 MPa).

To evaluate the residual thermal stress generated in specimen during processing (due to discrepancies between the thermochemical properties of the fibers, the interphase and the matrix), an analytical model similar to the one used by Michaux [START_REF] Michaux | Young's modulus, thermal expansion coefficient and fracture behavior of selected Si-B-C based carbides in the 20-1200°C temperature range as derived from the behavior of carbon fiber reinforced microcomposites[END_REF] was employed. The main difference is that the evolution of the thermochemical properties with temperature was taken into account: longitudinal and transverse Young's modulus and coefficient of thermal expansion, Poisson's ratio. In this model, the sample is assimilated to a microcomposite (one fiber + interphase + matrix) of infinite length. Three different components of the residual thermal stresses are calculated and evaluated: longitudinal (σ zz ), radial (σ rr )

and circumferential (σ θθ ). All the thermochemical properties as a function of temperature were fitted with Eq. ( 3):

(3)

A, B, C, D, E, F and G are constants, T the temperature (°C) and Y the thermochemical property. Table 3 summarize the constants used for the calculations. All the lacking constants are equal to zero. and are the longitudinal and transverse Young's moduli, respectively, and are the longitudinal and transversal coefficients of thermal expansion and Poisson's ratios ( and ) are supposed to be similar for a same material, whatever the direction. The latter assumption is true for isotropic materials (SiC fiber and matrix) but is an estimation for pyrocarbon. Anyway, variation of Poisson's ratios do not highly influence the calculation. The different parameters are presented in Table 3 [START_REF] Buet | Influence of surface fibre properties and textural organization of a pyrocarbon interphase on the interfacial shear stress of SiC/SiC minicomposites reinforced with Hi-Nicalon S and Tyranno SA3 fibres[END_REF].

Table 3

One should notice that this model cannot be rigorously applied to the tubular 2D shaped composites processed. However, the stress levels are representative of what is achieved after processing. Moreover, for these macroscopic composites, the stresses can be locally highly affected by the material microstructure (presence of pores) [START_REF] Gélébart | Effects of porosity on the elastic behaviour of CVI SiC/SiC composites[END_REF].

The evaluated thermal stresses are plotted in Fig. 14. The matrix volume fraction was taken equal to that of the fibers, given that this is almost the case for macroscopic composites. The fiber is slightly under tensile stress in all directions, whereas the matrix is under longitudinal and circumferential compression stresses along with low tensile stresses in the radial direction. Higher stress levels are observed in the pyrocarbon interphase with elevated circumferential tension and longitudinal compression. However, those relatively strong stresses in the interphase (up to 170 MPa) are not sufficient to damage the pyrocarbon interphase during the cool down to room temperature [START_REF] Sauder | The tensile properties of carbon matrices at temperatures up to 2200°C[END_REF].

The thermomechanical properties of the HNS fibers being close to the SiC matrix one, the longitudinal residual thermal stresses remain low as expected. A 35 ± 5 MPa longitudinal residual thermal stress was experimentally determined by the crossing of the curves asymptotes at the end the loading sequences of the cycled tensile tests ( § 3.B). Those calculations on a microcomposite gave a 30 MPa residual thermal stresses. Therefore, it is noted that even by applying this simple model, the residual thermal stresses determined experimentally are probably representatives. Fig. 14.

The cumulative AE count is proportional to the number of cracks generated in the composite [START_REF] Morscher | Fiber effects on minicomposite mechanical properties for several silicon carbide fiber-chemically vapor-infiltrated silicon carbide matrix systems[END_REF]. Thus, the linearity of the AE count could evidence the lack of matrix multicracking saturation for these SiC/SiC samples, on the contrary to the behavior observed of some other similar SiC/SiC composites [START_REF] Rohmer | Mechanical properties of SiC/SiC braided tubes for fuel cladding[END_REF]. It should be noted that the acquisition device only allows a simple event counting over a predefined threshold. The analysis of the AE energy signal (rather than AE cumulative count) for similar tests (analogous materials and mounting) exhibit a saturation above a stress of 200 MPa and a 0.5 % longitudinal strain [START_REF] Bernachy-Barbe | Anisotropic damage behavior of SiC/SiC composite tubes: Multiaxial testing and damage characterization[END_REF]. However, deviation from linearity at the beginning of the tensile test stress/strain curve appears before the first AE signal for all the samples and is not due to the beginning of matrix multicracking (Fig. 15). Table 4 summarizes the values (minimum, maximum and mean) of the stresses and strains reached either at the end of the linear elastic domain or at the first count of the AE signal, revealing the onset of matrix cracking. A mean longitudinal strain and stress of 0.012 % and 34 MPa are respectively measured for the beginning of the deviation from linearity, instead of 0.053 % and 112 MPa for the first count of AE signal. This notable difference was observed neither on similar 1D [START_REF] Sauder | Patent: Method for producing a composite including a ceramic matrix[END_REF] [44] or 2.5D SiC/SiC composites plates [START_REF] Kostopoulos | Fatigue behaviour of 3-d SiC/SiC Composites[END_REF] [49], nor on 2.5D braided SiC/SiC tubes [START_REF] Rohmer | Mechanical properties of SiC/SiC braided tubes for fuel cladding[END_REF], even though they share the same interface and fiber type. This specific behavior is then a characteristic of the process used in this study. Therefore, a phenomenon, non-detectable by AE, occurs which leads to a slight rigidity loss before the onset of the matrix cracking.

Table 4

This may originate from pre-existing cracks in the material as it could be observed in C/C [START_REF] Siron | Modelling of the stress/strain behaviour of a carbon/carbon composite with a 2.5 dimensional fibre architecture under tensile and shear loads at room temperature[END_REF][START_REF] Siron | Damage and failure mechanisms of a3-directional carbon/carbon composite under uniaxial tensile and shear loads[END_REF] or C/SiC materials [29]. The estimated residual thermal stresses due to mismatch between the fibers, pyrocarbon interphase and the matrix ( § 3.B.) are very low and cannot be the source of pre-existing cracks in the matrix after processing. An untested SiC/SiC tube specimen was cut in half along its axis of revolution, enrobed in an epoxide resin, polished and analyzed along its longitudinal section by optical microscopy (Fig. 16 (a)). The sample shows clearly the presence of longitudinal cracks of small opening (along the tube axis of revolution). The same observation was carried out on a tube after the consolidation process, i.e. after the interphase and few micrometers of matrix deposition (Fig. 16 (b)).

Again, the same type of cracks is observed. Therefore, it can be concluded that they are generated during this first CVI processing step. Even though the following process steps (densification) diminish their opening, these microcracks remain after the last step of the SiC/SiC tubes manufacture.

Fig. 16.

This result is related to the specific processing route employed. Indeed, the texture shaping is manufactured on a silica glass mandrel ( § 2.A.a.) and the consolidation step is carried out at about 1000°C. Then, during the cooling step to room temperature of the sample, residual thermal stresses are generated in both the silica glass mandrel and the composite, which have different thermomechanical properties. To estimate those stress values, the model previously used, was employed, considering a one layer microcomposite. In this case, the fiber was replaced by the silica glass mandrel of an external diameter of 8 mm and the composite represents the layer (0.75 mm) in contact with the glass. The composite thermomechanical characteristics at the consolidated state being unknown, it was decided to use the thermal expansion and Poisson's coefficients of the HNS fiber. An elastic modulus of 120 GPa was retained, given the low densification at the end of this processing step (high volume fraction of porosity). Eq. ( 3) was again used to determine the evolution of the thermomechanical properties as a function of temperature. The constants used for this calculation are given in Table 5. The silica mandrel thermochemical properties were extracted from [START_REF]Vof Micro to Nano, Technical information for type GE 124 fused quartz[END_REF]. The SiC/SiC ones in the consolidated state were extrapolated from those of the manufactured SiC/SiC by taking into account the level of densification.

Table 5

The simulation result is plotted in Fig. 17. The composite obtained after the consolidation step encounters high residual thermal stresses, especially in circumferential and longitudinal directions.

Those high residual thermal stresses after the consolidation step explain the origin of the matrix initial cracks observed in Fig. 17. Bernachy et al. [START_REF] Bernachy-Barbe | Anisotropic damage behavior of SiC/SiC composite tubes: Multiaxial testing and damage characterization[END_REF] highlighted that, for this type of bi-axial solicitation (tensile -internal pressure), the microcracks are perpendicular to the principal solicitation axis (internal pressure loading), which is consistent with the cracks observed in Fig. 16 (a) and (b), following the tube axis (perpendicular to the internal pressure loading generated by the mismatch in the thermomechanical properties). Fig. 17.

Conclusions

The nuclear reactors cladding may be subjected to strong thermomechanical stresses during the set up of the fuel, operation and especially in transient or accidental states. In order to assess the viability of SiC/SiC composites as cladding material, its mechanical behavior under uniaxial tensile solicitation was characterized. Even though the matrix was deposited by CVI, the final material porosity volume fraction remains low (6.6 %). The strain -stress curves show a high reproducibility of the mechanical behavior, with a pseudo-elastic behavior and relatively high longitudinal strain and stress levels at fracture (resp.

1.01 % and 279 MPa). The elastic modulus strongly decrease after the first microcracks are generated in the matrix but its value tends to an asymptote over 150 -200 MPa. However, the area of the cycles, relevant of the friction mechanisms within the composite, still increase at the last cycle before ultimate fracture. This result was confirmed by the analysis of the crack density that evolves along with the cumulated acoustic emission signal since the first microcracks are generated in the material. Therefore, the composites of this study do not present any matrix multicracking saturation. Thanks to a small mismatch between the thermomechanical properties of the Hi-Nicalon S fibers and the matrix, the manufactured composites have low residual thermal stresses, which was confirmed by both modelling and the cycled tensile tests analysis. A loss of linearity before the appearance of the first AE signal was observed, which was not observed on 1D or 2.5D similar composites. This phenomenon was attributed to the presence of small cracks in the as fabricated composite, which originated from the first CVI processing step (before the mandrel withdrawal) and is the consequence of the use of silica as mandrel for fibrous reinforcement structure processing. This assessment of the mechanical properties of the manufactured SiC/SiC cladding tubes will be take into consideration to evaluate the influence of nuclear environments on the materials, such as the liquid sodium at nominal operating temperature of SFR, in further studies. Coefficients of the analytic model for the Young's moduli, Poisson's ratio and coefficients of thermal expansion [START_REF] Buet | Influence of surface fibre properties and textural organization of a pyrocarbon interphase on the interfacial shear stress of SiC/SiC minicomposites reinforced with Hi-Nicalon S and Tyranno SA3 fibres[END_REF]. Coefficients of the analytic model for the calculation of the silica mandrel -consolidated composite thermal stresses [START_REF]Vof Micro to Nano, Technical information for type GE 124 fused quartz[END_REF].

Silica glass mandrel

Fig. 1 .

 1 Fig. 1.

Fig. 2 .

 2 Fig. 2.

Fig. 3 .

 3 An epoxide structural glue (3M Scotch-Weld TM 9323 B/A) allows the fixation of the test specimens with the end collars adjusted to the composites section. The whole, i.e. the tubular test specimen and the end collars, was installed on the mechanical testing machine (INSTRON 2404). The upper part of the sample was screwed on the 25 kN load cell (HBM U10M), fixed on the displaceable cross-rail. The lower part was glued on the machine stationary base. The longitudinal strain was measured with an INSTRON 2620-603 extensometer (± 1 mm course) with a 25 mm gauge length. The diametric strain was measured with a MTS 632.19F-20

Fig. 4 .Fig. 5 .

 45 Fig. 4.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Fig. 7. Table 2

Fig. 10 .Fig. 11 .

 1011 Fig. 10. Fig. 11.

Fig. 12 .Fig. 13 .

 1213 Fig. 12. Fig. 13.

Fig. 15 .

 15 Fig. 15.

Fig. Captions

  Fig. Captions

Fig. 1 .

 1 Fig. 1. SiC/SiC composites tubes as processed.

Fig. 2 .

 2 Fig. 2. X-ray tomography of the manufactured SiC/SiC clad cross-section perpendicular to (a) and along (b) its axis of revolution, respectively.

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. Experimental device employed for tensile testing, without diametric extensometer

Fig. 6 .Fig. 7 .

 67 Fig. 6. Typical tensile strain -stress curves (envelope of the cycled tests) along with the evolution of normalized AE on a reference sample (REF 3) and the fracture surface after mechanical solicitation up to failure.

Fig. 8 .

 8 Fig. 8. Typical evolution of tensile stress and normalized AE signal as a function of longitudinal and radial strains for tensile test.

Fig. 9 .

 9 Fig. 9. Evolution of the reduced modulus as a function of the maximum applied stress reached before unloading.

Fig. 10 .Fig. 11 .

 1011 Fig. 10. Tangents at the end of the reloading step allowing the determination of the stress and thermal residual strains (a). Anelastic, sliding and thermal residual strains as a function of the stress reached before unloading (b).

Fig. 12 .

 12 Fig. 12. Surface matrix cracking along the tube axis of revolution.

Fig. 13 .Fig. 14 .

 1314 Fig. 13. Evolution of crack density on the internal (filament winding structure) and external (2D braiding structure) sides and comparison with acoustic emission (b) as a function of the maximal axial stress reached.

Fig. 15 .Fig. 16 .

 1516 Fig. 15. Discrepancies in the beginning of the matrix multicracking and the end of the strain -stress linearity for a SiC/SiC composite tube specimen during uniaxial tensile test.

Fig. 17 .

 17 Fig. 17. Evaluation of the thermal residual strains between the silica mandrel and the consolidated composite.

CVI

Table 1 2

 1

	.2. Mechanical characterization
	2.2.1. Introduction
	Tubular test specimens were cut at 65 mm length before uniaxial tensile tests. The used procedure is
	described in the ISO 20323 standard [29] and is shown in

Table 4

 4 Strains and stresses at the end of the linearity domain and at the beginning of the matrix multicracking during uniaxial tensile tests.

		End of the linear elastic domain	Onset of matrix cracking (first Acoustic
		on the stress-strain curve	Emission signals)
		Longitudinal strain (%)	Stress (MPa)	Longitudinal strain (%)	Stress	(MPa)
	Min	0.009	24	0.047	102
	Max	0.016	44	0.059	122
	Average	0.012	34	0.053	112
	SD	0.002	4	0.005	5

Table 5

 5

Tables


Dawei Liu

Zhenyu Wang

Nicolas Valérie

Mika Delphine

Vandecasteele Grégoire

Rodolphe Fischmeister

Brenner Catherine
email: catherine.brenner@u-psud.fr

Dr Catherine Brenner

PDE2 regulates membrane potential, respiration and permeability transition of rodent subsarcolemmal cardiac mitochondria

Keywords: ∆Ψm, mitochondrial membrane potential, Bay, Bay 60-7550, Cil, Cilostamide, CsA, cyclosporine A, FCCP, Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, cilostamide, Cil, HCO3 -, bicarbonate, HF, heart failure, IFM, interfibrillar mitochondria , IM, inner membrane, OXPHOS, oxidative phosphorylation, sAC, soluble adenylyl cyclase, SSM, subsarcolemmal mitochondria, PDE, phosphodiesterase, Ro, Ro 20-1724, calcium, cAMP, mitochondrial membrane potential, PDE, respiration

Cyclic adenosine monophosphate (cAMP) production regulates certain aspects of mitochondria function in rodent cardiomyocytes, such as ATP production, oxygen consumption, calcium import and mitochondrial permeability transition (MPT), but how this cAMP pool is controlled is not well known. Here, expression, localization and activity of several cAMP-degrading enzymes, i.e. phosphodiesterases (PDEs), were investigated in isolated rodent cardiac mitochondria. In contrast to the heart ventricle where PDE4 is the major PDE, in cardiac mitochondria, cGMP-stimulated PDE2 activity was largest than PDE3 and PDE4 activities. PDE2 expression was mainly detected in subsarcolemmal mitochondria in association with the inner membrane rather than in interfibrillar mitochondria. PDE2, 3 and 4 activities were further confirmed in neonatal rat cardiomyocytes by real time FRET analysis. In addition, the pharmacological inhibition or the cardiac-specific overexpression of PDE2 modulated mitochondrial membrane potential loss, MPT and calcium import. In mitochondria isolated from PDE2 transgenic mice with a cardiac selective PDE2 overexpression, the oxidative phosphorylation (OXPHOS) was significantly lower than in wild-type mice, but stimulated by cGMP. Thus, cAMP degradation by PDEs represents a new regulatory mechanism of mitochondrial function.

Introduction

The heart is a highly energy-dependent organ with most of its energy provided by mitochondrial oxidative phosphorylation (OXPHOS). Mitochondria also play a role in many other essential cellular processes including metabolite synthesis, redox balance, calcium (Ca 2+ ) homeostasis and cell death, notably trough a sustained mitochondrio-nuclear dialog [START_REF] Brenner | Apoptosis. Mitochondria--the death signal integrators[END_REF][START_REF] Weinberg | Targeting mitochondria metabolism for cancer therapy[END_REF][START_REF] Vandecasteele | Mitochondrial calcium homeostasis: mechanisms and molecules[END_REF] . Therefore, maintaining a functional population of mitochondria is critical for cardiac function and identification of novel regulatory mechanisms of mitochondrial function is a crucial challenge. Recently, a soluble adenylyl cyclase (sAC) stimulated by bicarbonate (HCO3 -) and calcium (Ca 2+ ) has been revealed to serve as a local source of the second messenger cAMP in the mitochondrial matrix. In liver and brain, mitochondrial cAMP stimulates ATP production by OXPHOS in response to HCO3 -and/or Ca 2+ 4, 5 . In endothelial cells, HCO3 -is transported into mitochondria leading to cAMP production by sAC, which indirectly modulates the cell fate trough apoptosis inhibition 6 7 . As a result, this pathway serves as a mechanism for metabolic adaptation and could be a potential novel target to treat genetic mitochondrial diseases [START_REF] Acin-Perez | Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation[END_REF] . In line with these studies, we recently showed a protective role for cAMP against cell death, apoptosis as well as necrosis in primary rat neonatal cardiomyocytes. Moreover, upon stimulation with HCO3 -and Ca 2+ , a truncated form of sAC (sACt) localized in mitochondria produces cAMP, which in turn stimulates oxygen (O2) consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. In addition, cAMP is rate-limiting for matrix Ca 2+ entry via the exchange factor Epac1 and the mitochondrial Ca 2+ uniporter and, as a consequence, prevents MPT [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . Cyclic nucleotide phosphodiesterases (PDEs) are a class of key cellular enzymes, which degrade cAMP and/or cGMP by hydrolysis of phosphodiester bonds [START_REF] Bender | Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use[END_REF] . Thereby, they regulate intracellular levels of these ubiquitous second messengers. Cyclic nucleotides play an important role in the regulation of a host of cellular targets involved in cardiac function but also in the progression of cardiac diseases such as heart failure [START_REF] Sharma | Phosphodiesterases: Regulators of cyclic nucleotide signals and novel molecular target for movement disorders[END_REF] . In mammals, PDEs constitute a large superfamily which contains 11 PDE families (PDE1 to PDE11) and nongeneric PDEs such as the protein human Prune, PDE2 and PDE3 being respectively activated and inhibited by cGMP [START_REF] Conti | Biochemistry and physiology of cyclic nucleotide Phosphocliesterases: Essential components in cyclic nucleotide signaling[END_REF][START_REF] Omori | Overview of PDEs and their regulation[END_REF][START_REF] Middelhaufe | Domain mapping on the human metastasis regulator protein h-Prune reveals a C-terminal dimerization domain[END_REF][START_REF] Angelo | Prune cAMP phosphodiesterase binds nm23-H1 and promotes cancer metastasis[END_REF] .

In isolated mitoplasts from mouse liver, cAMP catabolic activity is fully inhibited by 3isobutyl-1-methylxanthine (IBMX, a non-selective PDE inhibitor), suggesting the presence of intra-mitochondrial PDE activity [START_REF] Acin-Perez | Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation[END_REF] . It has been reported that PDE4B co-localized with mitochondria, apparently through an interaction with the disrupted in schizophrenia 1 (DISC1) protein [START_REF] Millar | DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling[END_REF] . More recently, mitochondrial PDE2A was shown to be located in the mitochondrial matrix and the unique N terminus of PDE2A isoform 2 was shown to be responsible for mitochondrial localization of this isoform in liver and brain [START_REF] Acin-Perez | A phosphodiesterase 2A isoform localized to mitochondria regulates respiration[END_REF][START_REF] Modis | Hydrogen sulfidemediated stimulation of mitochondrial electron transport involves inhibition of the mitochondrial phosphodiesterase 2A, elevation of cAMP and activation of protein kinase A[END_REF] . Thus, mitochondrial PDE2 may form a local signalling system with sAC in the matrix, to regulate the activity of the respiratory chain [START_REF] Acin-Perez | A phosphodiesterase 2A isoform localized to mitochondria regulates respiration[END_REF] . PDE2 can also be found at the outer and inner mitochondrial membranes regulating mitochondrial morphology and apoptotic cell death via local modulation of a cAMP/PKA signalling module facing the cytosol [START_REF] Monterisi | PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling[END_REF] . In Drosophila, the cyclic nucleotide PDE Prune was also found to localize to the mitochondrial matrix.

Knocking-down Prune reduces mitochondrial transcription factor A (TFAM) and mitochondrial DNA levels, suggesting that Prune stabilizes TFAM and promotes mitochondrial DNA replication through down-regulation of mitochondrial cAMP signalling [START_REF] Zhang | The cAMP phosphodiesterase Prune localizes to the mitochondrial matrix and promotes mtDNA replication by stabilizing TFAM[END_REF] .

To gain new insights into the control and degradation of the cardiac mitochondrial pool of cAMP, we assessed the presence and contribution of various isoforms of phosphodiesterases (PDEs) to cAMP degradation in isolated rat or mouse heart mitochondria in the presence and absence of cGMP. Our results show that PDE2A, 3A, 4A, 4B and 4D can be detected within mitochondria. These PDEs control cAMP level to regulate Ca 2+ accumulation and certain mitochondrial functions. Our findings unravel the role of cAMP degradation by mitochondrial PDEs, which represents a new regulatory mechanism of mitochondrial function.

Materials and Methods

Unless specified, all reagents and chemicals are from SIGMA Aldrich and of analytical grade.

Animals

All animal care and experimental procedures conformed to the European Community guiding principles in the care and use of animals (Directive 2010/63/EU of the European Parliament) and authorizations to perform animal experiments according to this decree were obtained from the French Ministry of Agriculture, Fisheries and Food (No. D-92-283, 13 December 2012). All studies involving rats are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals [START_REF] Kilkenny | Animal research: reporting in vivo experiments: the ARRIVE guidelines[END_REF] .

Isolation of rat and mouse cardiac mitochondria

Subsarcolemmal mitochondria (SM) were isolated from the heart of adult Wistar rats at 8-10 weeks of age (275-375 g; Janvier, Le Genest St Isle, France) and from mice heart at 15-20 weeks of age (Janvier, Le Genest St Isle, France) as described [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . Briefly, over-night fasted rats were decapitated and mouse killed by cervival elongation in accordance with the rules of our animal facility. The heart and its atria were rapidly removed, rinsed to discard blood traces, and weighted. Then, ventricles were placed into a cold homogenization buffer containing 0.3 M sucrose, 0.2 mM EGTA, 5 mM TES (pH 7.2). The heart was grinded with Polytron fastly and homogenized by using a Potter on ice. The homogenate was centrifuged at 500 g for 10 min, 4°C. Then the supernatant was carefully removed and centrifuged again at 12,000 g for 15 min, 4°C. The pellets were washed in the homogenization buffer supplemented with 1 g/L bovine serum albumin (BSA), called buffer H-BSA, and mitochondria were kept on ice until use. To isolate interfibrillar mitochondria, the proteolytic enzyme trypsin (5 mg/g of tissue)

was added to the homogenate fraction and immediately centrifuged at 12,000 g for 15 min at 4°C. Then, the pellet was suspended in buffer H-BSA and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was centrifuged at 12,000 g for 15 min, 4°C and mitochondria were kept on ice until use.

Mitoplast preparation

Freshly isolated mitochondria were incubated in H2O containing 15 mM KCl for 30 min at 4°C to induce an osmotic shock and then collected by centrifugation at 3,000 g for 10 min, 4°C.

Measurement of mitochondrial transmembrane potential and swelling in isolated mitochondria

Isolated mitochondria (25 µg proteins) were incubated with Ca 2+ and drugs in 96 well microtiter plates [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . Mitochondrial transmembrane potential was measured using the fluorescent probe, rhodamine 123 (Rhod123, Excitation = 485 nm and Emission = 535 nm, Enzo Life Sciences, Villeurbanne, France) in a buffer containing 200 mM sucrose, 10 mM MOPS, 200 µM EGTA, 1 mM H3PO4, 5 mM succinate and 2 µM rotenone (pH 7.4) using Tecan Infinite 200 spectrofluorimeter. In parallel, matrix swelling was measured via absorbance at 540 nm [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . Raw results were exported to an Origin8 (OriginLab Corporation) files and half-time of effects were calculated using nonlinear curve fitting with a logistic fit.

Measurement of mitochondrial Ca 2+ in isolated mitochondria

Isolated mitochondria (25 µg proteins) were incubated with 5 µM Rhod-2 (Enzo Life Sciences, Villeurbanne, France) in the buffer containing 200 mM sucrose, 10 mM MOPS, 200 µM EGTA, 1 mM H3PO4, 5 mM succinate and 2 µM rotenone for 30 min in dark at room temperature. The mitochondria were then washed 2 times and were treated with various drugs for 10 min before applying Ca 2+ . Fluorescence was measured in real time for 60 min at room temperature (RT) in 96-well plates using Tecan Infinite 200 (excitation at 552 nm; Emission at 581 nm).

cAMP measurements by ELISA

Cyclic AMP measurement was performed according to the manufacturer's instructions using monoclonal anti-cAMP antibody based direct cAMP ELISA Kit (R&D, Lille, France) on freshly isolated mitochondria from rat hearts (500 μg proteins per sample) treated or not by HCO3 -, PDE inhibitors and 5µM cGMP for 20 min at RT before centrifugation and lysis 8 .

Cyclic AMP-PDE activity assay

The cAMP-PDE activity was measured in the supernatant according to a modification of the two-step assay procedure method described by Thompson and Appleman 20 , using 20 µg of heart homogenate proteins / 200 µg of mitochondria proteins in a total volume of 200 µl of buffer A, which includes 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 5 mM β-mercaptoethanol and 1 µM cAMP and supplemented with 10 5 cpm [ 3 H]-cAMP, as detailed previously [START_REF] Mehel | Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts beta-Adrenergic Responses in Cardiomyocytes[END_REF] . To evaluate PDE family-specific activities, the assay was performed in the absence or presence of either one or a combination of several selective PDE inhibitors: 100 nM Bay 60-7550 (Bay) for PDE2, 1 µM Cilostamide (Cil) for PDE3, 10 µM Ro 20-1724 (Ro) for PDE4 and 500 µM IBMX as a non-selective PDE inhibitor. In some experiments, 5 µM cGMP was added to the buffer A to activate PDE2 and inhibit PDE3.

Oxygen consumption analysis in isolated mitochondria

The XF e 96 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA, USA) was used to measure mitochondrial bioenergetic function [START_REF] Coletta | Endothelial dysfunction is a potential contributor to multiple organ failure and mortality in aged mice subjected to septic shock: preclinical studies in a murine model of cecal ligation and puncture[END_REF] . Respiration by the mitochondria (0.5 μg/well) was sequentially measured in a coupled state with substrate (basal respiration, state 2). State 3 initiated with 2 mM ADP, state 4 induced with the addition of 2.5µg/ml oligomycin and 2 µM FCCP-induced maximal uncoupler-stimulated respiration (state 3u) were sequentially measured. To activate PDE2 and inhibit PDE3, 5 µM cGMP was added at the beginning of the experiment to the isolated mitochondria during 20 min at RT. At the end of the experiment, the complex III inhibitor, antimycin A was applied at 4 µM to completely inhibit the mitochondrial respiration. This 'coupling assay' examines the degree of coupling between the electron transport chain (ETC), and OXPHOS.

SDS PAGE and Western blotting

Total heart homogenate and total mitochondrial proteins were resolved on 4-15% Tris-glycine SDS-PAGE gels and electroblotted onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Marnes La Coquette, France). Following electrotransfer, membranes were blocked for 1 h at room temperature in 5% BSA-PBST (10 mM Tris HCl, pH 8.0/150 mM NaCl/0.1% Tween 20). Next, membranes were incubated overnight at 4°C with primary antibody. Then, the membranes were washed six times with PBST and incubated with peroxidase-conjugated secondary antibody at room temperature for 1 h. Peroxidase activity was detected with enhanced chemiluminescence (ECL Advance Western blotting detection kit; Thermo Scientific, Villebon sur Yvette, France). For protein detection, the following antibodies were used: anti PDE2A (G-12, Santa Cruz, CA, USA), anti PDE3A (generous gift from Pr. Chen Yan, University of Rochester, USA), anti PDE4A, PDE4B and PDE4D (generous gifts from Pr. Marco Conti, University of California, San Francisco, USA), anti GAPDH (Cell signalling, Danvers, MA, USA) and anti voltage-dependent anion channel (VDAC) (Genosphere, Paris, France).

Neonatal cardiomyocyte isolation and adenoviral infection

Rat neonatal cardiomyocytes were isolated as previously described [START_REF] Morel | cAMPbinding protein Epac induces cardiomyocyte hypertrophy[END_REF] . For FRET experiments, neonatal cardiomyocytes were plated on 35 mm laminin-coated culture dishes (10 µg/ml) at a density of 4 × 10 5 cells per dish. On the following day, cells were infected with an adenovirus encoding the cAMP fluorescent reporter 4mt-Epac-S H187 in Opti-MEM® (Life technologies, St Aubin, France) for 48h with 100 of MOI (multiplicity of infection) . The construction of adenoviruse encoding 4mt-Epac-SH187 has been described previously [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . USA). The donor (mTurquoise2) [START_REF] Klarenbeek | Fourthgeneration epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity[END_REF] was excited during 300 ms by a Xenon lamp (Nikon) using a 440/20BP filter and a 455LP dichroic mirror. Dual-emission imaging of donor and acceptor was performed using a Dual-View emission splitter equipped with a 510 LP dichroic mirror and BP filters 480/30 and 535/ 25 nm, respectively.

Fluorescence resonance energy transfer measurements of cAMP levels

Immunostaining

Cultured mouse cardiomyocytes were rinsed with PBS and fixed with 4% formaldehyde.

Triton X-100-permeabilized cells were incubated with anti PDE2 antibody (Santa Cruz, CA, USA) or anti alpha actinin (A7811, Sigma). Cells were then incubated with, respectively, AlexaFluor goat anti-mouse secondary antibody (ThermoFisher scientific, Courtaboeuf, France) or AlexaFluor anti-rabbit secondary antibody (ThermoFisher scientific), and viewed under a fluorescent microscope in inverted confocal laser scanning microscope LSM 510-Meta (Carl Zeiss, Germany) using a Plan-Apochromat 63X/1.4 objective lens, equipped with an argon (488 nm excitation wavelength) and a helium neon laser (543 nm excitation wavelength). Sections stained by immunofluorescence were visualized by confocal microscopy and intensity profile obtained. To calculate the Pearson correlation coefficient, we used the plugin JACop in ImageJ software [START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF] . The coefficient is equal to -1 when there is a negative relationship between two parameters, +1 when there is a positive relationship and 0 when there is no relationship.

Electron microscopy

Isolated mitochondria were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1M sodium phosphate buffer pH 7.4 during 2h, followed by 4% paraformaldehyde for 3h.

Samples were gradually dehydrated in ethanol (30-100%) on ice and embedded in LRW resin, polymerised for 24h at 60°C. Thin sections (80 nm) were collected onto 300 mesh nickel grids, and processed for immunochemistry with anti-PDE2 antibodies. As a control, we checked that the gold nanoparticle conjugated antibody gives no background on the section of isolated mitochondria. The observation was processed with a Philips CM12 transmission electron microscope at 80kV.

Statistical data analysis

Results are expressed as mean ± standard error of mean (s.e.m). The Origin software and Graphpad Prism 6 were used for statistical analysis. Differences between 2 groups have been analyzed by one-way ANOVA and differences between groups of two genotypes were

Results

PDE2, PDE3 and PDE4 isoforms are expressed in cardiac mitochondria

To identify PDE expression pattern in cardiac mitochondria, we isolated mitochondria from rat heart ventricles by differential centrifugation and extensive washes to minimize cross contamination by subcellular fractions [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . The analysis of rat heart homogenate (H) and of isolated mitochondria (M) revealed several bands at apparent molecular weights comprised between 95 kDa and 130 kDa for PDE2A, PDE3A, PDE4A, PDE4B and PDE4D subtypes in western-blots (Fig. 1A). Because PDE2A was reported to be localized in the mitochondrial matrix in liver and brain 4 16 , whereas it was proposed to be localized mainly at the OMM and IMM in neonatal rat cardiomyocytes [START_REF] Monterisi | PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling[END_REF] , we next focused on PDE2 and explored its localization in mitochondrial subcompartments using fractionation followed by osmotic shock to prepare mitoplasts. Fig. 1B shows that a PDE2 band was detected in the mitoplast fraction but not in the post-mitoplast fraction, which corresponds to the intermembrane space and the outer membrane (Fig. 1B). To confirm this, we analyzed the mitochondrial population by transmission electron microscopy and immunolabelling. As shown in Fig. 1, we observed that not all mitochondria are labelled and found that the total mitochondria population (labelled+unlabelled, subsarcolemmal mitochondria (SSM) + interfibrillar mitochondria (IFM) exhibits a mean of 0.8 dots/mitochondrion (Fig 1D). In labelled mitochondria, PDE2

was mainly found to be localized in the inner membrane and the matrix (Fig. 1C-D).

Interestingly, PDE2 was ~2-fold more expressed in SSM than in IFM (Fig. 1D), and ~3-fold more present in the inner membrane than in the matrix (Fig. 1E). PDE2 was barely detected at the outer membrane, suggesting a low level of association with this membrane or the inaccessibility of epitopes due to PDE2 conformation or interaction with other proteins (Fig. 1E). In the following experiments, we used exclusively SSM mitochondria to investigate the role of PDEs.

cAMP-PDE activity in cardiac mitochondria

To evaluate the cAMP-hydrolysing activity of the three different PDE families that we identified in SSM mitochondria, a radioenzymatic assay was conducted in the absence or presence of selective PDE inhibitors [START_REF] Mehel | Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts beta-Adrenergic Responses in Cardiomyocytes[END_REF] : Bay 60-7550 (Bay, 100 nM) for PDE2, cilostamide (Cil, 1 µM) for PDE3 and Ro 20-1724 (Ro, 10 µM) for PDE4, as well as in the absence or presence of 5 µM cGMP to stimulate PDE2 and inhibit PDE3, respectively. As shown previously [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on ß-adrenergic cAMP signals[END_REF] , PDE4 was the major PDE in rat left ventricle (Fig. 2A & 2B), representing ~63% of total cAMP-hydrolyzing activity at 1 µM cAMP and ~74% in the additional presence of 5 µM cGMP, this difference being due to decreased contribution of the cGMPinhibited PDE3 to the total activity (Fig. 2B, Table 1). In ventricle, PDE2 accounted for only ~6% of total cAMP-hydrolyzing activity with cAMP alone, and increased to 12% in the presence of cGMP (Fig. 2B, Table 1). In contrast, in cardiac mitochondria, PDE2 activity represented about 22% of total cAMP-hydrolytic activity with cAMP alone and 63% in the presence of cGMP, indicating that in the presence of cGMP, PDE2 constituted the largest cAMP-PDE activity in cardiac mitochondria [START_REF] Martins | Purification and characterization of a cyclic GMPstimulated cyclic nucleotide phosphodiesterase from bovine tissues[END_REF] (Fig. 2C,D, Table 1). In line with the PDE activity assay, quantitative measurements of mitochondrial cAMP levels by ELISA showed that stimulation by HCO3 -, a known activator of sAC, increased the level of cAMP produced compared to untreated controls and that this increase was minored in the presence of cGMP (Fig. 2E). This result indicates that cGMP can directly affect cAMP catabolism in isolated mitochondria, most probably by activation of PDE2. Moreover, concomitant pharmacological inhibition of PDE2, 3 and 4 families by a combination of Bay, Cil and Ro also led to a significant increase in cAMP level. Finally, there was no impact of cGMP on HCO3 -and the three PDEs inhibitors (Fig. 2E).

FRET measurements of cAMP signals in cardiac mitochondria

To determine whether the different PDEs regulate mitochondrial cAMP in primary cardiomyocytes, neonatal rat cardiomyocytes were transduced with an adenovirus encoding a cAMP sensitive FRET sensor (Epac-S H187 24 ) fused with a 4mt sequence. The localization of 4mt-Epac-S H187 in mitochondria was shown by co-localization of its green fluorescence with MitoTracker red fluorescence [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] 3C). We next evaluated the effect of the different PDE families on cAMP degradation following sAC stimulation with HCO3 -. HCO3 -alone caused a 15% increase in CFP/YFP ratio and a subsequent application of Bay or Cil resulted in a further increase in mitochondrial cAMP (Fig. Fig. 3D andE). Inhibition of PDE4 had larger effect on mitochondrial cAMP levels than inhibition of PDE2 or PDE3 (Fig. 3F).

PDEs regulate Ca 2+ -induced mitochondrial permeability transition (MPT)

MPT is an irreversible process of membrane permeabilisation that is characterized by a sudden matrix swelling and a loss of mitochondrial membrane potential (ΔΨm) in response to Ca 2+ overload. MPT is thus an hallmark of dysfunctional mitochondria, since ΔΨm is critical for allowing the respiratory chain activity to generate ATP [START_REF] Zoratti | The mitochondrial permeability transition[END_REF] . Using spectrofluorimetry to measure ∆Ψm and matrix swelling, we and others previously showed that MPT can be elicited by 10 µM Ca 2+ and prevented by 5 µM cyclosporin A (CsA) in cardiac isolated mitochondria [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . We also showed previously that cAMP elevation protects mitochondria from Ca 2+ -induced MPT [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . We now examined whether PDEs could modulate the Ca 2+ -induced MPT response. For that, we used a robust miniaturized assay 8 29 to concomitantly measure ∆Ψm level and swelling induced by Ca 2+ as a function of time in the presence or absence of selective PDE inhibitors and cGMP. As shown in Fig. 4, PDE inhibition did not affect ∆Ψm loss or swelling induced by Ca 2+ under basal condition. However, upon stimulation of soluble adenylyl cyclase with HCO3, both ∆Ψm loss and swelling were delayed. Inhibition of PDE2, 3 and 4 further slowed down both processes compared to HCO3 -alone (Fig. 4). In our experimental conditions, both processes were not affected by 5 µM cGMP pre-treatment in the presence of Ca 2+ or not (not shown).

Cardiospecific PDE2 transgenic overexpression influences mitochondrial function

To examine more deeply the role of PDE2 in mitochondria, we took advantage of the heart specific PDE2A3 transgenic mouse line we generated and characterized previously [START_REF] Vettel | Phosphodiesterase 2 protects against catecholamine-induced arrhythmia and preserves contractile function after myocardial infarction[END_REF] . First, we checked the expression of PDE2 specifically in mitochondria isolated from wild type (WT) and transgenic (TG) mice by subcellular fractionation and western-blot (Fig. 5A andB).

The PDE2A level was about 20 times higher in SSM from PDE2 TG mice than in WT mice.

Moreover, we observed by immunolabelling and confocal microscopy that overexpression of PDE2 in mice leads to a major expression of the protein at the plasma membrane of cardiomyocytes (Fig 5C). Since the labelling of mitochondria by MitoTracker Red shows a distribution of the organelles within the whole cell, a fraction of overexpressed PDE2 could in situ colocalize with SSM near the plasma membrane (Fig. 5C). In addition, the Pearson's correlation coefficient between PDE2 green fluorescence and red MitoTracker fluorescence, which is 0.418, is in favor of a partial colocalization of PDE2 and mitochondria (Fig. 5C).

Altogether, the results of western-blot and immunostaining allow us to propose that in transgenic PDE2A3 mice, the protein is mainly located at the plasma membrane and that a fraction of the protein has a subsarcolemmal mitochondrial localization.

Then, we measured the mitochondrial Ca 2+ accumulation with Rhod 2 in SSM mitochondria from WT and TG mice. Interestingly, we observed that mitochondrial Ca 2+ accumulation is faster in isolated mitochondria from TG than in WT mice (Fig. 5D) and modulated by HCO3 - and the soluble AC inhibitor, 2 hydroxyestradiol (2HE). HCO3 -was less efficient in TG mice and WT to stimulate Ca 2+ accumulation (Fig. 5E). cGMP had no effect on Ca 2+ accumulation (not shown). Accordingly, the overexpression of PDE2 also influenced MPT induction, since SSM isolated from PDE2 TG mice showed a faster Ca 2+ -induced ∆Ψm loss and swelling compared to mitochondria isolated from WT mice (Fig. 6A-D). This was observed in basal condition as well as in conditions of sAC stimulation by HCO3 -and inhibition by 2HE (Fig. 6A-D). Of note, we observed no modulation of bicarbonate effect by 100 nM Bay 60-7550 in TG mitochondria (not shown). This dose is the classical dose used that doesn't elicit unspecific effects on other PDEs, and thus, our results reveal the limitation of the PDE2 TG model for pharmacological study of PDE2 function in mitochondria. Finally, we examined the bioenergetic function of mitochondria from WT and TG mice. We assessed mitochondrial respiration using the Seahorse XF e 96 machine, which allows real-time assessment of oxygen consumption rates (OCR) in isolated mitochondria. Thus, succinate was used as a substrate to feed electrons to complex II and basal respiration was measured. State 3 respiration was determined in the presence of ADP. Oligomycin (Oligo) addition blocked ATP synthase and so evoked state 4o respiration. Maximal respiration (state 3u) was measured in the presence of the uncoupler FCCP. Finally, antimycin A (AA) was added to inhibit complex III. Compared to mitochondria isolated from WT mice, those from PDE2 TG mice exhibited a lower rate in complex II-driven basal respiration, in ADP-driven state 3 respiration and in FCCP-induced state 3u respiration (Fig. 7A andB). Moreover, we studied the effect of 5 µM cGMP stimulation as a pretreatment of mitochondria (see material and methods). We observed no difference in OCR in basal conditions in WT and TG in the presence of cGMP. Similarly, we observed no effect of cGMP on state 3 and 3u respiration in WT mitochondria but in contrast, cGMP stimulated the state 3 and 3u respiration in TG mitochondria.

This led us to propose a scheme indicating that mitochondrial cAMP level can be increased by sAC and decreased by PDE2 activity. This cAMP acts as a molecular switch controlling mitochondrial calcium entry, and then, favoring OXPHOS and/or MPT sensitivity (Fig. 7C).

Discussion

Our results show the expression of various PDEs in cardiac mitochondria and demonstrate their role in the control of cAMP level inside mitochondria, which regulates mitochondrial functions such as ∆Ψm, respiration and Ca 2+ -dependent MPT. Among the 3 PDEs identified by western-blot (i.e. PDE2, 3 and 4), PDE2 was demonstrated to locate predominantly in the SSM in association with the inner membrane by electron microscopy and to limit mitochondrial level of cAMP.

Cyclic nucleotide signalling pathways regulate a vast number of physiological processes, including cell proliferation and differentiation, gene expression, apoptosis and metabolic pathways such as steroidogenesis, insulin secretion and glycogen synthesis [START_REF] Conti | Biochemistry and physiology of cyclic nucleotide Phosphocliesterases: Essential components in cyclic nucleotide signaling[END_REF][START_REF] Francis | Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions[END_REF][START_REF] Keravis | Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in various diseases and perspectives for future therapeutic developments[END_REF] . Once synthesized by adenylyl cyclases or guanylyl cyclases, respectively, cAMP and cGMP transduce signal-encoded information by acting through a number of cellular effectors. Then, cAMP and cGMP signals are hydrolyzed by PDEs, allowing to restore cell homeostasis [START_REF] Conti | Biochemistry and physiology of cyclic nucleotide Phosphocliesterases: Essential components in cyclic nucleotide signaling[END_REF][START_REF] Francis | Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions[END_REF][START_REF] Keravis | Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in various diseases and perspectives for future therapeutic developments[END_REF] .

Recently, the existence of a mitochondrial cAMP signalling pathway has been evidenced in various organs such as brain, liver and heart. It was also shown that cAMP increases ATP production in cells and isolated mitochondria [START_REF] Acin-Perez | Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation[END_REF][START_REF] Benedetto | Mitochondrial Ca2+ uptake induces cyclic AMP generation in the matrix and modulates organelle ATP levels[END_REF] . A decrease in mitochondrial cAMP level by sAC inhibition decreases ATP production, O2 consumption, and cytochrome oxidase (COX) activity, whereas sAC activation by HCO3 -and Ca 2+ stimulates oxidative phosphorylation [START_REF] Benedetto | Mitochondrial Ca2+ uptake induces cyclic AMP generation in the matrix and modulates organelle ATP levels[END_REF][START_REF] Acin-Perez | Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation[END_REF] . Different PDE families and subtypes were found in various subcellular compartments including nucleus, endoplasmic reticulum, sarcoplasmic reticulum and Golgi [START_REF] Lugnier | Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents[END_REF][START_REF] Maurice | Advances in targeting cyclic nucleotide phosphodiesterases[END_REF] . However, little was known about mitochondrial PDEs. Until now, only PDE4 and PDE2 have been found in mitochondria [START_REF] Acin-Perez | A phosphodiesterase 2A isoform localized to mitochondria regulates respiration[END_REF][START_REF] Millar | DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling[END_REF][START_REF] Modis | Hydrogen sulfidemediated stimulation of mitochondrial electron transport involves inhibition of the mitochondrial phosphodiesterase 2A, elevation of cAMP and activation of protein kinase A[END_REF] . Acin-Perez et al. found that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform in rodent liver and brain [START_REF] Acin-Perez | A phosphodiesterase 2A isoform localized to mitochondria regulates respiration[END_REF] . PDE4B was reported to partially colocalize with DISC1 in the mitochondria of SH-SY5Y and LAN5 cells [START_REF] Millar | DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling[END_REF] .

Asirvatham et al. showed that mitochondrial AKAP149 binds to PKA and PDE4A comprising regulatory modules to maintain cAMP homeostasis in the T cell [START_REF] Asirvatham | Akinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines[END_REF] . In this study, we found PDE2A, 3A, 4A, 4B and 4D expression in mitochondrial matrix from rat heart isolated by western blot and precised the PDE2 inner membrane anchorage by transmission electron microscopy. In addition, we detected heterologous PDE2A3 and endogenous PDE2 isoforms in SSM by immunolabelling and confocal microscopy, despite the absence of in silico predictable mitochondrial targeting sequence. Indeed, MitoFates software was unable to predict a cleavable mitochondrial presequence in PDE2A3, but identified the short sequence LQRAV, which is a TOM20 interaction domain, TOM20 being a component of the TOM (translocase of outer membrane) receptor complex responsible for the recognition and translocation of cytosolically synthesized mitochondrial preproteins (not shown), raising the possibility of a mitochondrial localization of PDE2A3.

Acin-Perez et al. showed that PDE2A was present in brain and liver mitochondrial fractions and was responsible for cAMP-hydrolysis. In contrast, PDE4 had no significant contribution to cAMP-hydrolysis in liver mitochondria, but showed almost the same activity as PDE2 in brain mitochondria [START_REF] Acin-Perez | A phosphodiesterase 2A isoform localized to mitochondria regulates respiration[END_REF] . In isolated rat heart mitochondria, we showed that PDE2 represented the largest mitochondrial cAMP-degrading PDE activity, followed by PDE3 and PDE4 and that PDE2 can be activated by cGMP (see table 1). Our cardiac mitochondrial PDE2 activity correlated with Acin-Perez's data, thus suggesting that PDE2 is the main PDE in heart, liver and brain mitochondria. However, in neonatal cardiomyocytes infected with 4mt-Epac-S H187 , inhibition of PDE2 and 3 caused about 2.5% increase in FRET signal whereas inhibition of PDE4 resulted in 25% increase in basal condition. Upon HCO3 -stimulation, i.e. upon increased cAMP levels, inhibition of PDE2 and 3 caused 40% increase in cAMP, whereas it reached up to 80% with PDE4 inhibition. This might be due to a difference in PDE2 activity in isolated mitochondria from adult rat heart versus neonatal cardiomyocytes, which is not observed for PDE3 and PDE4 and remains to be confirmed.

In cardiac mitochondria, we have shown that mitochondrial cAMP stimulates O2 consumption and ATP production [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . Consistent with this, Acin-Perez et al. reported that inhibition of PDE2 caused an increase in O2 consumption and ATP production in mouse brain mitochondria.

Here, using PDE2 TG mice, we showed that SSM isolated from PDE2 TG mice had a lower O2 consumption rate than WT mice, but higher when mitochondria are stimulated by cGMP.

This suggest a complex metabolic regulation of mitochondrial energetic metabolism by PDEs depending on the ratios of PDE2 and 3 isoforms and the level of cGMP by an unknown mechanism. The investigation of this mechanism might provide new insights on the regulation of cardiac cell function and dysfunction, which are energy-demanding (i.e. contraction, arrhythmias). In addition, we previously reported that cAMP produced within mitochondria regulates Ca 2+ accumulation and MPT [START_REF] Wang | A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death[END_REF] . Here, we found that upon HCO3 - stimulation to increase cAMP, inhibition of PDE2, PDE3 and PDE4 delayed these processes, although inhibition of PDEs did not have an effect in basal condition. Moreover, SSM isolated from PDE2 TG mice showed a faster ∆Ψm loss and swelling compared to mitochondria isolated from WT mice confirming the results obtained in isolated mitochondria treated with PDE2 inhibitor. Since increased PDE2A expression was reported in human heart failure [START_REF] Mehel | Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts beta-Adrenergic Responses in Cardiomyocytes[END_REF] , PDE2A inhibition could be seen as a therapeutic strategy to improve heart metabolism and protect from apoptotic cell death, as recently suggested [START_REF] Monterisi | PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling[END_REF] . However, no adverse effects were observed so far in heart-specific PDE2 TG mice [START_REF] Vettel | Phosphodiesterase 2 protects against catecholamine-induced arrhythmia and preserves contractile function after myocardial infarction[END_REF] . On the contrary, PDE2 TG mice are protected against arrhythmias and show an improved contraction force after severe ischemic insult [START_REF] Vettel | Phosphodiesterase 2 protects against catecholamine-induced arrhythmia and preserves contractile function after myocardial infarction[END_REF] . Thus, further work is needed to explore the pathophysiological roles of PDE2 in models of heart disease as well as to look more closely at its action on distinct cellular cAMP and cGMP pools [START_REF] Wagner | Phosphodiesterase 2: antiadrenergic friend or hypertrophic foe in heart disease? Naunyn-Schmiedeberg[END_REF] .

In conclusion, our present work demonstrates that cAMP degrading PDEs form a mitochondrial signalling pathway controlling cAMP produced locally by sAC, and regulate mitochondrial Ca 2+ accumulation and MPT. PDE2, 3 and 4 are localized in cardiac SSM and contribute to the hydrolysis of mitochondrial cAMP, regulate Ca 2+ accumulation and MPT and, eventually, cell survival and MPT-mediated cell death. Given the role of ATP and Ca 2+ in the heart, our findings may have implications for the metabolic control of cardiac function.

Fluorescence

  resonance energy transfer (FRET) imaging experiments were performed 48h after infection of rat neonatal cardiomyocytes with the 4mt-Epac-S H187 adenovirus. Cells were bathed in Hepes-buffered Ringer's solution containing: 125 mM NaCl, 25 mM Hepes, 10 mM glucose, 5 mM K2HPO4, 1 mM MgSO4, and 1 mM CaCl2, pH 7.4. For sAC activation by HCO3 -, the medium was the Krebs-Henseleit solution containing: 120 mM NaCl, 2.09 mM K2HPO4, 0.34 mM KH2PO4, 24 mM NaHCO3, 1 mM MgSO4, 1 mM CaCl2, and 10 mM Dglucose. Krebs-Henseleit solution was gassed continuously with 95% O2/5% CO2 to maintain a pH of 7.4. Real-time FRET experiments were performed at room temperature. Images were captured every 5 s using the 40× oil immersion objective of an inverted microscope (Nikon, Champigny sur Marne, France) connected to a Cool SNAP HQ2 camera (Photometrics, Tucson, AZ, USA) controlled by the Metafluor software (Molecular Devices, Sunnyvale, CA,

  analysed by two way ANOVA, Sidak's multiple comparisons. Statistical significance is indicated as following: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The number of animals, cells and independent experiments performed is indicated in the figure legends.

Figure 1 .

 1 Figure 1. Expression of PDE isoforms in rat heart and mitochondria. (A) Expression of

Figure 2 .

 2 Figure 2. cAMP-PDE activity and

Figure 3 .

 3 Figure 3. Effect of PDE inhibition on mitochondrial cAMP in the absence or presence of

Figure 4 .

 4 Figure 4. PDEs regulate Ca 2+ -induced mitochondrial permeability transition (MPT).Evaluation of ∆Ψm loss (A) and mitochondrial swelling (B) induced by calcium in response

Figure 5

 5 Figure 5.

Figure 6 .

 6 Figure 6. PDE2 regulates Ca 2+ -induced mitochondrial permeability transition (MPT).Evaluation of ∆Ψm loss (A) or swelling (B) induced by calcium in cardiac mitochondria isolated from WT and PDE2 transgenic mice treated or not by 15 mM HCO3 -and 15 µM

Figure 7 .

 7 Figure 7. Bioenergetic analysis in isolated heart mitochondria from WT and PDE2 TG

Table 1 . Relative proportion of PDE activities in ventricle homogenate vs mitochondria

 1

			Other PDEs	PDE2	PDE3 (%) PDE4 (%)
			(%)	(%)
	Heart	No cGMP	9	6	22	63
	Ventricle	5 µM	7	12	7	74
	Homogenate	cGMP
	Mitochondria No cGMP	19	22	28	31
		5 µM	14	63	9	14
		cGMP

  . In basal condition, inhibition of PDE2 with Bay (100 nM, Fig

3A) and PDE3 with Cil (1 µM, Fig 3B

)

, respectively, caused a slight increase in mitochondrial cAMP level (2.5% increase in CFP/YFP ratio). Inhibition of PDE4 with Ro (10 µM) resulted in a much stronger increase in mitochondrial cAMP (25% increase in CFP/YFP ratio, Fig.

Acknowledgments

This work has been funded by INSERM (CB, DM, GV, RF), the Investment for the Future program ANR-11-IDEX-0003-01 within the LABEX ANR-10-LABX-0033 (CB, DM, GV, RF), ANR-13-ISV1-0001-01 (CB), ANR JCJC ANR-16-CE14-0014 grant (DM) and ANR13BSV10003-02 (GV). We thank C. Longin for TEM microscopy (platform MIMA2, INRA Jouy en Josas, France) and Dr B. Manoury for his expertise in statistical analysis. Zhenyu WANG and Dawei LIU were supported by a fellowship from the China Scholarship Council. The Seahorse apparatus was co-funded by CORDDIM (Investissement, 2015) and IPSIT, Châtenay-Malabry, France.

Conflict of Interest

The authors declare no conflict of interest.