Dr Gaelle Delaizir

Dr Virginie Viallet

Abdelmaula Aboulaich

Dr Renaud Bouchet
email: renaud.bouchet@univ-amu.fr

Dr Laurence Tortet

Dr Vincent Seznec

Dr Mathieu Morcrette

Jean-Marie Tarascon

Dr Patrick Rozier

Dr Mickael Dollé

Prof J.-M Tarascon

Introduction

Lithium-ion batteries are the focus of much research around the world, due to their high volumetric and gravimetric energy density. This technology has conquered the market of lightweight electronic devices such as lap-top computers, cellular phones and personal digital assistants (PDAs). However, safety and cost are the main concerns to their application in the potential mass market of electric vehicles and other particular applications that require a high temperature (oil drilling and military applications). The most important safety issue deals with the inherent instability of the liquid electrolyte, which requires the development of a non-fl ammable electrolyte with a high ionic conductivity ( > 10 -3 S cm -1 ). [ 1 , 2 ] Solid-state electrolytes, such as polymer electrolytes or ceramic electrolytes, are perfect answers to this safety issue. [ 3 ] For inorganic, all-solid-state batteries, considerable effort has been devoted to the development of microbatteries for their application in microelectronics. [ 4 ] These batteries, which consist of a stack of three, thin, dense components, namely a positive electrode, a solid electrolyte and a negative electrode, are fabricated using expensive, multistep processes based on thin-layer-deposition techniques, such as chemical vapour deposition, [ 5 ] RF-sputtering [ 6 -8 ] or pulsed vapor deposition. [ 9 , 10 ] Typically, these microbatteries have a total thickness around 15 μ m including the protective packaging, and their specifi c capacities lie between 5 and 100 μ A h cm -2 , depending on the thickness of the electrodes. [ 11 ] Increasing the thickness of the electrodes further has been a long-lasting dream that has failed so far because of the formation of microcracks between the components, due to stress generated at the solid/solid interfaces due to the volume change of the active material during cycling. Furthermore, strong kinetic limitations have been observed when an upscale in the third dimension is done, due to the low mobilities of the ions/electrons in solid-state active materials. [ 4 ] The main challenge facing all-inorganic Li-ion batteries is the increase of the stored capacity per square centimeter [ 2 ] (i.e., the thickness of the electrode). It is a sizeable challenge to combine the advantages of all-solid-state batteries (safety, mechanical integrity, etc) and the large volumetric and gravimetric energy density of lithium-ion batteries.

A solution lies in the use of composite electrodes, which are made from a multifunctional material: they should present a high content of an electrochemically active material to get a high energy density, as well as contain electronic-and ionic conductor additives to ensure effi cient and homogeneous transfer of electrons and ions in the electrode volume. Composite electrodes should also display a good mechanical aspect to allow easy handling and to ensure cell lifetime. The main diffi culty is

The Stone Age Revisited: Building a Monolithic Inorganic Lithium-Ion Battery

A new path for the design of safe and effi cient, all-solid-state Li-ion batteries by spark plasma sintering (SPS) is considered. To reach a good electrochemical performance from such batteries, several parameters are investigated, such as the composite-electrode formulation (active material/electrolyte/ carbon ratio) and the infl uence of the sintering parameters on their compactness. The formulation is optimized to ensure good ionic and electronic percolation through the composite electrode's volume. The compactness has to be suffi cient to guarantee a good mechanical aspect, while the residual porosity in the composite electrode allows electrode-volume changes upon insertion and deinsertion, preserving the electrode/electrolyte interfaces, which are crucial in such technology. Based on these investigations, an all-solid-state battery with a surface capacity of 2.2 mA h cm -2 is assembled by SPS, displaying a promising electrochemical performance at 80 ° C. DOI: 10.1002/adfm.201102479 the electrolyte and the composite electrodes. Therefore, we show how the electrical properties (ionic and electronic) can be used as a tool to guide the optimization of the formulation of the electrode, the microstructure of the ceramics and, consequently, the sintering process conditions, while preserving a good mechanical stability. The optimized electrode formulation and sintering protocol was then used to assemble battery prototypes in one step, which were cycled at 80 ° C.

Results and Discussion

Structure of Initial Powders and Processed Ceramics

The LAG and LVP starting powders were formed by agglomerates with rather inhomogeneous sizes ( < 50 μ m) ( Figure 1 a,d). At higher magnifi cation, these agglomerates were found to consist of primary rectangular particles, in the case of LAG material, and ovoid particles, in the case of LVP material; both had particles sizes of around 1-2 μ m (Figure 1 b,e). The microstructures of the dense LAG and LVP ceramics processed by SPS (680 ° C, 100 MPa, 2 min) are presented respectively in Figure 1 c,f. The LAG ceramic shows a limited grain growth, due to the fast heat treatment (Figure 1 c), as well as less porosity in comparison with the LVP ceramic (Figure 1 f).

to assemble laminated ceramics (composite anode/electrolyte/ composite cathode) in such a way that intimate interfaces can be generated, both between the solid grains of the materials in each of the three parts, and between the components themselves. It is often very diffi cult to sinter such a stacking of different materials suffi ciently, because the different layers generally require different optimal sintering temperatures and present different thermal expansion coeffi cients, which may lead to the formation of cracks and delamination at the interfaces. In this perspective, this paper proposes an original methodology to design and optimize multifunctional ceramics, allowing the building of a safe, self-supported monolithic battery with a high surface energy by addressing the key critical point of making a "piece of stone" that is able to store electrical energy reversibly. First of all, the development of bulk-type, all-solid-state batteries requires the material selection to be based not only on electrochemical criteria to ensure a high performance, but also on criteria linked to the sintering process, like the thermal properties and chemical compatibilities. To show the feasibility of thick, all-solid-state batteries (surface capacities higher than a few mA h cm -2 and electrode thicknesses higher than 200 μ m), two phosphate compounds were selected on the basis of their high stability upon sintering, as recently confi rmed. [ 12 , 13 ] The sodium superionic conductor (Nasicon) ceramic, Li 1.5 Al 0.5 Ge 1.5 (PO 4 ) 3 (LAG), was chosen as the solid electrolyte, as it offers a high ionic conductivity (3 × 10 -4 S cm -1 at 25 ° C with a Li + -transport number of 1) and an excellent stability at potentials up to 7 V. [ 14 ] However, at low potential, the germanium is reduced in the presence of Li + ions to form Li x Ge intermetallic compounds, destroying the Nasicon structure, which implies a careful selection of the negative electrode. [ 15 ] Monoclinic Li 3 V 2 (PO 4 ) 3 (LVP) was chosen as both the negative-and positive electrode material in order to build symmetric composite-LVP/LAG/composite-LVP batteries. LVP presents a reversible capacity of 80 mA h g -1 at 1.8 V versus Li + /Li (corresponding to insertion/deinsertion of 1.2 Li + ions per mole of LVP) within the stability window of the LAG solid electrolyte. As a positive electrode, it presents reversible capacities of 120 mA h g -1 between 3 and 4.2 V, and 140 mA h g -1 between 3 and 4.65 V, versus Li + /Li. [ 16 ] As shown recently, monolithic, all-solid-state batteries can be obtained in a few minutes using the spark plasma sintering (SPS) technique to sinter the composite electrodes and the electrolyte simultaneously. [ 17 ] Such an approach has allowed the assembly of battery prototypes that were cycled at least 30 times with only a small capacity fading, demonstrating the possibility of developing a viable, thick, solid-state technology. With regard to this result, the present paper is aimed at understanding the reasons for this interesting behaviour. The assembly of symmetric LVP/LAG/LVP batteries by SPS is addressed specifically, and the optimization of the composite-electrode formulation (LVP/LAG/carbon) is highlighted. Considering different materials (electrode and electrolyte materials), the principal challenge was fi rst to produce a multimaterial stack composed of two, thick, composite ceramic electrodes, separated by an ionic conducting ceramic, with the whole structure being able to withstand the mechanical stresses induced by the volume changes of the active material during lithium intercalation and deintercalation. Then, regarding the electrochemical concerns, the principal limitation comes from charge transport through charge-transport numbers for ions and electrons in the same range (these results were confi rmed by DC measurements that are not shown here). From an Arrhenius plot of the conductivity as a function of temperature, an activation energy of 0.59 eV was obtained, and the effective conductivity above 80 ° C was higher than 10 -5 S cm -1 .

Composite Electrodes

The optimization of the electrode formulation is based on a compromise of several parameters, the two main ones being:

1 The composite electrode must present a good ionic and electronic percolation to ensure transport of ionic species and electrons. The electrode composition (LVP/LAG/C) is therefore critical. 2 The compactness of the composite electrode (and the allsolid-state battery) has to be high enough to ensure a good mechanical handling, as well as ionic/electronic percolation, while preserving enough "free space" to compensate the appearance of mechanical strains inside the composite electrode and to preserve the interfaces.

Therefore, with regard to these considerations, the sintering protocol has to be adjusted, while ensuring that no decomposition or reaction between the LAG, LVP and carbon occurs upon heat treatment.

These different points were thus considered, starting with the chemical compatibilities between the different materials during the sintering process. X-ray diffraction (XRD) analysis ( Figure 3 ) of composite electrodes sintered by SPS at different temperatures showed that no reaction occurred at temperatures up to 680 ° C (100 MPa/2 min dwelling time). This temperature was then set as the maximum to obtain composite electrodes.

With the electronic conductivity within the composite electrode being mainly ensured by the amount of carbon (Csp), the electrical behaviour of composite electrodes with 5, 10 and 15 wt% (8, 15, 23 vol%, respectively) of carbon was investigated in a 1/1 LVP/LAG-ratio formulation. The results reported in Figure 4 a show that, to reach values higher than 1 S cm -1 , at least 10 wt% of Csp was necessary. Then, for a constant ratio of carbon (10 wt%), the infl uence of the LVP/LAG ratio (1/2, 1/1 and 2/1) was also scrutinized. The results obtained for composites with formulations 30LVP/60LAG/10C, 45LVP/45LAG/10C and 60LVP/30LAG/10C (in wt%) show that the electronic conductivity was increased with the decrease of the LVP/LAG ratio. The combination of the two results leads to the optimal formulation of 25LVP/60LAG/15C (wt%) to be defi ned.

Figure 4 b shows the compactness as a function of the sintering parameters for two electrode formulations. The compactness of the composite electrode for similar sintering parameters (680 ° C, 100 MPa, 2 min dwell time) was higher in the case of the 25LVP/60LAG/15C electrode composition (compactness of 86%) in comparison with the 45LVP/45LAG/10C electrode composition (compactness of 70%). As observed, the compactness of the composite electrodes, as well as the electronic conductivity, increased systematically when the amount of LAG increased.

Electrical Properties of the Materials

LAG Electrolyte

Different sintering temperatures from 500 ° C to 850 ° C during the SPS experiments (75 MPa or 100 MPa and 2 min dwell time) were used to obtain LAG ceramics, leading to different compactnesses, from 57% to 93%, as shown in Figure 2 a (the compactness being dimensionless and defi ned as the ratio of the experimental density to the theoretical density).

At high frequencies, the impedance spectrum, in Nyquist coordinates, (Figure 2 b) was composed of two loops; the fi rst one, at the highest frequencies, is related to the grains and the second to the grain boundaries. The sum of these two contributions gives the effective resistance of the sample. The effective conductivity of the LAG ceramic was calculated using the geometric parameters of the ceramics. The evolution of the LAGceramic conductivity as a function of temperature for different compactnesses is given in Figure 2 c. The activation energy calculated from the slope of the curves is close to 0.39 eV and independent of the compactness, contrary to the conductivity, which increases with the compactness. From the isothermal variation of the conductivity as a function of the LAG compactness at 20 ° C (Figure 2 d), the value of the conductivity of a 100% dense LAG ceramic was extrapolated to 2.8 × 10 -4 S cm -1 . Both values are in good agreement with the literature. [ 14 ] However, the impact of the electrolyte compactness is very important, since a drop by a factor of 6.4 for each loss of 10% of compactness was obtained. The modelling of this effect with a brick layer model, presented in a forthcoming paper, indicates that a compactness of at least 80% is necessary to reach a conductivity of 10 -5 S cm -1 . As this value is the minimum to guarantee good electrochemical properties at moderate temperatures, the minimal sintering temperature should be set to 650 ° C.

LVP Active Material

Following the same procedure, different SPS sintering protocols were applied for the preparation of LVP ceramics, for which the compactnesses were ≈ 70% at 680 ° C (100 MPa, 2 min dwell time) and ≈ 75% at 750 ° C (100 MPa, 2 min dwell time).

The characteristic impedance spectrum obtained at 130 ° C from an LVP ceramic with a compactness of 76% is given in Figure 2 e in Nyquist coordinates. A semicircle can be observed at high frequencies, which can be attributed to the LVP ceramic resistance; then, there is a small, fl at contribution at medium frequencies, which may correspond to interfacial electron charge transfer and, fi nally, at low frequencies, the beginning of a loop that starts with a straight line, with a characteristic angle approaching 45 ° with the real axis can be seen. This last contribution is due to a diffusion process inside the LVP. In the case of a pure ionic conductor (even in the case of a mixed anion and cation conductor), the gold electrodes return a vertical straight line (capacitive effect see Figure 2 b) at low frequencies, which is characteristic of ion-blocking electrodes. On the contrary, for a pure electronic conductor, the low frequency part is only a single point on the real axis, corresponding to the electronic resistivity. Thus, the impedance spectra suggest that the LVP ceramic is a mixed ionic/electronic conductor with To understand this result, the microstructures of processed ceramics with LVP/LAG ratios of 1/2, 1/1 and 2/1, and 10 wt% of carbon, were analyzed. The pellet microstructures corresponding to LVP/LAG ratios equal to 1/2 or 1/1 ( Figure 5 a,b) were quite similar and were made of large (10 to 20 μ m), dense electrolyte regions surrounded by porous regions composed of smaller LAG and LVP grains covered by carbon (black regions in Figure 5 ), which ensure electronic percolation. The compactness of the LAG and LVP pellets after sintering at 680 ° C (100 MPa/2 min dwell time) were, respectively, 84% and 70%. This clearly indicates that the LAG electrolyte was sintered before the active material and acts as cement in the composite electrodes, ensuring both mechanical integrity and ion transport in the electrode. During the sintering of the LAG, the LVP and carbon were segregated in the porosity of the LAG network (Figure 5 a (zoom) andd). Therefore, the LVP grains were found to be surrounded by carbon beads with many connections to the LAG network. This local environment of the LVP appears to be an important key for the relaxation of the interfacial stress during electrochemical cycling. The microstructure of the composite electrode with an LVP/LAG ratio equal to 2/1 was, as expected, more porous for similar sintering parameters, as shown in Figure 5 c. It was mainly constituted of small LVP grains surrounded by a porous network of carbon and isolated LAG grains. It is also noteworthy that the increase of the electronic conductivity as the amount of LAG increased is consistent with literature relating to inorganic solid-state electrodes thicker than a few micrometers. [18][19][20][21][22][23] The formulation of the composite electrodes to ensure ionic and electronic percolation within the electrode volume lies generally in the range of 25-30% of active materials and 55-65% of electrolyte, with the rest being the electronic conductor (10-20%). However, the intrinsic electrical properties of the active material are very important, as was the case for olivine-type active materials, which are very poor electronic conductors and thus need a carbon coating to deliver their energy. [ 24 ] According to our data, LVP is a mixed ionic/electronic material, which may assist electron and ion charge transport through the volume of the composite electrode. Finally, a higher amount of active material could be obtained in the formulation notably by playing on the electrolyte grain size and improving the mixing of the precursor powders, allowing both the homogenization of the chemical compound distribution into the bulk of the electrode, and, therefore, the effi ciency of electronic and ionic percolation, to be improved.

As a summary of the criteria deduced from these studies, it appears that to sinter the composite electrode and then the battery, the temperature should be selected to sinter the electrolyte (LAG) fully, in order that it forms a continuous network in the composite electrodes, ensuring both the mechanical framework and ion transport in the whole battery. According to the microstructure of the starting powders, the optimized formulation contains at least 10 wt% of Csp and an LVP/LAG ratio close to 1/2. A composite electrode compactness of around 85% seems to be a good compromise, allowing good mechanical handling, as well as good electronic percolation. Furthermore, the residual porosity and local composite architecture allows the connection of LAG and LVP grains only at some localized points (Figure 5 d), allowing the "structure" to move and dissipate mechanical constraints as the ionic species will insert into and be removed from the LVP structure during cycling.

Battery Assembly

Based on our results, the optimized composite electrode formulation (25LVP/60LAG/15Csp (wt%)) and the optimized sintering parameters (680 ° C, 100MPa, 2 min dwell time) were used to build a symmetric prototype with a positive-electrode/ negative-electrode mass ratio close to unity. Figure 6 a shows a picture of one all-solid-state battery prototype, assembled by SPS. Figure 6 b demonstrates the good mechanical properties of this all-ceramic bulk-type battery, which has clean and welldefi ned interfaces (Figure 6 c).

The electrical properties of the battery, measured by impedance spectroscopy (Figure 2 b), were used to deduce the compactness of the electrolyte between the two composite electrodes using Figure 2 d as a reference graph. A compactness of 92% was deduced based on the measured ionic conductivity (red arrows in Figure 3 d), which is in good agreement with the geometrical estimation. The composite electrode's compactness of about 80-85%, as expected, was lower than the compactnesses of pure LVP and pure LAG pellets, about 70% and 84%, respectively, at 680 ° C. The electrode and electrolyte thicknesses were all around 400 μ m.

The galvanostatic cycling of the prototype at 80 ° C, at rates of C/50 or C/25, is shown in Figure 6 d. The results show good cycling performances with surface capacities of 1, 2 and 2.2 mA h cm -2 , depending on the selected cut-off voltage, for 1.75, 2.35 or 2.45 V, respectively.

Conclusions

This work provides a new path worth pursuing for the design of inorganic Li-ion batteries capable of operating completely safely over a wide temperature range, and with a surface capacity comparable to that of classical lithium-ion batteries. To design effi cient, monolithic, all-solid-state batteries, several parameters need to be considered: the choice of materials, the granulometry of the starting powder, the composite electrode formulation, and the sintering conditions affect the ionic and electronic percolation paths through the volume of the composite electrode and thus the electrochemical performance. The optimization of the composite electrodes requires the correlation of their microstructure with their electrical properties. Finally, a solid-state battery prototype was assembled by SPS considering the optimized parameters. The fi rst results show good cycling and a promising electrochemical performance (surface capacity of ≈ 2.2 mA h cm -2 with a cut-off voltage of 2.45 V). The local architecture of the composite electrode is believed to be a key parameter, allowing stress relaxation during cycling.

Experimental Section

Materials : The active material, A-Li 3 V 2 (PO 4 ) 3 (LVP) powder, was obtained by initial homogenization of the precursors (stoichiometric amounts of NH 4 VO 3 and LiH 2 PO 4 ) dissolved in an aqueous solution (1 mol L -1 ), followed by the slow evaporation of H 2 O at 100 ° C and that XRD was performed using a D8 Bruker instrument in the range 10-50 ° (2 θ ), using Cu K α radiation.

The conductivities of the LVP and LAG starting materials and of the composite electrodes were characterized in the temperature range 30-130 ° C using electrochemical impedance spectroscopy (Solartron 1260) in the frequency range from 1 mHz to 10 MHz, with an excitation voltage of 20 mV. Thin gold layers were deposited by physical vapor deposition (PVD) on each face of the ceramics obtained by SPS as (ion-) blocking electrodes. Then, the metallized ceramics were dried over 5 d in a glove box (H 2 O < 3 ppm), before being mounted in tightened swagelock type cells to perform the electrochemical measurements outside the glove box. The impedance data were modelled using the Z-View software (Scribner inc.). Finally, the electrochemical performance of one battery prototype was determined at 80 ° C and at a current density ranging from 40 μ A cm -2 (C/50, C/1 or C being defi ned as one Li + (resp. 1 electron) exchanged in 1 h) to 80 μ A cm -2 (C/25), using a battery-test device (Solartron 1470).

of volatile species at 400 ° C under Ar/H 2 . Then, a second annealing was conducted at 750 ° C (12 h) under Ar/H 2 gas fl ow (10%) to achieve reduction of V (V) to V (III). [ 16 ] The solid electrolyte, Li 1.5 Al 0.5 Ge 1.5 (PO 4 ) 3 (LAG), was synthesized as follows. Germanium and aluminum alkoxide reactants, Ge(OC 2 H 5 ) 4 and Al(OC 4 H 9 ) 3 , were dissolved in tetrahydrofuran (THF) and lithium and ammonium salts (pure CH 3 COOLi•2H 2 O and pure NH 4 H 2 PO 4 ) were dissolved in water. After dissolution of the precursors, the water solution was added drop by drop to the alkoxide solution. Then, the excess of solvent was removed by heating at 80 ° C while stirring the solution, and an appropriate heat treatment (2 h at 850 ° C) was performed to generate the pure phase with a well-controlled microstructure.

Carbon black (Csp) (Ketjen Black from Timcal) was used as the additive that imparts electronic conductivity to the solid-state composite electrode.

Sintering of the Composite Electrodes, the Electrolyte, and the Battery Assembly by Spark Plasma Sintering : The pellets were obtained in 10 min thanks to the use of the SPS technique. Prior to the one-step battery assembly, different thermomechanical cycles for the SPS were tested on the electrolyte (the LAG or the active material (LVP) alone, and the composite electrodes (LVP/LAG/C)), leading to ceramics with different compactnesses.

The composite electrodes were prepared by the hand mixing of the LVP, LAG and Csp. The same formulation was used for the negative and positive electrodes, and the battery was assembled under vacuum by SPS in a graphite die with an inner diameter of 8 mm. Typically, the battery prototypes had a diameter of 8 mm and a thickness of about 1.2 mm.

Microstructure, Impedance Spectroscopy, and Electrochemical Measurements : The microstructures of the starting powders (LVP, LAG)

Figure 1 .

 1 Figure 1 . a-f) SEM images of the initial LAG powder (a,b), the LAG ceramic processed by SPS (c), the initial LVP powder (d,e), and the LVP ceramic processed by SPS (f) (SPS sintering parameters: 680 ° C, 100 MPa, 2 min dwell time).

Figure 2 .

 2 Figure 2 . a) Infl uence of the sintering temperature and pressure (constant dwell time of 2 min) on the compactness of the LAG electrolyte ceramics. b) Electrochemical impedance diagram obtained at room temperature for the thick, solid-state lithium-ion battery LVP/LAG/LVP (open squares), and the LAG electrolyte (open triangles) sintered by the SPS technique. c) Temperature dependence of the ionic conductivity of LAG ceramics with different compactnesses (open symbols). d) Isothermal variations of the effective conductivity of the LAG ceramics as a function of compactness. This curve was used as a reference graph to obtain the "electrical" compactness of the LAG electrolyte in battery prototypes directly (open circles). e) Characteristic impedance spectrum of gold-metallized LVP ceramics with a compactness of 76% at 130 ° C. Some of the characteristic frequencies are indicated.

Figure 3 .

 3 Figure 3 . XRD diffractograms of the starting LAG powder (a), the starting LVP powder (b), and the composite electrode (LVP/LAG/Csp) processed by SPS (680 ° C, 100MPa and 2 min dwelling time) (c).

Figure 4 .

 4 Figure 4 . a) Electronic percolation of LVP/LAG/Csp composite electrodes sintered with the optimized SPS conditions. b) Infl uence of the SPS sintering temperature and pressure (constant dwell time of 2 min) on the compactness of composite electrodes (LVP/LAG/Csp) with different formulations.

Figure 5 .

 5 Figure 5 . a-c) Back-scattered electron images of the polished ceramics processed by SPS (680 ° C, 100 MPa, 2 min dwell time). LVP/LAG ratios (10 wt% C): 1/2(a), 1/1 (b), and 2/1(c). d) SEM photograph of a broken LVP/LAG/Csp (25/60/15) composite electrode sintered by SPS (680 ° C, 100 MPa, 2 min dwell time).

Figure 6 .

 6 Figure 6 . a-b) Pictures of an LVP/LAG/LVP battery obtained by the SPS technique. c) SEM image of the interface electrolyte/composite electrode in a broken solid-state battery (SPS sintering parameters: 680 ° C, 100 MPa, 2 min dwell time). d) Charge/discharge profi les obtained at 80 ° C and C/50 or C/25 for one symmetrical LVP/LAG/LVP battery prototype (electrode composite composition: 25LVP/60LAG/15Csp)

Acknowledgements

The authors express their sincere gratitude to the French National Research Agency (Project CeraLion-ANR-07-Stock-E-04) and the Aerospace Valley competitiveness cluster for their fi nancial support.


Celine Guilbeau-Frugier

Marie Cauquil

Clement Karsenty

Camille Dambrin

Bruno Payre

Hervé Cassard

Claudie Josse

Marie-Hélène Seguelas

Sophie Allart

Maxime Branchereau

Ce ´line Guilbeau-Frugier

Cle ´ment Karsenty

Olivier Lairez

Herve ´cassard

Marie-He ´le `ne Seguelas

Christophe Heymes

Franck Mandel

Marie-Bernadette Delisle

Atul Pathak

Etienne Dague

Jean-Michel Se ´nard

Ce ´line Gale
email: celine.gales@inserm.fr

Structural evidence for a new

Keywords: Cardiomyocyte, Lateral membrane, Cardiac tissue architecture, Subsarcolemmal mitochondria, Electron-atomic force microscopy, Sarcomere relaxation

Introduction

The adult cardiac tissue cohesion necessary for the continuous cyclic mechanical strain mainly relies on a substantial three-dimensional (3D) interaction between contractile cardiomyocytes (CMs) and the extracellular matrix (ECM). Interestingly, CMs undergo different morphological adaptations during heart development. Throughout embryonic and early postnatal developmental stages, CMs exhibit a plastic phenotype with a round or spindle-fibroblastic shape that correlates with their proliferative state and contributing to the increase of heart mass through hyperplasia. At approximately Day 5 postnatal in rodents, CMs stop proliferating and initiate a maturation step to acquire their typical adult rod-shape. [START_REF] Yang | Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes[END_REF] Within the tissue, this rod-shaped cell morphology is stabilized through specific and direct CM-CM interactions via the intercalated disk (ID) localized at the bipolar ends of the CM, while the lateral cell surface is classically believed to interact with the ECM through receptors (integrin/dystroglycan) on the costamere, without physical contacts between proximate CMs. [START_REF] Samarel | Costameres, focal adhesions, and cardiomyocyte mechanotransduction[END_REF] Both surface structures control the CM contraction with the ID underlying the physical anchorage of the contractile myofibrils and contraction synchronization via gap junctions and the lateral membrane, relaying ECM extracellular signals to the myofibrils for contractile adaptation to the cardiac pressure load.

More recently, we and other groups have identified that cell-cell communication transmembrane proteins, i.e. Ephrin-B1 [START_REF] Genet | Ephrin-B1 is a novel specific component of the lateral membrane of the cardiomyocyte and is essential for the stability of cardiac tissue architecture cohesion[END_REF] and Claudin-5 [START_REF] Sanford | Claudin-5 localizes to the lateral membranes of cardiomyocytes and is altered in utrophin/ dystrophin-deficient cardiomyopathic mice[END_REF] have an atypical expression on the lateral sarcolemmal membrane of adult CMs and act independently on the contractile apparatus, thus suggesting that the functions of the lateral membrane are not completely understood. These findings subsequently prompted us to explore the lateral membrane at a high resolution in mice, and using atomic force microscopy (AFM), we identified a highly organized cell surface architecture of the CMs with the presence of periodic stiff crests related to the presence of subsarcolemmal mitochondria (SSM), which were lost following ischaemia. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] Similar highly organized crest structures and their disorganization in heart failure have been reported indirectly by analysing the CM ionic surface profile via scanning ion conductance microscopy. [START_REF] Nikolaev | Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation[END_REF][START_REF] Lyon | Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart[END_REF] However, to date, these studies have mainly relied on the analysis of isolated adult CMs. Whether the presence of well-defined crest structures at the CM surface is a general feature of all heart compartments and how they organize within the cardiac tissue remain unknown.

Herein, we explored the lateral membrane of CMs in cardiac tissue samples from different heart compartments that originated from various mammalian species using high-resolution scanning or cryo-scanning and transmission electron microscopy (TEM), as well as AFM. We first identified a compartment-and species-dependent crest architecture at the CM surface. More strikingly, we noted the presence of intermittent tight junctions between crests from adjacent CMs in mammalians including humans, thus establishing for the first time proof of the concept of existence of a physical interaction between lateral membranes of adjacent CMs. This discovery brings to the fore the complexity of the 3D-structure of the adult CMs in the tissue and should open a new research area to decipher the functional relevance of these lateral contacts in cardiac physiology and pathophysiology.

Methods

Animal models and cardiac tissue processing

Studies were performed on 2-month-old male C57BL/6JOlaHsd mice and WistarRccHan rats (purchased from Envigo, Huntingdon, UK). The experimental animal protocols were carried out in accordance with the European and French regulation guidelines for animal experimentation and were approved by the local ethical committee. Mice and rats were euthanized through an intraperitoneal injection of pentobarbital sodium (160 mg/kg), while 50 mg/kg was used for the KCl experiments.

To stop the heart in diastole, KCl (1 M) was directly injected as a bolus in the inferior vena cava in anaesthetized and thoracotomized mice. After arrest, the hearts were immediately removed, and sections ($1 mm 3 ) were fixed in 2% glutaraldehyde in cold Sorensen's buffer for further electron microscopy analysis.

For cardiac tissue processing on ice, immediately after removal, the heart was plunged in cold PBS for several seconds, thus instantly stopping the heartbeat. The heart was then cautiously sliced ($1 mm 3 ) on an ice bed without torsion or pressure and fixed in 2% glutaraldehyde in Sorensen's buffer at 4 C for further electron microscopy analysis.

For transcardial fixative perfusion, immediately after removal from the chest, the heart was plunged in cold PBS for several seconds thus instantly stopping the heartbeat. The aorta was then first retroperfused with cold phosphate buffer saline with a peristaltic pump (0.3 mL/min) for 3 min followed by 3 min of retroperfusion with cold 2% paraformaldehyde/0.1% glutaraldehyde. The heart sections ($1 mm 3 ) were subsequently fixed in 2% glutaraldehyde in cold Sorensen's buffer for further electron microscopy analysis.

For the cow study, two 8-year-old Blonde d'Aquitaines (weight 770 kg) were analysed in collaboration with the Veterinary National School of Toulouse (ENVT). They were euthanized with a barbiturate injection due to a bilateral pyelonephritis and a chronic abscess on the paws. Immediately after bleeding, the heart was removed and sections ($1 mm 3 ) were fixed in 2% glutaraldehyde in Sorensen's buffer at 4 C for further electron microscopy analysis.

Transaortic constriction (TAC) and echocardiographic assessment

Two-month-old male C57Bl/6JCrl mice were used for these experiments. The mice were housed with a 12:12 light-dark cycle and fed ad libitum with standard chow and water. Thoracic aortic constriction (TAC) was performed on mice anaesthetized with 1% isoflurane. After endotracheal intubation, the mice were connected to a rodent ventilator. The transverse aorta was isolated and TAC was performed by tying a nylon suture ligature against a 26-gauge needle, the latter being promptly removed to induce pressure overload cardiac hypertrophy. After aortic constriction, the chest was closed and the pneumothorax was evacuated. After extubation, the mice were allowed to recover from the anaesthesia. Sham operated animals (controls) underwent the same operation except for the performance of aortic constriction. Fifteen days after surgery, animals underwent non-invasive transthoracic echocardiography using a Vevo V R 2100 device (VisualSonics) under 1% isoflurane anaesthesia. Cardiac wall thickness and ejection fraction were measured at least three times for each animal in Mmode images and in long-axis B-mode, respectively. All measurements were obtained by an investigator blinded to the genotype of the animals.

Human tissue samples

Human cardiac appendages from the tissue collection of the Toulouse University Hospital (registered according to French regulations for clinical research-DC 2009-989) were analysed after patients consented to their use. All patients were Caucasian and had undergone extracorporeal circulation for cardiac surgery performed at Toulouse University hospital (Table 1). Within 1 h of the removal, the appendages were isolated and 1 mm 3 pieces were directly fixed in 2% glutaraldehyde in

Cardiomyocyte purification from adult rats

For the AFM study, CMs were isolated from adult rats using the Langendorff perfusion method as previously described. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] Beating hearts were quickly removed from the chest after intra-peritoneal injection of pentobarbital (160 mg/kg) and Langendorff perfused with Krebs solution (in mM: NaCl 113, kCl 4.7, KH2PO4 0.6, NaHPO4 0.6, MgSO4 1.2, NaHCO3 12, KHCO3 10, HEPES 10, taurin 30, 2,3-butanedione monoxime (BDM) 10, glucose 5.5, pH 7.4) containing 0.5 mM CaCl2 at 37 C for 6 min. Perfusion was then switched to a decalcifying solution (Krebs solution containing 0.1 mM EGTA) at 37 C for 2 min. Finally, the perfusion was switched to an enzymatic solution (Krebs solution containing 2.2 mg/mL Type II collagenase-Worthington Biochemical corporation) and 12.5 lM CaCl2 at 37 C for 20 min. Following digestion, the left and right free wall ventricles were separated and dissected into 1 mm 3 pieces. The myocytes were mechanically dissociated using Pasteur pipette in resuspension solution (Krebs solution containing 10% bovine calf serum and 12.5 lM CaCl2). The myocyte suspension was then filtered through a 200 lm mesh and harvested after sedimentation. The isolated myocytes were plated for 10 min on specific laminin (mouse laminin, Life technology)-coated culture dishes (40 Â 11 mm, TPP-Tissue Culture treated dishes) in the resuspension solution at 37 C (5% CO 2 ). CMs were then fixed in 2% glutaraldehyde in Sorensen's buffer for 10 min at room temperature, washed three times with PBS and immediately used for the AFM experiments in PBS.

Transmission and scanning electron microscopy (TEM-SEM)

Heart samples were fixed by immersion in 2% glutaraldehyde in Sorensen's buffer at 4 C and processed for TEM or scanning electron microscopy (SEM) as previously described. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] All crest height and sarcomere length measurements were performed on TEM micrographs. The crest heights were measured from the top of the crest to the first myofibril top layer using FiJi software and the SSM number was quantified for each crest. The sarcomere lengths were measured similarly on the first myofibril layer below the CM surface crests.

Cryo-scanning electron microscopy

After fixation, as indicated for the SEM procedure, the samples were incubated for 12 h in cryo-protectant glycerol and rapidly frozen in nitrogen slush at -220 C. The frozen samples were transferred under vacuum to the cryo-chamber apparatus (Quorum PP3000T) at -140 C and fractured with a cold knife integrated in the cryo-chamber. The temperature was increased to -95 C and maintained for 10 min of sublimation and the specimen was then coated with platinum by sputtering (60 s, 10 mA) and moved in the high vacuum mode into the SEM observation chamber of a FEI Quanta 250 FEG scanning electron microscope (5 kV). The specimen was maintained at a temperature less than -140 C at all procedure steps.

Dual beam (SEMþFIB) 3D reconstitution microscopy

The 3D-characterization of the CM lateral membrane was carried out using Focused Ion Beam (FIB) tomography. This method was developed using the dual beam system Helios NanoLab 600i (FEI company). The instrument incorporates both a FIB and a field emission scanning electron microscope (FEG-SEM).

The myocardial heart sample was processed similarly as the SEM until resin embedding and then introduced into the FIB-SEM chamber. The sample was covered with a conductive layer of platinum to avoid surface charging and protect it from the ion beam (30 kV, 2.5 nA). Dual-beam scanning was conducted on 300 sequential sample slices (50 nm thick) with SEM image acquisition for each slice (5 kV voltage, 0.69 nA current, TLD detector). Three-dimensional reconstitution of the heart myocardium sample was conducted using AVIZO software (FEI).

Atomic force microscopy

Surface images of fixed isolated rat CMs were recorded in PBS buffer in Quantitative ImagingTM mode [START_REF] Chopinet | Imaging living cells surface and quantifying its properties at high resolution using AFM in QI mode[END_REF] with MLCT AUWH (Bruker) cantilevers (nominal spring constant of 0.01 N/m) and using an AFM Nanowizard III (JPK Instruments, Berlin, Germany). The applied force was maintained at 3 nN and the Z length to 5 mm. Therefore, the loading rate was 2 500 000 pN/s (acquisition frequency of the force curves is 25 Hz). The cantilever spring constants were determined by the thermal noise method. For all results presented in this study, the silicon nitride AFM tips were bare. AFM topographical images were analysed using the JPK data processing software. The crest heights were measured based on a manual cross-section delimitation perpendicular to the CM myofilaments and considering the base line as the position of the plasma membrane in the absence of crests. Membrane holes, due to fixative artefacts, were not considered for height measurements.

Immunofluorescence imaging

All imaging data was obtained using frozen cardiac tissue sections fixed in acetone, except for collagen III-SIM imaging (paraffin-embedded heart sections). Heart sections were immunostained using standard protocols with the following primary antibodies incubated overnight at 4 C: anti-laminin 2a (Abcam, ab11576), anti-claudin-5 (Acris, DP157), anti-collagen III (Acris, BP8014), anti-a-actinin (Abcam ab68167), anti-cardiac Troponin T (Life Technologies, MA5-12960). Heart sections were rinsed three times with PBS and labelled with secondary antibodies (Oregon Green 488 goat anti-rabbit IgG, Life Technologies O11038; AlexaFluor 594 goat anti-rat IgG, Life Technologies A11007; STAR RED goat anti-rabbit, Sigma 41699) and Texas-Red (Life Technologies, W21405)-or Oregon Green 488 (Life Technologies, W6748) conjugated Wheat-Germ-Agglutinin for 1 h at room temperature. Coverslips were finally mounted on microscopy slides using Mowiol.

Confocal imaging was carried out using a Zeiss LSM 780 confocal microscope and Zen 2011 software (Carl Zeiss).

Super-resolution structured illumination microscopy (SR-SIM) was carried out on aELYRA PS.1 (Carl Zeiss), lasers 488, 561, and/or 642 nm with a Plan Apochromat 63Â/NA 1.46 oil objective and a grid size of 32 or 42 lm equipped with a sCMOS PCO Edge camera. Image stacks of several micrometer thicknesses were taken with 0.110 lm z-steps, five phases and three rotations per z-section. Reconstruction of images was performed using Zeiss Zen 2.1 software and a Noise Filter.

Super-resolution stimulated emission depletion (SR-STED) microscopy images were acquired using a Leica SP8 STED 3Â microscope (Leica Microsystems, Germany) and a 100Â NA: 1.4 oil immersion objective. To optimize the resolution without bleaching in 3D, the 775 nm STED laser line was applied at the lowest power that can provide sufficient improvement in resolution compared with confocal. Z-stack series were acquired sequentially with the pulsed 532 nm laser line and the pulsed 635 nm laser line. With such settings, lateral and axial resolutions of 130 nm could be reach for both acquisition channels. For image acquisition, we used the following parameters: 43 nm pixel size, two time average par line, 400 Hz scan speed. STED images were subsequently deconvolved with Huygens Professional (SVI, USA) using the CMLE algorithm, with a signal to noise ratio (SNR) of 7. Three-dimensional visualization and reconstruction using Imaris software (Bitplane, Switzerland).

Quantification of CM area

For in situ quantification of the CM surface area, deparaffinized heart slides were stained with Texas Red-conjugated-WGA (Life technologies, W21405) and both CM area and density were measured in heart crosssections by manually tracing the cell contour on images of whole hearts acquired on a digital slide scanner NanoZoomer (Hamamastu) using NDP.view2 viewing software. The experimenter was blinded to the mouse group.

Statistics

All bar graphs represent means ± s.e.m. Statistical analyses were performed using Prism v5 software. Statistical significance was calculated using Student's t-test (Figures 2B,C, H, 3B, E, 5B), two-way ANOVA (Figure 2D), Spearman correlation test (Figure 2E, G), or Fisher's exact test (Figure 5C). *P < 0.05, **P < 0.01, ***P < 0.001.

Results

Intricate 3D-CM surface crest architecture in cardiac tissues of different mammalian species and technical pitfalls

In general, the cardiac tissue structure is inferred from a histological analysis commonly performed on formol-fixed hearts and paraffin embedded sections, thus preventing appreciation of the CM 3Dstructure and organization. Here, we first processed samples from the left ventricles (LV) of adult male mouse hearts (2 months old) using SEM to visualize at a high resolution, the 3D-morphology of the CM surface within the tissue. In the left ventricular myocardium, we found that the circumference of the CM lateral surface is covered by highly organized periodic rows of bulged structures in contact with the ECM (Figure 1A, upper panels). These elongated structures are orientated perpendicularly with regard to the longitudinal intracellular myofibrils. In a second set of experiments, samples were processed for cryo-SEM to enable visualization of the CM intracellular components through cell transversal sectioning. In this context, the CM surface noticeably appears as a periodic alternation of crests filled with SSM tightly attached to the endoplasmic reticulum, as previously described using TEM [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] (Figure 1A, lower panels). Similar structures were identified in the cow-LV myocardium (Figure 1B, upper left panel and Supplementary material online, Figure S1) as well as in the rat-LV and human-LV (Supplementary material online, Figure S1). Consistent with these observations of surface bulged rows, sequential 3D-reconstitutions of the SEM images by dual-beam microscopy in the mouse indicated that SSM adopt an elongated morphology beneath the sarcolemma (Figure 1C). Noticeably, the periodicity of the crest rows relies on their lateral anchorage to the Z-lines that delimit each sarcomere from the outer myofibril layer (Figure 1B, upper middle panel and Supplementary material online, Figure S1, Cow-LV), more visible in TEM (Figure 1D), which we previously described as CM surface 'hollows'. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] It is important to note that the visualization of CM surface crest structures and intracellular SSM analysis using TEM are extremely dependent on the handling procedure of the cardiac tissue. First, the animal euthanasia method is fundamental. Specifically, cervical dislocation, as recently described for SSM observation, [START_REF] Hendgen-Cotta | Mouse cardiac mitochondria do not separate in subsarcolemmal and interfibrillar subpopulations[END_REF] leads to a complete SSM loss (not shown), most likely as a consequence of cardiac fibrillation, thus precluding its use. Euthanasia using potassium chloride intra-cardiac injection, classically recommended to observe the sarcomere ultrastructure, also destroys the crest architecture (Supplementary material online, Figure S2A), most likely due to its substantial impact on sarcomere relaxation, in some cases associated with cardiac spasms. Another critical step is the delay between tissue handling after cardiac arrest and fixation for electron microscopy that must be as short as possible since, in contrast with interfibrillar mitochondria (IFM), SSM are highly prone to shrinking during tissue removal. Intra-aortic paraformaldehyde/glutaraldehyde fixative perfusion, considered to efficiently preserve soft tissues, tends to increase the lateral interspace between two CMs because of vasculature dilatation, even at the lowest perfusion flow rate, and conversely decreases crest height ('flattened-like' crests) due to substantial reductions in the SSM volume (Supplementary material online, Figure S2B). However, this latter technique is ideal for visualizing the SSM ultrastructure. Finally, in accordance with the well-known sensitivity of the heart to both protease activity and temperature, the optimal procedure for crest-SSM observation in TEM relies on the use of an ice-cold buffer immediately after tissue removal and on tissue dissection on ice, even if sarcomeric details, such as the I-band of the Z-lines cannot be systematically visualized due to arbitrary heart arrest in a contractile or relaxed state (Supplementary material online, Figure S2C). These technical pitfalls indicate that crest-SSM observation within the tissue requires specific handling of cardiac biopsies, which most likely account for the chaotic organization of the CM surface and SSM often described in the literature, as well as the discrepancies in observations between studies.

CM surface crest architecture in cardiac tissue is species-dependent

We subsequently investigated whether the CM surface crest architecture in the LV was similar in different mammalian adult species. TEM was thus conducted on different biopsies from LVs obtained from adult mice, rats, cows, and humans (Figure 2A). In the two latter cases, the management was less flexible than in rodents since the biopsies could not be handled in optimized conditions for crests-SSM observation. However, despite these technical pitfalls, clear differences between species were identified and quantified. When initially considering cardiac LV samples from mice, we quantified the crest height and SSM number/crest. The TEM measurements indicated average crest heights of approximately 1 mm (0.87 mm ± 0.07; Figure 2B) with a majority of the crest populations with 1-2 SSM per crest (Figure 2C and D) and the crest height correlated with the SSM number (Figure 2E). In contrast, the crests from rat left ventricular biopsies can reach heights up to 1.5 mm (1.31mm ± 0.13; Figure 2B) and exhibited a higher average number of SSM per crest (Figure 2C) with a majority of the crest population with 2-3 SSM (Figure 2D and Supplementary material online, Figure S3). Even if in the minority, we also identified some crests with 4-6 SSM in the rats that were never observed in the mice. Interestingly, despite statistical quantification due to low sample numbers, cow and human left ventricular biopsies demonstrated higher crest heights within the tissue than in mice or rats and proportionally less crest populations with 1 SSM to the advantage of populations with 2, 3 and a higher SSM number (Figure 2F). Thus, in the LV, the CM surface crest heights and associated SSM number depend on the mammal species.

The insertion of CM surface crests on either Z-line side of the sarcomere highly suggested that the crests could have a role on the sarcomere contractile state. Supporting this hypothesis, and based on the MET images quantification in mice and rats, we found a significant correlation between the crest height and the sarcomere length (Figure 2G). Given that the cardiac biopsies are indifferently removed in the diastolic or systolic phase of the cardiac cycle for the MET observations, the crest heights should thus be better quantified, relative to the sarcomere length, to compensate for this contingency. As shown in Figure 2H, the correction of the crest height measurements confirmed the difference that we previously described between the mouse and rat LV (Figure 2B).

CM surface crest architecture is cardiac compartment-dependent

In cell biology, the cell structure is generally dedicated to its function. Given that each heart compartment harbours different and specific functions, we questioned the potential difference in the CM surface crest architecture in between compartments. Cryo-SEM and TEM were conducted on biopsies from the right ventricle (RV) and right atrium (RA) or the appendage (Ap) from mouse, rat, cow, and human hearts, while accurate quantifications were performed in rats. Noticeably, the 3D-CM surface relief architecture was less pronounced in the RV than in the LV as illustrated in the mice and cows in TEM-SEM micrographs (Figures 3A,2A, 1A and B and Supplementary material online, Figure S4). In agreement, within the tissue, the CM crest heights were significantly lower in the rat's RV than in the LV (Figure 3B) and correlated with the presence of low number of SSM/crest (Figure 3C). In particular, we measured a significant increase in the crest populations with 1 SSM/crest (RV: $68%; LV: $46%) and we also identified an important percentage of SSM-free-crests (0 SSM; RV: 15%; LV: 4.5%). In contrast, the crest populations with 2 SSM were significantly reduced (RV: $14%; LV: $32.5%), while the crest populations above 4 SSM could not be detected compared with the LV. Similar lower crest heights were identified in the RV from cows (Supplementary material online, Figure S4A).

To confirm this in situ observation, we quantified surface crest heights using AFM on CMs isolated from the rat RV or LV. Due to the natural de-differentiation of purified adult living CMs in culture and the relatively rapid loss of crest structures in living CMs during the AFM acquisition time we previously described, [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] we analysed CMs fixed immediately after purification. Different fixators have been tested, however, only a short fixation in 2% glutaraldehyde was consistent with the overall surface crest preservation. In particular, we identified surface bulged rows as visualized in cryo-SEM tissue sections in the CMs from both the LV and RV (Figure 3D). The AFM-based quantifications of the crest height in the CMs isolated from the RV or LV were also highly consistent with the MET quantifications in tissue with significantly higher crests in the LV than in the RV (Figure 3E). However, the raw values highly differed between MET-(tissue) and AFM-(isolated CMs) quantifications. This discrepancy most likely relies on the stabilization of CM crests within the 3D-tissue, while isolated CMs rapidly derived and lost this structure ('SSM shrinking') in culture. This observation reinforces the notion that isolated adult CMs harbour different features than those within the tissue.

With respect to the different cardiac compartments, the CMs from mouse RA exhibited a flat and smooth sarcolemmal surface (Supplementary material online, Figure S4B, upper panels). Accordingly, at the subcellular level, although the surface crests could be perceived in TEM by their lateral anchorage to the Z-lines, they were free of 3D relief due to the lack of SSM. In contrast, the cow-RA constantly exhibited a 3D relief at the CM surface with 1-2 SSM/crest, thus resembling the RV (Supplementary material online, Figure S4B, lower panels). Finally, biopsies from human right appendages (RAps) displayed considerably higher crest reliefs with 6-7 SSM/crest (Supplementary material online, Figure S4C).

Overall, these results unveiled the existence of a compartmentdependence of the CM surface crest relief.

CMs establish lateral communications with neighbour CMs through crest-crest direct physical interactions

Another intriguing question was the organization of the lateral surface crest architecture between neighbour CMs. Unexpectedly, lateral crest interlocking was identified in the mouse (Figure 4A) and cow-LV biopsies (Supplementary material online, Figure S5). When screening numerous TEM micrographs in the LV from mice, cows, and humans and in contrast Figure 2 Continued number/crest in mouse LV was assessed using Spearman test. (F) Qualitative comparison of crest subpopulations according to their SSM number content in mouse, rat, cow, and human LV (n = 7 mice, 3-7 CMs/mouse, n = 5 rats, 3-10 CMs/rat, n = 2 cows, 14-16 CMs/cow, and n = 1 human, 16 CMs). (G) Linear correlation between crest height and sarcomere length in mouse or rat LV was assessed using Spearman test. (H) Crest heights measured in (B) were quantified in mouse and rat LV relative to the sarcomere length. The statistical significance between mouse and rat LV was assessed using unpaired Student's t-test (*P < 0.05).

. . . . . . . . . . . to the assumption, we identified electron dense structures, typical of tight junction between two adjacent lateral sarcolemma, thus indicating direct physical interactions between lateral nearby CMs (Figure 4B and Supplementary material online, Figure S6). Distinct crest structures could not be visualized in these pictures due to the general poor quality of the human biopsies due to deterioration (delayed fixation and no ice use). Compared with the ID, the tight junctions at the CM lateral side are scattered and intermittent. Indeed, when first depicted on a 70 nm-thick

AFM-Topography

Rat-LV Rat-RV MET micrograph, the next tight junction could be found only 40 serial micrographs ($2.8 mm) further. Tight junction structures, indicative of a direct physical communication in between cells, are generally only accepted from the ID standpoint in the CM. However, the existence of lateral CM interactions through tight junctions reconciles the presence of specific tight junctional proteins like Claudin-5 or zonula occludens-1 (ZO-1) that we and others have previously described at the CM lateral membrane. [START_REF] Genet | Ephrin-B1 is a novel specific component of the lateral membrane of the cardiomyocyte and is essential for the stability of cardiac tissue architecture cohesion[END_REF][START_REF] Sanford | Claudin-5 localizes to the lateral membranes of cardiomyocytes and is altered in utrophin/ dystrophin-deficient cardiomyopathic mice[END_REF][START_REF] Mays | Claudin-5 levels are reduced in human end-stage cardiomyopathy[END_REF][START_REF] Swager | Claudin-5 levels are reduced from multiple cell types in human failing hearts and are associated with mislocalization of ephrin-B1[END_REF] It also corroborates the presence of Ephrin-B1, a cell-cell communication protein from the Eph-Ephrin system, that we previously reported as a direct partner of Claudin-5 at the CM lateral face. [START_REF] Genet | Ephrin-B1 is a novel specific component of the lateral membrane of the cardiomyocyte and is essential for the stability of cardiac tissue architecture cohesion[END_REF] To ascertain the direct lateral physical interactions between CMs and to get a better overview of these lateral tight junctions on a larger 3D cardiac tissue image, we conducted super-resolution microscopy on mouse heart cryo-sections using SIM (Structured Illumination Microscopy) or STED (Stimulated Emission Depletion Microscopy) microscopy, allowing 100 and 70 nm xy resolution respectively when compared with the classical 250 nm resolution in confocal microscopy. Both Claudin-5 and Laminin-2, a specific basement component of the CM lateral membrane, have been examined in immunofluorescence microscopy. When first visualized through classical confocal microscopy (Supplementary material online, Figure S7A), some wavy lateral structures (yellow arrows) could be observed at the surface of CMs in the mouse myocardium, in agreement with the presence of the crests. Compared with Laminin-2, the Claudin-5 staining was punctuated, with some Claudin-5-Claudin-5 interactions in trans that could be perceived in tissue zones with interlateral space enlargement (Claudin-5 zoom).

When considering higher resolution using SIM microscopy (Figure 4C), the CM surface crests could be better appreciated and Claudin-5-Claudin-5 interactions could be clearly depicted in between two adjacent CMs. These interactions were irregular along the CM surface in a 2D image, thus reminiscing a zip. We also confirmed the presence of a continuous basement membrane layer as reflected by the Laminin-2 staining at the CM surface by opposition to the intermittent Claudin-5 expression. STED imaging further confirmed these 2D observations (Figure 4D) but allowed us to go deeper in the z resolution imaging of the 3D lateral space between two CMs. The reconstituted 3D images of a 2 mm thick tissue (Supplementary material online, Video and Figure 4E) highlighted direct interactions between two Claudin-5 in trans via their extracellular part in the inter-lateral membrane space between two adjacent CMs. The bottom view of the CM surfaces also indicated the interruption of the Laminin-2 staining at Claudin-5 contacts throughout the surface (Supplementary material online, Figure S7B). These intermittent Claudin-Claudin interactions thus create an intricate lateral meshwork at the sarcolemmal surface of the CMs.

CMs exhibit loss of surface crest architecture and lateral crest-crest interactions in cardiac diseases

We next questioned the organization of lateral crests at the CM surface in the cardiac pathophysiology.

Using TEM, we examined the CM surface crests in Ap collected from patients (Methods). As shown in Figure 5A (upper panels), the RAp from Patient 3, which demonstrated a dilated atrium but a preserved ejection fraction, partially preserved the CM surface crest relief with the presence of SSM (left panel), although some of the crests were SSM-free (presence of autophagosome) (right panel). In contrast, Patient 5 with both left atrium (LA) and left ventricular dilation (Figure 5A, left lower panels) or Patient 4 with LA dilatation and heart failure (Figure 5A, lower panels) mostly lost their CM surface crests (flat surface) with only residual atrophic (SSM shrinking) or giant SSM (SSM fusion). Thus, the loss of CM surface crests could be a general feature in cardiac pathophysiology which could entail a subsequent loss of the lateral CM contacts and a lateral space enlargement. However, given the poor biopsy sampling, the advanced disease state but also the associated medication of these patients, it was impossible to conclude on the impact of a particular parameter on the crest disruption in such multifactorial context.

To better evaluate a potential link between the pathological cardiac state and its impact on crest contacts, we conducted an experimental model of pressure overload-induced hypertrophy in mice (Transverse Aortic Constriction, TAC-model) (Figure 5B). Fifteen days after TAC, the mice developed a significant hypertrophy as shown by echocardiography (Figure 5C) and CM area quantification (Figure 5D) with no major contractile dysfunction (Figure 5C). The SIM microscopy of heart biopsies revealed some interstitial fibrosis as indicated by collagen III immunostaining in between adjacent CMs in the left ventricular myocardium (Supplementary material online, Figure S8). Correlatively, we found that in TAC mice, all the CMs partially or totally lose their surface crests compared with sham mice as indicated by the MET quantifications (Figure 5E). It is thus tempting to conclude on a role of hypertrophy on the disruption of lateral crest-crest contacts. However, it is difficult to ascertain this conclusion since TAC generally leads to concomitant hypertrophy and fibrosis development. Given that the surface crests anchored to Z-lines and that the crest-crest contacts ensure tight lateral CM-CM connections, the loss of the CM surface crests could affect the overall cardiac tissue cohesion but also the myofibril and myofiber lateral alignment (Supplementary material online, Figure S9). Consistent with this hypothesis, the cardiac tissue from TAC mice demonstrated a substantial Z-lines misalignment as indicated by the a-actinin staining as well as a myofibril and myofiber disorganization reflected by the Troponin-T imaging (Supplementary material online, Figure S10).

Discussion

We have characterized, at a high resolution, the 3D-CM surface organization within cardiac tissue. The salient findings are: (i) the compartmentand species-dependent coverage of the CM lateral surface by periodic bulged rows filled with SSM, (ii) the physical interaction of the bulged rows with those of adjacent CMs through Claudin-5-dependent tight junction strands. These results reconcile the identification of atypical proteins from cell-cell communication at the CM lateral membrane, that we and others previously reported. [START_REF] Genet | Ephrin-B1 is a novel specific component of the lateral membrane of the cardiomyocyte and is essential for the stability of cardiac tissue architecture cohesion[END_REF][START_REF] Sanford | Claudin-5 localizes to the lateral membranes of cardiomyocytes and is altered in utrophin/ dystrophin-deficient cardiomyopathic mice[END_REF][START_REF] Mays | Claudin-5 levels are reduced in human end-stage cardiomyopathy[END_REF][START_REF] Swager | Claudin-5 levels are reduced from multiple cell types in human failing hearts and are associated with mislocalization of ephrin-B1[END_REF]

Lateral crest structure-function: cardiac tissue cohesion hypothesis

The existence of spatially distinct subpopulations of mitochondria in the CM is accepted. [START_REF] Hollander | Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies[END_REF] Despite several attempts to determine the specific role of SSM, [START_REF] Hollander | Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies[END_REF] given their extreme sensitivity to cardiac tissue handling procedures as reported here or the CM isolation previously described [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] and the lack of a stringent marker to distinguish SSM from IFM, the question remains open. The existence of SSM beneath the CM lateral plasma membrane is established but was never quantified, and the incidence of such peculiar cell surface architecture at the cellular and tissue 3D-scale has not been previously explored. In contrast to the initial assumption of spherical mitochondria, 12 3D-reconstitution beam images indicated that SSM exhibit a similar elongated morphology to IFM. However, they are orientated perpendicularly to the IFM rows and lie on the outer myofibril layer. The SSM end-to-end arrangement shapes the overall lateral surface of the CM into periodic bulged rows. This particular 3D-organization of the CM surface has not been depicted in living CMs in AFM. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] In contrast, periodic crest/hollow structures were observed rather than rows. This most likely arises from a technical issue because SSM are highly prone to shrinking once the CM separated from the cardiac tissue. Thus, in isolated CMs, elongated SSM tend to shrink and adopt a more round morphology, thus leading to periodic ball structures at the CM surface as previously indicated by AFM. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] More strikingly, the discovery of lateral physical connections between adjacent CMs through surface crest-crest interaction changes our view on the spatial architecture of the cardiac tissue because to date, CM-CM direct interactions have been restricted to the ID. Thus, while the ID most likely supports the longitudinal stretch of intracellular myofibrils, the lateral stretch provided by crest-crest interactions could ensure their stacking during contraction. Indeed, the CM surface crests are directly anchored to the Z-lines of the outer myofibril layer and the lateral crest-crest contacts could thus stretch and allow the alignment of the Zlines from the overall myofibrils (Supplementary material online, Figure S9A). From a tissue standpoint, lateral CM interactions could ensure the alignment between myofibers (Supplementary material online, Figure S9B), thus providing and strengthening the overall tissue cohesion which is necessary to adapt to the physical constraints during cyclic heart beats. In agreement with this hypothesis, the loss of CM surface crests was observed in the barometric stress-mouse model. Interestingly, this could influence the compliance of the cardiac compartments during the cardiac cycle by generating a 3D meshing more or less closely dependent on the crest height. It could thus underlie the well-known higher compliance of the RV with lower crest relief than the LV with higher crests. [START_REF] Walker | The right ventricle: biologic insights and response to disease[END_REF] 4.2 Lateral crest structure-function: sarcomere relaxation control hypothesis An intriguing finding is the dependence of the relief of CM surface crest on the cardiac compartment among the different mammalian species and on the species for a compartment. CM crest relief could correlate with the compartment pressure with, for example, higher crest relief in the LV than in the RV. However, the observation of crests in compartments with almost no pressure, such as cow atria and human appendage, does not fit with this hypothesis. From the species perspective, higher crest reliefs were identified in higher mammalians (mouse < rat < cow < human) and were inversely correlated with the heart rate. In agreement, the decrease in the heart rate during postnatal cardiac maturation [START_REF] Ostchega | Resting pulse rate reference data for children, adolescents, and adults: United States, 1999-2008[END_REF] tightly correlates with SSM and cell surface crest maturation. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] At the sarcomere level, the longitudinal movements of myofibrils control both heart contraction (systole) and relaxation (diastole). In this study, we found a significant correlation between the crest height and the sarcomere length, consistent with the crest anchorage to the Z-lines and most likely arguing for a role of the crests in the control of the contractile state of the sarcomere. To date, the lateral function of the sarcomere was only attributed to the cell surface costamere connecting the extracellular barometric stress to the intracellular and compensatory myofibril contraction. [START_REF] Samarel | Costameres, focal adhesions, and cardiomyocyte mechanotransduction[END_REF] Intuitively, when considering the crest-crest lateral interactions and mechanical forces involved, they should not interfere with sarcomere contraction (Figure 6). In contrast, lateral forces generated by the crest-crest interface may restrain the sarcomere relaxation length during diastole to preserve crest-crest interactions. A mechanosensing mechanism could control this phenomenon because the CM is highly recognized as a mechanosensor machinery. [START_REF] Dostal | Mechanosensing and regulation of cardiac function[END_REF] Thus, the crest height could dictate the sarcomere relaxation state with higher crests enabling a maximal relaxation length and time (Figure 6). In such hypothetical model, crests could thus impact the isovolumic relaxation time (IVRT), the dynamic phase of the diastole. In agreement, the LV-IVRT is longer in higher mammalian species (horse: 90 ± 17 [START_REF] Verheyen | Cardiac changes in horses with atypical myopathy[END_REF] ms, human: $74 ± 7 ms, [START_REF] Nagueh | Recommendations for the evaluation of left ventricular diastolic function by echocardiography[END_REF] pig: 80 ± 15 ms 18 ) than in smaller animals (cat: 48.0 ± 6.8 ms, 19 rat: 25.5 ± 6.26 ms 20 ; mouse: 14 ± 0.6 ms [START_REF] Horgan | Murine models of diastolic dysfunction and heart failure with preserved ejection fraction[END_REF] ) and we showed that CMs from human-Ap or cow-LV myocardium harbour higher surface crest relief than those of rats or mice. The IVRT theory is also consistent with the compartment-dependence of the crest relief. Indeed, even if the existence of a dynamic phase of atrial diastole, despite recent suggestions, [START_REF] Rai | The dynamic function of the atrial diastole of the heart and motion of venous valves in humans[END_REF] is not clearly established, we found that the mouse crests in atrium < RV < LV. This also suggests that the human atrial IVRT should be substantially lower or nonexistent than the RV (53.7 ± 9.16 ms [START_REF] Elnoamany | Right ventricular myocardial isovolumic relaxation time as novel method for evaluation of pulmonary hypertension: correlation with endothelin-1 levels[END_REF] ) or LV (IVRT $74 ± 7 ms 17 ) Finally, the IVRT and thus the active diastolic control of the sarcomere could coordinate all CMs from a compartment in a similar relaxation length to synchronize the subsequent passive diastolic phase. Noticeably, the diastolic sarcomere length-crest height relationship is further reinforced by the indirect correlation between the CM surface crest height 5 and diastolic function, [START_REF] Zhou | Developmental changes in left and right ventricular diastolic filling patterns in mice[END_REF] which both concomitantly maturate within the first three weeks postnatal in rodents. In the future, animal models that specifically lack SSM and CM surface crests should help validate this hypothesis. The CM surface crest structure in animal models of diastolic dysfunction with a preserved ejection fraction and specific IVRT lengthening would also be interesting to investigate.

Disruption of the lateral crest architecture in cardiac pathologies

From a pathological standpoint, human data clearly highlight CM surface crest defects in patients with dilated cardiomyopathies in the absence of systolic dysfunction and their worsening with an altered ejection fraction. Moreover, in an experimental mouse model of hypertrophy with no major contractile function defects, we also identified a major loss of CM surface crests.

Specifically, we noted here and previously in hearts from mice with post-ischaemic myocardial dysfunction, 5 considerable SSM shrinking, SSM fusion and mitophagy events, which are all indicative of mitochondria dysfunctions. [START_REF] Ong | Mitochondrial morphology and cardiovascular disease[END_REF] Interestingly, mitochondria shrinking has long been described as a metabolic adaptation of the mitochondria to its environment. [START_REF] Packer | Metabolic and structural states of mitochondria. I. Regulation by adenosine diphosphate[END_REF] Given the dramatic changes in cardiac metabolism that occur in most cardiac diseases, SSM localized in the frontline at the CM surface could thus be affected first. Consequently, SSM shrinking could be indicative of pathological metabolic changes. Similarly, specific changes of cardiac metabolism have been described in mice depending on the anaesthesia-euthanasia procedure [START_REF] Overmyer | Impact of anesthesia and euthanasia on metabolomics of mammalian tissues: studies in a C57BL/6J mouse model[END_REF] which could support the substantial euthanasia-SSM sensitivity reported in this study. These results also confirm that the CM surface crest defects could be a more general event that occurs in heart failure as previously reported. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF][START_REF] Lyon | Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart[END_REF] It also suggests that the crest loss at the CM surface could be an initial event participating to the later development of heart failure. In line with this hypothesis, we previously demonstrated in isolated CMs from failing mouse hearts (ischaemic hearts) that the early loss of surface crests evolves over time to a substantial CM plasma membrane fragility that could lead to its ultimate disruption. [START_REF] Dague | Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure[END_REF] The loss of CM crests could thus participate to the CM death through necrosis, a specific process relying on the CM plasma membrane fragility but also a specific hallmark of patients with end-stage heart-failure. [START_REF] Silver | Cardiovascular Pathology[END_REF] Moreover, we also previously showed that such crest loss precedes T-Tubule disorganization in an ischaemic heart model, which is considered as a general hallmark of endstage heart failure from different origins. [START_REF] Ibrahim | The structure and function of cardiac t-tubules in health and disease[END_REF] Thus, given that T-Tubule network reflects intracellular invaginations of the CM lateral membrane, the initial crest-crest contact disruption and crest loss could constitute an initial common hallmark that evolves over time to the setting of heart failure. Finally, whether crest loss could specifically underlie the initial

  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sorensen's buffer at 4 C prior to inclusion and further electron microscopy analysis.

Figure 1 Figure 2

 12 Figure 1 Lateral surface of adult cardiomyocytes (CM) exhibits crest architecture in left ventricular myocardial tissue. (A and B) Scanning electron microscopy (SEM) (A) or SEM after cryo-fracture (cryo-SEM) (B) micrographs of mouse-(A) or cow-(B) left ventricular cardiac tissue show covering of CM surface by rows of elongated structures filled with SSM. (C) Dual beam-SEM and 3D-reconstruction images of CM lateral surface (over 15 mm thick) from mouse-LV cardiac tissue show elongated SSM morphologies. (D) TEM of mouse-LV shows SSM in cell surface crests anchored to lateral Z-lines of sarcomeres.

Figure 3

 3 Figure 3 CM lateral surface crest relief differs in heart compartments. (A) Cryo-SEM or TEM micrographs showing CM surface crest relief of mouse (upper panels) or rat RV myocardium (lower panels). Red arrows indicate CM surface crests. (B) Quantification of crest heights on TEM micrographs (n = 5 rats, VG/3-10 CMs/rat/237 total crests; VD/5-15 CMs/rat/239 total crests). The statistical significance between rat LV and RV was assessed using unpaired Student's t-test (**P < 0.01). (C) Quantification of crest subpopulations in rat LV and RV according to their SSM number content (n = 5 rats) (left panel). The statistical significance between rat LV and RV was assessed using two-way ANOVA followed by a Bonferroni post-test (*P < 0.05; ***P < 0.001). (Right panel) Pie chart representation of CM surface crest subpopulations between rat LV and RV. (D) Representative image of a 3D AFM-based topography height map of CM surface from rat LV or RV. (E) Quantification of crest heights on AFM-height maps (n = 3 rats, 2-4 CMs/rat). The statistical significance between rat LV and RV was assessed using unpaired Student's t-test (**P < 0.01).

Figure 4

 4 Figure 4 Neighbour CMs establish lateral physical contacts through tight junction-dependant crest-crest interactions. (A) Cryo-SEM or TEM micrographs of mouse left ventricular myocardium showing close interactions between lateral surface crests of two adjacent CMs. (B) TEM micrographs of human RAp (Patient 1) showing longitudinal electron-dense tight junctions between lateral membranes of two adjacent CMs (red arrows). (C and D) High-resolution SIM

Figure 4

 4 Figure 4 Continued (C) and STED (D) immunofluorescence imaging of Laminin-2 and Claudin-5 at the lateral surface of CMs in the cardiac tissue from mouse myocardium and highlighting cell surface crests (wavy surface) and Claudin-5/Claudin-5 interactions in trans. (E) 3D-reconstitution of STED imaging showing Claudin-5 interactions in the CM-CM lateral space and connecting the two lateral faces of neighbour CMs reflected by the Laminin-2 staining (See also Supplementary material online, movie).

Figure 5

 5 Figure 5 CM surface crest defects in cardiac diseases. (A) TEM micrographs of human Ap from hearts with various cardiac defects. (Upper panels) Dilated RAp with preserved ejection fraction (EF) (Patient 3) showing small surface crests with small SSM (yellow arrows) and autophagosomes (AP-blue arrows) in the smallest SSM-free crests. (Left lower panels) Dilated left appendage (Lap) with left ventricular dilatation with normal EF (Patient 5) showing total loss of

Figure 5 Figure 6

 56 Figure 5 Continued surface crests (red arrows) and absence of SSM or, in some cases, very small but giant elongated SSM (yellow arrows) and the loss of Z-line crest anchorage. (Right lower panel) Dilated LAp with heart failure (Patient 4) showing flat CM lateral surface (red arrows) and absence or very small SSM (yellow arrows). In all cases, the loss of lateral physical contacts between CMs is observed. (B) Experimental mouse model of barometric stress-induced cardiac hypertrophy through transaortic constriction (TAC). (C) Echocardiography-based analysis of diastolic interventricular septal thickness (IVST, d), diastolic left ventricular posterior wall thickness (LVPW, d), and EF measured 15 days after TAC. (D) (left panel) In situ quantification of CM area (Sham n = 3 mice, TAC n = 6 mice; 100-150 CMs/group). (E) The partial or total crest loss were quantified in TEM micrographs and data are expressed as percentage of the total CMs observed (Sham n = 4 mice; TAC n = 5 mice; 10 CMs/group). Statistical analysis was performed using Fisher's exact test, ***P < 0.001. (Right panels) Illustrative TEM micrographs of LV showing the partial or total loss of surface crests in the TAC model compared with sham mice. Some CM surface heaps related to the loss of the periodic lateral membrane anchorage to the Z-line and thus to the assembly of SSM from different neighbour crests were also noted.

Table 1

 1 ...................................................................................................................................................................................................................... Clinical data for human heart samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

	Patient	Gender	Age (years)	Cause of surgery	Collected tissue	LVEF (%)	Others
	1	Female	80	Coronary bypass	RAp	Normal	-
	2	Male	70	Mitral and tricuspid valves flail	LAp	Normal	Dilated RVPAH
	3	Male	68	Aortic valve stenosis	RAp	Normal	Dilated atria
	4	Female	60	Mitral valve stenosis	LAp	40%	Dilated LA
	5	Male	69	Mitral valves flail	LAp	Normal	Dilated LA and LV
	6	Male	59	Cardiac transplantation for arrhythmogenic	LV	Normal	Dilated RV
				right ventricular dysplasia
	LA, left atrium; LAp, left appendage; LV, left ventricle; LVEF, left ventricle ejection fraction; PAH, pulmonary arterial hypertension; RA, right atrium; RAp, right appendage; RV,
	right ventricle.

.

Downloaded from https://academic.oup.com/cardiovascres/article-abstract/115/6/1078/5133589 by SCD -Universite Toulouse III user on 30 August 2019

C. Guilbeau-Frugier et al. Downloaded from https://academic.oup.com/cardiovascres/article-abstract/115/6/1078/5133589 by SCD -Universite Toulouse III user on 30 August 2019

Acknowledgements

The authors thank E. Blasco and K. Ramondou for technical assistance and the ANEXPLO platform for help with echocardiography. We are also grateful to TRI Genotoul network facilities, specifically to Elodie Ve ´ga for technical assistance for SIM imaging (Cellular Imaging Facility-I2MC/TRI plateform), the Cellular Imaging Facility-CPTP/TRI platform for STED imaging, and the 'Centre de Microscopie Electronique Applique ´e a `la Biologie-Faculte ´de Me ´decine Rangueil'/TRI platform.

Funding

This work was supported by the 'Fondation Bettencourt Schueller', the 'Fondation pour la Recherche Me ´dicale' (FRM DEQ20170336733 to C.G.) and the «Fondation de France».

ventricular diastolic dysfunction based on our molecular hypothesis (Figure 6) and precede the systolic dysfunction will need future investigations.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.