Balkaran Singh Sran

Sanyog Sharma

Fabrice Pointillart

Olivier Cador

Geeta Hundal
email: geetahundal@yahoo.com

Field-induced Single Molecular Magnetism and Photoluminescence in Rare Cocrystals of Isomorphic Lanthanide (III) Coordination Compounds with Fully Substituted Pyridine-4-carboxamide Ligand

A series of four isomorphous, 1:2 (Complex :L) rare cocrystals of coordination compounds of Ln(III) ions as [Ln(L)(NO 3 ) 3 (H 2 O)] 2 [L] 2 (Ln(III) = Gd (1), Tb (2), Dy (3) and Ho (4)), were synthesized with N, N-diisobutylisonicotinamide (L) using metal-to-ligand ratio of 1:1. All compounds are dimeric in nature with two co-crystallised L molecules centro-symmetrically interspersed between two dimeric units with H bonded bridges between them to form interesting, self-assembled H-bonded tapes along the c axis. Detailed Shape analysis and Hirshfeld analysis are done to demonstrate geometry around the metal centres and various non-covalent interactions present in the systems, respectively. Magnetic studies show that compound 3 is a field-induced Single-Molecule Magnet (SMM) for which the magnetization relaxes through a combination of Orbach ( = 51 K and  0 = 3.1 x10 -7 s) and Raman mechanisms. Solid state luminescence studies reveal that compounds 1, 2 and 3 are photoluminescent in the visible range while 4 exhibits luminescence in the NIR region. Compound 3 shows cold white light emission with CIE (International Commission on Illumination) coordinates (0.31, 0.30) and CCT (Correlated Color Temperature) value of 6942 K.

Introduction:

A high impetus area, in solid-state chemistry dealing with isolation and characterization of various crystal forms of a given molecule, includes phenomena like polymorphism, isomerism and co-crystallisation [START_REF] Bernstein | Polymorphism -A Perspective Cryst[END_REF][START_REF] Aakeroy | Recent advances in crystal engineering[END_REF][START_REF] Bishop | Helical Tubuland Diols: A Synthetic and Crystal Engineering Quest Acc[END_REF][START_REF] Nangia | Supramolecular chemistry and crystal engineering[END_REF][START_REF] Habgood | Conformers, and Cocrystal Stoichiometry: Insights from the Crystal Energy Landscapes of Caffeine with the Hydroxybenzoic Acids[END_REF][START_REF] Moulton | From Molecules to Crystal Engineering: Supramolecular Isomerism and Polymorphism in Network Solids[END_REF][START_REF] Desiraju | Crystal engineering: a holistic view[END_REF][START_REF] Cruz-Cabeza | Facts and fictions about polymorphism[END_REF][START_REF] Stahly | Diversity in Single-and Multiple-Component Crystals. The Search for and Prevalence of Polymorphs and Cocrystals Cryst[END_REF][START_REF] Butterhof | Polymorphism in co-crystals: a metastable form of the ionic co-crystal 2 HBz•1 NaBz crystallised by flash evaporation[END_REF][START_REF] Clarke | Structure-Stability Relationships in Cocrystal Hydrates: Does the Promiscuity of Water Make Crystalline Hydrates the Nemesis of Crystal Engineering?[END_REF] which may impact the physicochemical properties and end up producing materials with improved performance in their applications. [START_REF] Karki | Improving Mechanical Properties of Crystalline Solids by Co-Crystal Formation: New Compressible Forms of Paracetamol[END_REF] Out of these three phenomena, co-crystallization entails the formation of a phase pure material composed of two or more compounds (molecular or ionic) in certain stoichiometric ratios, provided they are not simple salts or solvates [START_REF] Aitipamula | Salts, and Cocrystals: What's in a Name?[END_REF] and intentionally playing with positions of molecules in their crystal lattice, aiming for better products than the parent compounds. Co-crystallisation in organic compounds is well known to give better pharmaceuticals, agrochemicals, explosives and pigments [START_REF] Bolton | High Power Explosive with Good Sensitivity: A 2:1 Cocrystal of CL20: HMX[END_REF][START_REF] Sekhon | Co-crystals of agrochemical actives[END_REF][START_REF] Bucar | Advantages of mechanochemical cocrystallization in the solid-state chemistry of pigments: colortuned fluorescein cocrystals[END_REF][START_REF] Aitipamula | Trimorphs of a pharmaceutical cocrystal involving two active pharmaceutical ingredients: potential relevance to combination drugs[END_REF][START_REF] Aitipamula | Pharmaceutical Salts of Haloperidol with Some Carboxylic Acids and Artificial Sweeteners: Hydrate Formation, Polymorphism, and Physicochemical Properties[END_REF][START_REF] Banerjee | Saccharin Salts of Active Pharmaceutical Ingredients, Their Crystal Structures, and Increased Water Solubilities[END_REF][START_REF] Almarsson | Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-Crystals Represent a New Path to Improved Medicines?[END_REF] while material chemistry has reported co-crystallized materials with enhanced magnetism, luminescence and mechanical strength. [START_REF] Karki | Improving Mechanical Properties of Crystalline Solids by Co-Crystal Formation: New Compressible Forms of Paracetamol[END_REF][START_REF] Yan | A Co-Crystal Strategy to Tune the Luminescent Properties of Stilbene-Type Organic Solid-State Materials[END_REF][START_REF] Sonoda | Fluorinated Diphenylpolyenes: Crystal Structures and Emission Properties[END_REF][START_REF] Hori | Luminescence from π-π Stacked Bipyridines through Arene-Perfluoroarene Interactions[END_REF] Growth in organic cocrystals has been exponential owing to the similar crystallization kinetics and molecular structures of the components, connected through various non-covalent interactions. [START_REF] Allen | The Cambridge Structural Database: a Quarter of a Million Crystal Structures and Rising[END_REF][START_REF] Devogelaer | Cocrystals in the Cambridge Structural Database: a network approach[END_REF] However, such reports are rather limited in the case of coordination compounds [START_REF] Wang | Tuning the Lead Complexes Based on a Double 1,10-Phenanthroline Derivative with Versatile Coordination BehaviorbyDicarboxylates: from 0D Nano-Ring to an Unprecedented 0D + 3DCocrystal[END_REF][START_REF] Mukherjee | Ni 2 ), (Ni 3 ), and (Ni 2 + Ni 3 ): A Unique Example of Isolated and Cocrystallized Ni 2 and Ni 3 Complexes[END_REF][START_REF] Golbedaghi | II) Complexes of Three [2+2] Macrocyclic Schiff Base Ligands. Synthesis and X-ray Crystal Structure of the First Binuclear-binuclear Co-Crystal[END_REF][START_REF] Evers | Cocrystallization of Photosensitive Energetic Copper(II) PerchlorateComplexes with the Nitrogen-rich Ligand 1,2-Di(1H-tetrazol-5-yl)ethane[END_REF] as different coordination modes of the precursor metal complexes result in different lattice packing forces and hence dissimilar crystallization kinetics. Lesser yet are the reports of cocrystals of a coordination complex of a ligand along with the free ligand. Gao et al., reported [START_REF] Gao | Crystal Structures, Photoluminescence, and Magnetism of TwonNovel Transition-Metal Complex Cocrystals with Three-Dimensional H-Bonding Organic Framework or Alternating Noncovalent Anionic and Cationic Layers[END_REF] two, multi-component cocrystals of coordination complexes of Cu(II) and Zn (II) with the corresponding uncoordinated neutral ligand and anions. However, such cocrystallization with Ln(III) as metal ions finds only one report so far [START_REF] Chen | Dynamic Magnetic and Optical Insight into a High Performance Pentagonal Bipyramidal DyIII Single-Ion Magnet[END_REF] and is thus a very rare phenomenon, to the best of our knowledge and belief.

The interest in lanthanide (III) complexes for the past two decades emanates from their exceptional potential in the fields of luminescence and molecular magnets owing to their typical electronic structure. They are the most sought after metal ions in the pursuit of single-molecule magnets (SMM) due to their high spin values and magnetic anisotropy [START_REF] Rinehart | Exploiting single-ion anisotropy in the design of felement single-molecule magnets[END_REF] while wide range intense, narrow and long-lived emission bands of their complexes more than qualify them as the new age luminescent materials. More recently the combination of these two properties to produce multifunctional opto-magnetic Ln(III) complexes is the new craze for research in this area. [START_REF] Pointillart | Uncommon lanthanide ions in purely 4f Single-Molecule Magnets[END_REF][START_REF] Long | Recent advances in luminescent lanthanide based Single-Molecule Magnets[END_REF][START_REF] Wen | Multifunctional Lanthanide Complexes Based on Tetraazacyclolamidophenol Ligand with Field-Induced Slow Magnetic Relaxation, Luminescent and SHG Properties[END_REF][START_REF] Brunet | Exploring the dual functionality of an ytterbium complex for luminescence thermometry and slow magnetic relaxation[END_REF][START_REF] Chen | L A Piezochromic Dysprosium(III) Single-Molecule Magnet Based on an Aggregation-Induced-Emission-Active Tetraphenylethene Derivative Ligand[END_REF][START_REF] Shintoyo | Crystal Field Splitting of the Ground State of Terbium(III) and Dysprosium(III) Complexes with a Triimidazolyl Tripod Ligand and an Acetate Determined by Magnetic Analysis and Luminescence[END_REF][START_REF] Errulat | A Luminescent Thermometer Exhibiting Slow Relaxation of the Magnetization: Toward Self-Monitored Building Blocks for Next-Generation Optomagnetic Devices[END_REF][START_REF] Ren | Lanthanide phosphonates with pseudo-D 5h local symmetry exhibiting magnetic and luminescence bifunctional properties[END_REF][START_REF] Cucinotta | Magnetic Anisotropy in a Dysprosium/DOTA Single-Molecule Magnet: Beyond Simple Magneto-Structural Correlations[END_REF][START_REF] Pointillart | A Series of Tetrathiafulvalene-Based Lanthanide Complexes Displaying Either Single-Molecule Magnet or Luminescence. Direct Magnetic and Photo-Physical Correlations in the Ytterbium Analogue[END_REF][START_REF] Liu | Near-IR Luminescence and Field-Induced Single Molecule Magnet of Four Salen-type Ytterbium Complexes[END_REF][START_REF] Gálico | Triplet-State Position and Crystal-Field Tuning in Opto-Magnetic Lanthanide Complexes: Two Sides of the Same Coin[END_REF][START_REF] Pointillart | Luminescence and Single-Molecule Magnet Behavior in Lanthanide Complexes Involving a Tetrathiafulvalene-Fused Dipyridophenazine Ligand[END_REF] For designing of photoluminescent SMM complexes the choice of the metal ion is the most crucial in terms of the kind of anisotropy in its electron density (prolate or oblate) followed by its emitting state and sensitizing mechanism. The latter depends wholly on the choice of ligand that could both sensitize the ion as well as 2 A c c e p t e d m a n u s c r i p t produce a ligand field further augmenting the anisotropy of the metal ion. The relevant literature shows that for opto-magnetic materials Dy(III) hits the charts followed by Yb(III), Tb(III) and Er(III) essentially in that order [START_REF] Perfetti | Chapter 14: Luminescent Molecular Magnets, Molecular Magnetic Materials: Concepts and Application[END_REF][START_REF] Marin | Shining new light on multifunctional lanthanide single-molecule magnets[END_REF] due to its strong magnetic anisotropy [START_REF] Rinehart | Exploiting single-ion anisotropy in the design of felement single-molecule magnets[END_REF] and possibility of white-light emission. [START_REF] Chorazy | White Light Emissive Dy III Single-Molecule Magnets Sensitized by Diamagnetic [Co III (CN) 6 ] 3-Linkers[END_REF][START_REF] Guo | A zigzag Dy III 4 cluster exhibiting single-molecule magnet, ferroelectric and white-light emitting properties[END_REF][START_REF] Wang | Achieving white light emission and increased magnetic anisotropy by transition metal substitution in functional materials based on dinuclear Dy III (4-pyridone)[M III (CN) 6 ]3-(M = Co, Rh) molecules[END_REF] In the present studies, we are reporting crystal structures of a series of four isomorphous and iso-structural coordination complexes [Ln(L)(NO 3 ) 3 (H 2 O)] 2 [L] 2 (Ln(III) = Gd (1), Tb (2), Dy (3) and Ho (4)), where L is N, N-diisobutylisonicotinamide (Scheme 1), all of which have crystallized as rare cases (vide supra) of 1:2 co-crystals of a dimeric complex unit and uncoordinated ligands L. The presence of both coordinated and cocrystallised free ligands creates a possibility of enhanced antenna effect, both 'through bond' and 'through space' sensitization mechanisms. The dimers and the free ligands are arranged in linear tapes, extending into H-bonded 2D networks. All the complexes have been studied

for their magnetic and emission properties in the pursuit of opto-magnetic materials. L has evidently shown some antenna effect to produce sharp emission spectra for Dy(III) and Tb(III) and Ho(III) complex but not for Gd(III) complex where ligand-based emission dominates the spectrum. Dy(III) complex shows field-induced SMM behaviour with an effective barrier 51 K and also is a white light emitter with Commission Internationale de l'Eclairage (CIE) chromaticity coordinates 0.310, 0.300.

Procedure for synthesis of complexes:

A c c e p t e d m a n u s c r i p t Methods: Details about Instrumentation, X-ray data collection and structure solutions, Hirshfeld analysis, colour purity calculations can be found in the supporting information. A summary of the crystal data is given in Table 1.

Result and Discussion

Crystal and molecular structure of compounds:

As all compounds are isomorphous and isostructural therefore the detailed description of only 1 has been given below and the molecular structures of (2-4) and their important bond distances and bond angles are given in the supplementary information (ESI, Table S1, Figure S1-S3). A unit cell packing for complex 1 is shown in ESI Figure S7. Two co-crystallised L molecules are centro-symmetrically interspersed between two successive dimeric units forming a bridge between them via H bonding through their N6 (pyridine) and O11 (amide) atoms (Figure 2). Each dimeric unit is involved in this H-bonding using its water molecule, O1W which acts as a double H-bond donor to N6 (Py) and O11 (amide) of the free L molecules, forming H-bonded tape along the c axis (Figure 2, Table S5 for H-bonding distances and symmetries). This tape is further supplemented by C-H...O interactions between nitrate group and methylene protons of the free ligands. Further, these tapes are extended to a 2D network in the bc plane as these dimeric units are connected to each other Figure 4a shows the self-assembly of two centrosymmetric, co-crystallized ligands between two dimers, along the c axis which gives a robust crystal structure. The absence of these cocrystallised L molecules generates voids between two dimeric units, which are being efficiently filled by these two L molecules (Figure 4b). These free L molecules seem to have stabilized the crystal structure in the ratio M:L 1:2 despite the fact that the reactions were run in the M:L ratio of 1:1. IR spectroscopy: Compound 1 shows a strong band at 1595 cm -1 which describes that amide oxygen is participating in coordination. [START_REF] Lumb | Coordination chemistry of Cu(II), Co(II), Zn(II) and Ag(I) complexes of isomeric pyridine 2-and 4carboxamides and their biological activity evaluation[END_REF][START_REF] Nakamoto | Infrared and Raman Spectra of Inorganic and Coordination Compounds[END_REF] Presence of broad band for ν OH at 3400 cm -1 in 1

[Gd(L)(NO 3 )(H 2 O)] 2 [L 2 ]( 1
shows coordinating water. The ν C-N (amide) and ν C-H stretching bands are assigned to 1476 cm -1 and 2979, cm -1 , for 1. Three bands at 1423 cm -1 , 1281 cm -1 and 1016 cm -1 appear due to asymmetric NO 2 , symmetric NO 2 and N-O stretching modes of coordinating nitrate group (FigureS7). [START_REF] Gatehouse | Infrared Spectra of Some Nitratoco-ordination Complexes[END_REF] Similar bands have been observed for other compounds (2-4) and are given in the supplementary material (ESI, Figure S8-11, Table S3).

Powder X-ray Diffraction studies

To confirm the phase purity of the bulk material correspond to the single crystal structure of complexes (1-4), the powder X-ray data were collected at room temperature. The

Magnetic Properties

Static magnetic measurements: Static magnetic measurements were performed for all compounds (1-4) in temperature range 2-300K are shown in Figure 7. For compound 1  M T is equal to 15.9 cm 3 K mol -1 at room temperature which is in good agreement with the expected value (15.8 cm 3 K mol -1 , 8 S 7/2 , S = 7/2, L = 0, g = 2) for two uncoupled Gd(III) ions. [START_REF] Kahn | Molecular Magnetism[END_REF] Towards lower temperatures the value remains constant up to 20 K after which it decreases abruptly to 14.3 cm 3 K mol -1 at 2 K. For compound 2  M T is equal to 23.5 cm 3 K mol -1 at room temperature which is in good agreement with the expected value (23.6 cm 3 K mol -1 , 7 F 6 , S = 3, L = 3, J = 6, g = 3/2) for two uncoupled Tb(III) ions. [START_REF] Kahn | Molecular Magnetism[END_REF] On lowering the temperature it decreases steadily up to 100 K after which there is an abrupt decrease to a value of 16.9 cm 3 K mol -1 at 2 K. [START_REF] Kahn | Molecular Magnetism[END_REF] For compound 3, room temperature  M T is 28.2 cm 3 mol -1 K which is in good agreement with the expected value (28.3 cm 3 K mol -1 , 6 H 15/2 , S = 5/2, L = 5, J = 15/2, g = 4/3) for two uncoupled Dy(III) ions.  M T remains constant down to 150 K while cooling and then starts decreasing to a minimum of 17.3 cm 3 K mol -1 at 2 K. For compound 4,  M T values are measured as 27.8 cm 3 mol -1 K which is close to the expected value (28.1 cm 3 K mol -1 , 5 I 8 , S = 2, L = 6, J = 8, g = 5/4) for two isolated Ho (III) ions. [START_REF] Kahn | Molecular Magnetism[END_REF] The compound also shows abrupt decrease below 100K and gives a value of 9.0 cm 3 K mol -1 at 2 K. For all the compounds, the decrease of the  M T can be attributed to the thermal depopulation of the m J levels and intermolecular antiferromagnetic dipolar interaction while antiferromagnetic exchange interactions are excluded, analysing the exchange pathway between the spin carriers. At 2 K, the field dependence of the magnetization shows a classical behaviour with values at 50 kOe (ESI, Figure S28-31) far away from the expected saturation values in the multiplet ground state framework. This is due to the presence of magnetic anisotropy and/or low-lying excited states. [START_REF] Jeletic | M An Organometallic Sandwich Lanthanide Single-Ion Magnet with an Unusual Multiple Relaxation Mechanism[END_REF] A c c e p t e d m a n u s c r i p t S4, ). [START_REF] Dekker | Activated dynamics in a two-dimensional Ising spin glass: Rb 2 Cu 1 -xCo x F 4[END_REF][START_REF] Cole | Dispersion and Absorption in Dielectrics I. Alternating Current Characteristics[END_REF] Relaxation time distribution parameter, α is large (0.33-0.52) in the low-temperature range (2-3.0 K) which suggests multiple relaxation processes occurring in this temperature range. This may be due to the presence of dipolar interactions in the lower temperature region. [START_REF] Morita | Near-infrared luminescence and SMM behaviors of a family of dinuclear lanthanide 8-quinolinolate complexes[END_REF] Further, we fitted these relaxation times to the following equation 2 to find the relaxation pathway.

 -1 = 0 -1 exp(-/T)+ CT n ----------2 13
A c c e p t e d m a n u s c r i p t

From the data fitting it is found that best parameters are obtained by Raman + Orbach pathway (Figure 9) for compound 3 which gives us an activation energy barrier around 35.3(2) cm -1 (51 K) and relaxation time 3.1(1)x10 -7 s that falls well within the limit of a good SMM [START_REF] Jia | Luminescent singlemolecule magnets based on lanthanides: Design strategies, recent advances and magneto-luminescent studies[END_REF] in a temperature range of 2-6 K. Constant value obtained for Raman process is C=1.034(2) and n=4.92(2) (where C and n are the Raman constant and exponential, respectively) which is greater than 4 and falls in the expected criteria for Raman relaxation pathway. [START_REF] Zadrozny | Slow magnetization dynamics in a series of two coordinate iron(II) complexes[END_REF] The absence of slow relaxation in compounds 2 and 4 can be explained on the basis of the fact that both of them (Tb(III) and Ho(III)) are non-Kramers ions. As it is well known that for a Kramers ion like Dy(III), there is always a bistable ground state, which has a necessary contribution to SMM behaviour, the lowest energy doublet has high |M J | and the complexity of the relaxation phenomena is related to the number of relaxation paths available. The latter could be anyone or all of the following i.e. reversal of magnetization in the lowest energy doublet via QTM, thermal mechanism via an excited state or quantum tunnelling of magnetization within an exciting doublet which is thermally activated. Comparing with Dy(III) ion, the Tb(III) and Ho(III) are non-Kramers ions, so their respective complexes will possess a bistable ground state only if they are presented in a highly axial symmetric ligand field. [START_REF] Mandal | Anion-driven structures and SMM behavior of dinuclear terbium and ytterbium complexes[END_REF][START_REF] Harriman | Magnetic Axiality: Design Principles from Molecules to Materials[END_REF] As the coordination geometry is the same in all the complexes being reported here (vide supra) therefore the ligand field shows same amount of distortion from spherical symmetry. This distortion can support an oblate ion to accentuate its magnetic anisotropy, albeit to a limited extent (due to the donor atom at the capping position). Therefore all three complexes (2-4) of oblate ions should show SMM properties but the absence of any such behaviour for 2 and 4 may be due to them being non-Kramers ions, with crystal field not being sufficiently axial. properties in the visible region and compound 4 exhibits luminescence properties in the NIR region. For Compound 1, upon excitation at 382 nm, its emission spectrum gives broadband around 509 nm (19646 cm -1 ) (Figure 10a), which has been assigned as an intra-ligand transition. This is done due to the fact that the free ligand (Figure S40, ESI) also shows a broad band in the same range. It is well known that for Gd(III) ion the energy gap between the ground state ( 8 S 7/2 ) and the excited state ( 6 P 7/2 ) is very large ≈ 32000 cm -1 which makes energy transfer impossible from the triplet state of ligand to the excited state of the metal ion. [START_REF] Jiménez | Silicon-induced UV Transparency in Phosphate Glasses and its Application to the Enhancement of the UV Type B Emission of Gd 3+[END_REF] For compound 2, peaks are clearly visible at 489 nm (20449 cm -1 ), 544 nm (18382 cm - 1 ), 583 nm (17152 cm -1 ), 622 nm (16077 cm -1 ) including less intense peaks at 650 nm (15384 cm -1 ), 668 nm(14970 cm -1 ), 679 nm (14727 cm -1 ), upon excitation wavelength 365 nm (Figure 10b). These correspond to [START_REF] Habgood | Conformers, and Cocrystal Stoichiometry: Insights from the Crystal Energy Landscapes of Caffeine with the Hydroxybenzoic Acids[END_REF] 10c). Compound 4, on excitation at 395 nm, shows emission at 970 nm (10309 cm -1 ) in the NIR region corresponding to 5 F 5  5 I 7 transitions (Figure 10d). Excitation spectra for compound 1-4 are given in ESI Figure S37. "Compound 2-4 show luminescence peaks as expected in compounds due to the successful energy transfer to the excited states of the lanthanides. [START_REF] Jia | Luminescent singlemolecule magnets based on lanthanides: Design strategies, recent advances and magneto-luminescent studies[END_REF] The excitation spectra (Figure S41)

suggests that both antenna effect from the organic ligand and direct f-f excitations are involved in the sensitization mechanisms. From the luminescence data it is expected that triplet state energy of ligand L will lie (2500-4500 cm -1 ) higher than the 5 D 4 (20449 cm -1 ), 4 F 9/2 (20746 cm -1 ) excitation state energies in Tb(III) and Dy(III), respectively and much lower than 6 P 7/2 (32000 cm -1 ) excitation level of Gd(III). [START_REF] Jiménez | Silicon-induced UV Transparency in Phosphate Glasses and its Application to the Enhancement of the UV Type B Emission of Gd 3+[END_REF] It is found that for compound 3 CIE coordinates (0.31, 0.30) lie close to NTSC standard value for white light (0.310, 0.316). [START_REF] Manzur | Tuning White Light Emission in Dinuclear Phenoxo Bridged Dy(III) Complexes[END_REF] Recent literature reports show that Dy(III) compounds are capable of producing white light as one component. [START_REF] Manzur | Tuning White Light Emission in Dinuclear Phenoxo Bridged Dy(III) Complexes[END_REF] In terms of colour purity compound 3 displays very low colour purity which is essential for good white light emitter. [START_REF] Alexander | Novel white light emitting dysprosium oxalate nanocrystals: Competing luminescence quenching by the features of Dy-Oxalate layered structure[END_REF] In general, the accepted CCT values should be greater than (> 5000 K) and (< 5000 K), for cold and warm light emission, respectively [START_REF] Singh | Investigation of structural, spectral and photometric properties of CaTiO 3 :Dy 3+ nanophosphors for the lighting applications Electron[END_REF] The CCT value observed for 3 is 6942 K hence it may serve as a cold, white light emitter in lighting applications. 73 (2-4) display enhanced photoluminescence of the metal ions owing to the efficient antenna effect of the L organic chromophore while 1 shows only L centered luminescence. The Dy(III) complex exhibits good opto-magnetic properties and is a promising multi-functional material. The complex gives cold-white light emission with CIE coordinates (0.31, 0.30) and CRT value around 6942 K which can be used for light-emitting material. The synthetic strategy using co-crystallization approach might be an efficient approach to enhance both luminescent and magnetic properties by i) increasing the amount of organic ligand in the crystal lattice or/and allowing through bond and through space energy transfer to the lanthanide ion and ii) by playing the role of spacer to provide efficient magnetic isolation to the spin carriers.

Scheme 1 .

 1 Scheme 1. Showing coordination mode of ligand (μ 2 -κ 2 , η 1 :η 1 )

  ):The asymmetric unit (ESI, FigureS4) of 1 shows a Gd(III) ion, coordinated by a bidentate, bridging ligand L, three nitrate ions, one water molecule and a non-coordinating L co-crystallized along with the complex. In the molecular structure of the dimeric compound 1 (Figure1) Gadolinium centres are separated from one another by a distance equal to 8.458 Å. Both Gd(III) metal ions are nine-coordinated with the ligand having a μ 2 -κ 2 , η 1 :η 1 mode (scheme 1) with Gd-N(Py), 2.611(4) Å and Gd-O(amide), 2.301(4) Å. Six positions around the metal ion are occupied by three bidentate chelating nitrate ions (av. Gd-O (nitrate) distance, 2.460 Å) and one remaining position is occupied by a water molecule (Gd-O, 2.344(5)Å). The arrangement around the metal centre leads to a coordination sphere with a spherical capped square antiprism (C 4v ), symmetry. (The deviation from ideal symmetries has been determined by SHAPE analysis[START_REF] Llunell | SHAPE Program for the Stereochemical Analysis of Molecular Fragments by Means of Continuous Shape Measures and Associated Tools[END_REF] , ESI, FigureS5TableS2).

A c c e p t e d m a n u s c r i p tFigure 1 .

 1 Figure 1. Showing the molecular structure of complex 1 along with the co-crystallized ligand.

6

 6

  A c c e p t e d m a n u s c r i p t

Figure 2 .

 2 Figure 2. Showing the hydrogen bonding of the dimeric unit with cocrystallized ligand along the c axis forming 1D chains.

Figure 3 .

 3 Figure 3. Showing the hydrogen bonding between dimeric units along b axis.

7 AFigure 4 .

 74 Figure 4. (a) Showing 1D representation of crystal packing along the c axis (cocrystalising L are shown in blue color (b) space-filling representation with and without free L along the c axis.

A c c e p t e d m a n u s c r i p t

  Thermogravimetric analysisThermogravimetric analysis was performed to check thermal stability of compounds (1-4) (FigureS1-S4). All complexes follow the same trend of decomposition, losing two coordinated water molecules up to 150 o C, endothermically. All L molecules (coordinating and free) are decomposed in the temperature range of 200-300°C, exothermally. Further they decomposition coordinating nitrates takes place up to 500 o C, leaving behind the metal skeleton residues after 700 o C. The detailed analysis for each compound is available in the ESI (Figure S12-S15).

  FigureS20) to N(pyridine) and O(amide) of the free L molecules. Similarly, this surface is connected with other dimeric units through hydrogen bonding from oxygens of nitrate group (color light red). In case(II), N(pyridine) and O(amide) from the free L are the potential interaction points (Figure5b, red spots) which are connected via water molecules to two different dimeric units holding them perfectly in a linear tape (ESI, FigureS21).

A c c e p t e d m a n u s c r i p tFigure 5 .

 5 Figure 5. d norm surfaces showing (a) Case I-O(Nitro)⋯H, H(water)⋯O(amide) and H(water)⋯N(pyridine) between the adjacent molecules for the dimeric unit (b) case II Showing the O(amide)⋯H, N(pyridine)⋯H interactions between the adjacent molecules for L. Red spots represent the strong point of interaction on the d norm surface.

Figure 6 .

 6 Figure 6. Showing 2D fingerprint plots for (a) case I (b) case II (c) case III

Figure 7 .

 7 Figure 7. Showing thermal dependence of  M T for 1 (Red), 2 (purple), 3 (Green) and 4 (blue).

A c c e p t e d m a n u s c r i p tFigure 8 .

 8 Figure 8. Frequency dependence of the out-of-phase (up) and in-phase (below) components of the ac magnetic susceptibility under applied field 2 kOe for compound 3.

A c c e p t e d m a n u s c r i p tFigure 9 .

 9 Figure 9. Temperature dependence of relaxation time log τ (black circles) with the best fitting curve (Red solid line) obtained by a combination of Raman and Orbach mechanisms with the parameters give in the text.

Figure 10 .

 10 Figure 10. Room-temperature emission spectra of (a) 1 at  em (nm) = 509 ( ex = 382 nm) (b) 2 at  em (nm) = 489 , 544, 583, 622 , Inset-less intense peaks at 650 nm, 668 nm, 679 nm ( ex = 365 nm) (c) 3 at  em (nm) =482 , 573, 668 ( ex = 360 nm) (d) 4 at  em (nm) = 970 ( ex = 395 nm).

16

 16

A c c e p t e d m a n u s c r i p t

  A schematic diagram representing the observed transitions and images for solid-state luminescence images under 365nm UV lamp for compound 1-3 are shown in Figure 11. Solid-state UV absorbance spectra for the ligand is shown in ESI Figure S42.

Figure 11 . 68 for 1 - 3 3 A c c e p t e d m a n u s c r i p tFigure 12 .

 116813312 Figure 11. Schematic representation of energy level (Jablonski, Tailbones) diagram of the energy transfer process involved in the luminescence of compounds 1-4. (Inset) Solid State luminescence for compounds 1-3 under 365nm UV lamp.

A c c e p t e d m a n u s c r i p tConclusions

  Four isomorphous and iso-structural complexes of L with Ln(III) having co-crystals with the free ligand L are being reported for the first time in the case of lanthanides. The selfassembly of two-component co-crystals is stabilized due to strong H-bonding interactions between them. Dynamic magnetic studies reveal that compound 3 is a field-induced SMM with an effective energy barrier around 51 K and with 3.1 x 10 -7 s relaxation time. Complexes

u s c r i p t 73 .

 73 Adhikari, G. C.; Thapa, S.; Zhu, H.; Grigoriev, A.; Zhu, P. Synthesis of CsPbBr 3 and Transformation into Cs 4 PbBr 6 Crystals for White Light Emission with High CRI and Tunable CCT. J. Phys. Chem. C. 2019, 123, 12023-12028. For TOC only A series of four isomorphous, 1:2 (Complex :L) cocrystals of dimeric coordination compounds of Ln(III) ions as [Ln(L)(NO 3 ) 3 (H 2 O)] 2 [L] 2 (Ln(III) = Gd (1), Tb (2), Dy (3) and Ho (4)), were synthesized with N, N-diisobutylisonicotinamide (L) along with two cocrystallised L molecules centrosymmetrically. Compound 3 turns out to be a promising optomagnetic material by being a field-induced Single-Molecule Magnet (SMM) ( = 51 K) and shows cold white light emission with CCT value of 6942 K.

27

 27

A c c e p t e d m a n u s c r i p t

NO 3 ) 3 (H 2 O)] 2 [L] 2 (1):

  Gd(NO 3 ) 3 •6H 2 O (68 mg, 0.2 mmol) in 20.0 mL of acetonitrile and ligand (L) (66mg, 0.2mmol) in 20.0 mL of methanol, were mixed together and refluxed with continuous stirring overnight, in a 50 mL round bottom flask. A clear solution was filtered out and its volume reduced to half by heating and then kept for slow evaporation. A

	viscous liquid appeared after a week. After 2 weeks crystals appeared in the viscous liquid.
	Crystals are separated from the liquid and washed with few drops of ice-cold methanol. Pale
	white block shaped crystals were obtained in 38% yield. IR (υ max /cm -1 ): 1595, 1281, 2967,
	Anal. Found: C, 40.36; H, 5.32; N, 11.17. Calc. for Complex 1: C, 40.13; H, 5.15; N, 11.22%.
	[Tb(

[Gd(L)(

L)(NO 3 ) 3 (H 2 O)] 2 [L] 2 (2): Compound 2 was

	synthesized in a similar way to 1, except that
	Gd(NO 3 ) 3 •6H 2 O was replaced by Tb(NO 3 ) 3 •6H 2 O (68 mg, 0.2 mmol). Pale white, block
	shaped crystals of 2 were obtained in 32% yield.IR (υ max /cm -1 ): 1595, 1284, 2974 .Anal.
	Found: C, 40.52; H, 5.59; N, 11.81. Calc. for Compound 2: C, 40.32; H, 5.68; N, 11.63%.

[Dy(L)(NO 3 ) 3 (H 2 O)] 2 [L] 2 (3): Compound 3 was

	synthesized in a similar way to 1, except that
	Gd(NO 3 ) 3 •6H 2 O was replaced by Dy(NO 3 ) 3 •6H 2 O ( 86 mg, 0.2 mmol). Pale white, block
	shaped crystals of 3 were obtained in 31% yield. (IR (υ max /cm -1 ): 1591, 1277, 2964, Anal.
	Found: C, 40.44; H, 5.58; N, 11.79. Calc. for Compound 3: C, 40.61; H, 5.51; N, 11.67%.

[Ho(L)(NO 3 ) 3 (H 2 O)] 2 [L] 2 (4): Compound 4 was

	synthesized in a similar way to 1, except that
	Gd(NO 3 ) 3 •6H 2 O was replaced by Ho(NO 3 ) 3 •6H 2 O (88 mg, 0.2 mmol). light orange, block
	shaped crystals of 4 were obtained in 36% yield. IR (υ max /cm -1 ): 1587, 1281, 2978, Anal.
	Found: C, 40.15; H, 5.54; N, 11.71. Calc. for Compound 4: C, 40.35; H, 5.61; N, 11.93%.

Table 1 .

 1 Crystallographic Data for compounds(1-4)

	Identification code	Compound 1	Compound 2	Compound 3	Compound 4
	Empirical formula	C56H92Gd2N14O24	C56H92Tb2N14O24	C56H92Dy2N14O24	C56H92Ho2N14O24
	Formula weight	1659.93	1663.27	1670.43	1675.29
	T(K)	150(2)	150(2)	150(2)	150(2)
	Crystal system	Monoclinic	Monoclinic	Monoclinic	Monoclinic
	Space group	P 21/c	P 21/c	P 21/c	P 21/c
	Absorption coefficient (mm -1 )	1.759	2.049	1.988	2.113
	Crystal Size (mm 3 )	0.22 x 0.18 x	0.19 x 0.16 x 0.12	0.18 x 0.16x 0.12	0.22 x 0.18 x 0.14
	a (Å)	0.16	13.433(2)	13.785(2)	13.758(2)
	b (Å)	13.926(5)	9.276(6)	9.5764(17)	9.5642(16)
	c (Å)	9.559(3)	28.901(2)	29.667(5)	29.606(5)

  D 4  4 F 6 , 5 D 4  4 F 5 , 5 D 4  4 F 4 , 5 D 4  4 F 3 and less intense (Figure 10b (inset)) 5 D 4  4 F 2 , 5 D 4  4 F 1 , 5 D 4  4 F 0 . For compound 3, excitation wavelength is -1 ), corresponding to 4 F 9/2  6 H 15/2 (blue band), 4 F 9/2  6 H 13/2 (Yellow band), 4 F 9/2  6 H 11/2 (Red Band) (Figure

	m a n u s c r i p t
	A c c e p t e d
	360 nm, which gives emission at 482 nm (20746 cm -1 ), 573 nm (17452 cm -1 ), 668 nm (14970

cm

Table 2 :

 2 Representing CIE coordinates, CCT and % purities for compounds 1-3.

	Compound	CIE x	CIE y	CCT	Purity %
	1	0.30	0.54	6164	53.5
	2	0.26	0.53	7021	41.8
	3	0.31	0.30	6942	11.6

Acknowledgement: BSS is thankful to UGC-BSR for a research fellowship and IFCPAR/CEFIPRA for Raman-Charpak fellowship, GH is thankful to CSIR (Research grant No. 01(2971)/19/EMR-II) for financial support. G.H. thanks the DST (Department of Science and Technology, India) for providing facilities under FIST and PURSE programs and the UGC (University Grants Commission) under CAS-II and UPE programs. SS is thankful to the CSIR for the fellowship of Research Associate. This work was supported by CNRS, Université de Rennes 1 and the European Commission through the ERC-CoG 725184 MULTIPROSMM (project n. 725184).


Delphine Mika

Pierre Bobin

Martine Pomérance

Patrick Lechêne

Ruth Westenbroek

William A Catterall

Grégoire Vandecasteele
email: gregoire.vandecasteele@u-psud.fr

Jérôme Leroy

Rodolphe Fischmeister
email: rodolphe.fischmeister@inserm.fr

Differential regulation of cardiac excitation-contraction coupling by cAMP phosphodiesterase subtypes

Keywords:

Aims

Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2-4) in cardiac excitation-contraction coupling (ECC).

Methods

Sarcomere shortening and Ca 2+ transients were recorded simultaneously in and results adult rat ventricular myocytes and ECC protein phosphorylation by PKA was determined by western blot analysis. Under basal conditions, selective inhibition of PDE2 or PDE3 induced a small but significant increase in Ca 2+ transients, sarcomere shortening, and troponin I phosphorylation whereas PDE4 inhibition had no effect. PDE3 inhibition, but not PDE2 or PDE4, increased phospholamban phosphorylation. Inhibition of either PDE2, 3 or 4 increased phosphorylation of the myosin binding protein C, but neither had an effect on Ltype Ca 2+ channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared to individual PDE inhibition but the most potent combination was obtained when inhibiting simultaneously PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal βadrenergic receptor stimulation increased ECC and this effect was potentiated by individual PDE inhibition with the rank order of potency PDE4=PDE3>PDE2. Identical results were obtained on ECC protein phosphorylation. Conclusions Our results demonstrate that PDE2, PDE3 and PDE4 differentially regulate ECC in adult cardiomyocytes. PDE2 and PDE3 play a more prominent role than PDE4 in regulating basal cardiac contraction and Ca 2+ transients. However, PDE4 becomes determinant when cAMP levels are elevated, for instance upon β-adrenergic stimulation or PDE3 inhibition.

Introduction

The cAMP pathway is critical for autonomic regulation of the heart. Sympathetic stimulation increases myocardial contractility mainly through stimulation of β-adrenergic receptors (β-ARs), elevation of intracellular cAMP ([cAMP]i) and activation of the cAMP-dependent protein kinase (PKA). PKA in turn phosphorylates key components of cardiac excitationcontraction coupling (ECC) such as the L-type Ca 2+ channel (Cav1.2), ryanodine receptor type 2 (RyR2), phospholamban (PLB), troponin I (TnI) and myosin-binding protein C (MyBP-C).

The phosphorylation of Cav1.2 and RyR2 leads to enhanced Ca 2+ influx and sarcoplasmic reticulum (SR) Ca 2+ release, contributing to enhanced Ca 2+ transients. PLB phosphorylation increases SR Ca 2+ uptake by the Ca 2+ -ATPase (SERCA2), thus accelerating cardiac relaxation but also increasing SR Ca 2+ load and consequently SR Ca 2+ release. The phosphorylation of TnI and MyBP-C reduces myofilament Ca 2+ affinity and increases crossbridge kinetics. Altogether, these events produce the typical inotropic and lusitropic effects of β-AR stimulation. 1 Intracellular cAMP levels result from the balance between cAMP synthesis by adenylyl cyclases and cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). PDEs are subdivided into 11 families among which four can hydrolyse cAMP in the heart: PDE1, which is activated by Ca 2+ /calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; and PDE4. 2 While PDE1 and PDE2 can hydrolyse both cAMP and cGMP, PDE3 preferentially hydrolyses cAMP and PDE4 is specific for cAMP. Another PDE, named PDE8A which specifically hydrolyses cAMP, was shown recently to control ECC in mouse cardiac myocytes. 3 All PDE isoforms except PDE8A [START_REF] Soderling | Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases[END_REF][START_REF] Fisher | Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase[END_REF] are inhibited by xanthine derivatives such as 3isobutyl-1-methylxanthine (IBMX), and a number of drugs have been developed as selective PDE inhibitors [START_REF] Lugnier | Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents[END_REF][START_REF] Bender | Cyclic nucleotide phosphodiesterases: from molecular regulation to clinical use[END_REF] : EHNA [START_REF] Méry | Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes[END_REF] and Bay 60-7550 [START_REF] Boess | Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance[END_REF] for PDE2; milrinone, cilostamide and other bipyridines for PDE3 [START_REF] Bender | Cyclic nucleotide phosphodiesterases: from molecular regulation to clinical use[END_REF] ; rolipram and Ro 20-1724 for PDE4. [START_REF] Bender | Cyclic nucleotide phosphodiesterases: from molecular regulation to clinical use[END_REF] There is currently no commercially available selective inhibitor of PDE1.

Direct monitoring of cAMP in living cardiac myocytes emphasized the importance of PDE isoforms 2 to 4 for the control of [cAMP]i. In rodent heart, a predominance of PDE4 over other PDEs for the control of cAMP signals generated by stimulation of β-ARs and other GsPCRs was observed. [START_REF] Rochais | Negative feedback exerted by PKA and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes. An in vivo study using adenovirus-mediated expression of CNG channels[END_REF][START_REF] Mongillo | Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases[END_REF][START_REF] Rochais | A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gscoupled receptors in adult rat ventricular myocytes[END_REF][START_REF] Nikolaev | Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching ß1-adrenergic but locally confined ß2-adrenergic receptor-mediated signaling[END_REF][START_REF] Leroy | Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases[END_REF] PDE4 was shown to associate with β-ARs and ECC proteins, either directly or through scaffold proteins such as β-arrestins or A-kinase anchoring proteins. [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: The role of cyclic nucleotide phosphodiesterases[END_REF][START_REF] Scott | A-kinase anchoring proteins: getting to the heart of the matter[END_REF] In mouse heart, a specific PDE4 variant (PDE4D3) was shown to control the phosphorylation of RyR2 and regulates diastolic SR Ca 2+ leak. [START_REF] Lehnart | Defective ryanodine receptor interdomain interactions may contribute to intracellular Ca 2+ leak: a novel therapeutic target in heart failure[END_REF] Similarly, a PDE4D variant was found to coimmunoprecipitate with SERCA2 in mouse and to control PLB phosphorylation and Ca 2+ transients decay kinetics. [START_REF] Kerfant | PI3KY is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes[END_REF] As Cav1.2 is a well characterized target of the cAMP/PKA pathway in heart, the cardiac L-type Ca 2+ channel current (ICa,L) has been frequently used as a functional index of the contribution of PDE isoforms to this pathway. Inhibition of PDE2, PDE3 and PDE4 was shown to increase ICa,L amplitude in a number of species. [START_REF] Rochais | A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gscoupled receptors in adult rat ventricular myocytes[END_REF][START_REF] Hartzell | Opposite effects of cyclic GMP and cyclic AMP on Ca 2+ current in single heart cells[END_REF][START_REF] Malecot | Biphasic contractions induced by milrinone at low temperature in ferret ventricular muscle: role of the sarcoplasmic reticulum and transmembrane calcium influx[END_REF][START_REF] Rivet-Bastide | cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes[END_REF][START_REF] Kajimoto | Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes[END_REF][START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF][START_REF] Vandecasteele | Cyclic GMP regulation of the L-type Ca 2+ channel current in human atrial myocytes[END_REF] Recently, we observed in mouse heart that a PDE4B variant associates with Cav1.2 and controls its activity upon β-AR stimulation. [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias[END_REF] However, important differences exist among species as to whether PDE3 or PDE4 is predominant and whether all three PDE isoforms control the ICa,L amplitude at basal or only upon β-AR stimulation. PDE3 inhibitors were also reported to increase SR Ca 2+ uptake and SR Ca 2+ content, [START_REF] Yano | Effect of milrinone on left ventricular relaxation and Ca 2+ uptake function of cardiac sarcoplasmic reticulum[END_REF][START_REF] Xiong | Diminished inotropic response to amrinone in ventricular myocytes from myopathic hamsters is linked to depression of high-gain Ca 2+induced Ca 2+ release[END_REF][START_REF] Beca | PDE3A regulates basal myocardial contractility through interacting with SERCA2a-signaling complexes in mouse heart[END_REF] an effect attributed to an increase in PKA-dependent phosphorylation of PLB.

While it is well established that PDE3 inhibition exerts a direct positive inotropic effect in the heart from large mammals, [START_REF] Harrison | Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs[END_REF][START_REF] Weishaar | Subclasses of cyclic AMPspecific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF][START_REF] Komas | Differential sensitivity to cardiotonic drugs of cyclic AMP phosphodiesterases isolated from canine ventricular and sinoatrial-enriched tissues[END_REF] the contribution of other PDE families to the regulation of cardiac contractility remains less clear and often controversial. [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF] For instance, while selective inhibition of PDE2 or PDE4 was shown to increase cardiac contractility following β-AR stimulation in some studies, [START_REF] Mongillo | Compartmentalized phosphodiesterase-2 activity blunts ß-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway[END_REF][START_REF] Muller | Involvement of rolipram-sensitive cyclic AMP phosphodiesterase in the regulation of cardiac contraction[END_REF][START_REF] Katano | Cyclic AMP metabolism in intact rat ventricular cardiac myocytes -Interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine[END_REF][START_REF] Katano | Differential effects of Ro 20-1724 and isobutylmethylxanthine on the basal force of contraction and beta-adrenoceptor-mediated response in the rat ventricular myocardium[END_REF][START_REF] Christ | Inotropy and L-type Ca 2+ current, activated by ß1-and ß2-adrenoceptors, are differently controlled by phosphodiesterases 3 and 4 in rat heart[END_REF] other studies failed to demonstrate an effect. [START_REF] Herzer | Effects of type IV phosphodiesterase inhibition on cardiac function in the presence and absence of catecholamines[END_REF][START_REF] Mj | Comparative actions of diazepam and other phosphodiesterase inhibitors on the effects of noradrenaline in rat myocardium[END_REF] cAMP PDEs and cardiac EC coupling -D. Mika et al.

5

In an attempt to reconcile these contradictory findings, we compared, in the same mammalian cardiac preparation (adult rat ventricular myocytes), the effect of a selective inhibition of PDE2, PDE3 and PDE4 on Ca 2+ transients, sarcomere shortening, and phosphorylation of five key ECC proteins (TnI, Cav1.2, RyR2, PLB and MyBP-C). Furthermore, we compared for each PDE isoform, the effects observed under basal condition and after sub-maximal β-AR stimulation with isoprenaline. Our study sheds new light on the respective contribution of the three major cardiac PDE isoforms in the regulation of cardiac ECC.

Methods

All experiments were carried out according to the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J n°L358, 18 December 1986), the local ethics committee (CREEA Ile-de-France Sud) guidelines and the French decree n°87-848 of October 19, 1987 (J Off Rép Fr, 20 October 1987, pp. 12,245-12,248). Authorizations to perform animal experiments according to this decree were obtained from the Ministère Français de l'Agriculture, de la Pêche et de l'Alimentation (nº 92-283, June 27, 141 2007).

Reagents

Bay 60-7550 (Bay, 2-[(3,4-dimethoxyphenyl)methyl]-7-[(1R)-1-hydroxyethyl]-4- phenylbutyl]-5-methyl-imidazo[5,1-f][1,2,4
]triazin-4(1H)-one) was from Alexis Biochemicals (Lausen, Switzerland): it blocks PDE2 with an IC50 value of 4.7 nM, [START_REF] Beavo | Cyclic nucleotide phosphodiesterases in health and disease[END_REF] and was used here at a 100 nM concentration to fully block the enzyme. 9 Cilostamide (Cil) was from Tocris Bioscience (Bristol, UK): it blocks PDE3 with an IC50 ranging from 5 nM 41 to 27 nM, [START_REF] Sudo | Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity[END_REF]

6

µM [START_REF] Rose | Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity[END_REF][START_REF] Rich | In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors[END_REF] and was used here at 10 µM. At these concentrations, Bay, Cil and Ro were shown to be selective for PDE2, PDE3 and PDE4 respectively (Supplemental Table 1). IBMX (3isobutyl-1-methylxanthine) and isoprenaline (Iso) were from Sigma Aldrich (Saint Quentin, France).

Cardiomyocyte isolation and culture

Male Wistar rats (250-300 g) were subjected to anesthesia by intraperitoneal injection of pentothal (0.1 mg/g) and hearts were excised rapidly. Individual adult rat ventricular myocytes (ARVMs) were obtained by retrograde perfusion of the heart as previously described. [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF] For enzymatic dissociation, the hearts were perfused retrogradely at a constant flow of Triton X100 supplemented with 1 mM Na3VO4, and a protease inhibitor mixture (1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1 mM benzamidine, 10 μg/mL soybean trypsin inhibitor, 10 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL antipain, 1 μg/mL pepstatin). The lysates were centrifuged at 10,000 g for 10 min and assayed for protein concentration using bicinchoninic acid-protein reagent (Sigma Aldrich, Saint Quentin, France).

For immunoblotting assays, proteins were resolved by 4-12% or 8-16% SDS-PAGE (Lonza Verviers, Verviers, Belgium), transferred onto nitrocellulose membranes, and stained with 3%

Ponceau S (Sigma Aldrich, Saint Quentin, France). The membranes were saturated with 3% bovine serum albumin. Phosphorylation of phospholamban (PLB) at Ser [START_REF] Scott | A-kinase anchoring proteins: getting to the heart of the matter[END_REF] and cardiac troponin I (TnI) at Ser 23/24 were detected using specific antibodies from Cell Signaling Technology (Beverly, MA, USA). Phosphorylation of ryanodine receptor (RyR2) at Ser 2809 was detected using a specific antibody kindly provided by Dr A. R. Marks (Columbia University, USA). Ltype Ca 2+ channel pore forming subunit (Cav1.2) phosphorylation at Ser 1928 was detected using the anti-CH3-P antibody. [START_REF] Hulme | ß-adrenergic regulation requires direct anchoring of PKA to cardiac Cav1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15[END_REF] Phosphorylation of myosin binding protein C (MyBP-C) at Ser 282 was detected using antibodies from Alexis Biochemicals (San Diego, CA, USA).

Immunoreactive proteins were revealed using a 1:30,000 dilution of anti-rabbit or anti-mouse IgG peroxidase conjugate antibodies (Promega Corp., Madison, USA), and were visualized with Supersignal West Pico reagent (Perbio Science, Vigneux, France). The membranes were cAMP PDEs and cardiac EC coupling -D. Mika et al.

stripped and reprobed with specific antibodies against calsequestrin (CSQ) (Affinity BioReagents, Ozyme, Saint-Quentin-Fallavier, France) used as a loading control. Each sample was normalized to CSQ (phosphorylated proteins/CSQ ratio) and double-normalized to the control ratio. Densitometric analyses of western blots were performed using the NIH ImageJ software.

Data Analysis

Cell contraction was assessed by the percentage of sarcomere shortening, which is the ratio of twitch amplitude (difference of end-diastolic and peak systolic sarcomere lengths) to enddiastolic sarcomere length (SL). Ca 2+ transient amplitude was assessed by the percentage of variation of the Fura-2 ratio, by dividing the twitch amplitude (difference of end-diastolic and peak systolic ratios) to end-diastolic ratio. Relaxation was assessed by measuring the time-to-50% relaxation from the time to peak shortening, and the Ca 2+ transient decay was evaluated by measuring the time-to-50% decay of the Fura-2 ratio from the time to peak ratio. These parameters were obtained by analyzing the 10 last contractions and calcium transients before addition of the next drug. All parameters were calculated offline on a dedicated software (IonWizard 6x, IonOptix). All results are expressed as mean±SEM. GraphPad Prism software (GraphPad software Inc., La Jolla, CA, USA) was used for statistical analysis. To determine statistical significance with multiple groups, we used a One-way ANOVA followed by a Newman-Keuls test for multiple comparisons. Differences with p values <0.05 were considered as statistically significant. The number of independent experiments performed is indicated in the figure legends. 2). Upon pacing at 0.5 Hz, the average peak amplitude of Ca 2+ transients was 22±0.1% above the diastolic Fura-2 ratio (Supplemental Table 3), whereas sarcomere length decreased by 0.78±0.05% (n=95) control). As shown in Supplemental Figure 1, the Iso response on Ca 2+ transients and cell shortening was stable during the 10 min incubation period suggesting that β-AR stimulation by 1 nM Iso does not induce desensitization of the receptors, in our experimental conditions.

Results

Regulation of ECC by PDE2, PDE3 and PDE4 in ARVMs

(
In a first series of experiments, the effects of PDE2 inhibition were tested (Fig. 1 and Supplemental Table 2 and 3) using the selective inhibitor Bay. As shown on the representative traces in Fig. 1A and on the summary data in Fig. 1B, 100 nM Bay increased basal Ca 2+ transient amplitude and basal cell contraction approximately 2-fold. Bay also accelerated the decay kinetics of both signals, as shown by a significant decrease in the t1/2 off values for both parameters (Fig. 1C). These effects were small when compared to that of 1 nM Iso. To examine whether PDE2 modulates ECC under β-AR stimulation, 100 nM Bay was applied on top of Iso.

As shown in Fig. 1A and on the Supplemental Table 2 and 3, the Iso effect was potentiated upon PDE2 inhibition. However, the decay kinetics of the signals were unchanged compared to those obtained under Iso stimulation (Fig. 1C).

We next studied the consequences of a selective PDE3 inhibition on Ca 2+ transients and cell contraction using Cil (Fig. 2 and Supplemental Table 2 and3). Application of 1 µM Cil alone induced a significant increase in Ca 2+ transient and sarcomere shortening amplitudes by approximately 2-and 3-fold, respectively (Fig. 2A and2B). Cil also significantly accelerated the decay kinetics of both signals (Fig. 2C). This was accompanied by an increase in SR Ca 2+ load with no change in SR fractional Ca 2+ release (Fig. 5). PDE3 inhibition also strongly potentiated the effect of Iso on both parameters, enhancing their amplitude by 30% for Ca 2+ transient and 70% for cell shortening (Fig. 2B), and accelerating their relaxation kinetics (Fig. 2C).

In contrast to the results obtained with PDE2 or PDE3 inhibitors, PDE4 inhibition by 10 µM Ro had no effect on Ca 2+ transients and sarcomere shortening under basal conditions (Fig. 3A and 3B and Supplemental Table 2 and3). However, Ro strongly potentiated the effects of Iso (1 nM), further increasing the Ca 2+ transients amplitude and sarcomere shortening by 60% (Fig. 3B). PDE4 inhibition also significantly accelerated the decay kinetics of both signals (Fig. 3C).

We next examined the functional consequences of combinations of selective PDE inhibitors on cell contraction under basal conditions (Fig. 4 and Supplemental Table 2). As shown in Fig. 4A andB, simultaneous inhibition of PDE2 and PDE4 by a combination of 100 nM Bay and 10 µM Ro (Bay + Ro) increased Ca 2+ transients and sarcomere shortening, and slightly accelerated the relaxation kinetics of the twitch. However, comparison with Fig. 1 shows that these effects were similar to that of individual PDE2 inhibition under basal conditions. In contrast, concomitant inhibition of PDE2 and PDE3 by 100 nM Bay and 1 µM

Cil strongly increased the amplitude of Ca 2+ transients and sarcomere shortening and these effects were significantly larger than when either PDE2 or PDE3 alone were inhibited (p<0.05

versus Bay and Cil alone). The relaxation kinetics were significantly accelerated as compared cAMP PDEs and cardiac EC coupling -D. Mika et al.

12 to control (Fig. 4C), t1/2 off values of sarcomere shortening were significantly smaller for Bay + Cil as compared to Bay alone (compare Fig. 1C and4C) but not as compared to Cil alone (compare Fig. 2C and4C). This suggests that PDE3 has a more prominent role in controlling cell relaxation than PDE2 under basal conditions. Concomitant inhibition of PDE3 and PDE4

by Cil + Ro had a major impact on ECC, increasing the amplitude of Ca 2+ transients 2.5-fold and sarcomere shortening 15-fold (Fig. 4B). This was accompanied by a major acceleration of Ca 2+ transients and cell shortening decay phases, which was significantly larger than with Bay + Cil (Fig. 4C). Furthermore, Cil + Ro doubled SR Ca 2+ load and drastically increased fractional Ca 2+ release from 40% in Ctrl or after PDE3 inhibition, to 80% when both PDE3 and PDE4

were inhibited (Fig. 5). Finally, application of the broad spectrum PDE inhibitor IBMX (100 µM) had similar functional effects as a concomitant inhibition of PDE3 and PDE4.

Regulation of the phosphorylation status of key ECC proteins by PDEs

To get some insights into the molecular mechanisms by which individual PDE families regulate ECC in ARVMs, we examined the consequences of PDE inhibition on the phosphorylation of key ECC proteins. For this, we performed Western blot analysis with phospho-specific antibodies directed against TnI, MyBP-C, PLB, RyR2 and Cav1.2 under basal conditions (Fig. 6) or after a non maximal β-AR stimulation by Iso (1 nM) (Fig. 7). In line with ECC measurements, Fig. 6 shows that global PDE inhibition with IBMX or concomitant inhibition of PDE3 and PDE4 by Cil + Ro led to a major increase in the phosphorylation of TnI (Fig. 6A), MyBP-C (Fig. 6B), PLB (Fig. 6C), RyR2 (Fig. 6D) and CaV1.2 (Fig. 6E). Individual inhibition of PDE2 and PDE3 as well as simultaneous inhibition of PDE2 and PDE3 or PDE2 and PDE4

slightly but significantly increased the phosphorylation of TnI, whereas inhibition of PDE4 alone did not (Fig. 6A). In contrast, MyBP-C phosphorylation was significantly increased by selective inhibition of PDE2, PDE3 or PDE4 and by their dual inhibition, especially in the case cAMP PDEs and cardiac EC coupling -D. Mika et al.

13

of PDE3 and PDE4 (Fig. 6B). Selective PDE3 inhibition by Cil or simultaneous inhibition of PDE2 and PDE3 also increased significantly PLB phosphorylation whereas individual or associated PDE2 and PDE4 inhibition did not (Fig. 6C). The phosphorylation of CaV1.2 and RyR2 was only increased by simultaneous blockade of PDE3 and PDE4 or global PDE inhibition with IBMX (Fig. 6D andE).

Fig. 7 shows that after a non-maximal -AR stimulation by Iso, we observed a significant increase in the phosphorylation of TnI, MyBP-C, PLB, RyR2 and CaV1.2. A maximal level of phosphorylation was obtained by treatment of cells by Iso followed by IBMX (Fig. 7). The effect of Iso on the phosphorylation of all of these proteins was potentiated by the selective inhibition of PDE4, in agreement with the results obtained on Ca 2+ transients, sarcomere shortening and relaxation kinetics. Similar results were obtained upon PDE3 inhibition, except that Cil only tended to increase the level of phosphorylation of TnI. In contrast, PDE2 inhibition only increased the phosphorylation of MyBP-C and CaV1.2 in the presence of Iso.

Discussion

It is well established that cardiac ECC is regulated by the cAMP/PKA pathway. Initiation of the pathway takes place at the sarcolemmal membrane by cAMP synthesis through the activity of adenylyl cyclases; termination of the pathway involves the activity of cyclic nucleotide PDEs which are responsible for the hydrolysis of cAMP into 5'-AMP, and phosphatase activity which is responsible for PKA substrate dephosphorylation. At least five PDE families are reported to degrade cAMP in heart. 2,[START_REF] Patrucco | PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and -independent effects[END_REF] Comparison of the functional role of individual PDE families is possible by using pharmacological agents that selectively inhibit their activity. Since selective inhibitors of PDE1 and PDE8 are still lacking, we focused our attention on PDE2, PDE3 and PDE4 for which selective inhibitors exist. Using Bay to inhibit PDE2, Cil to inhibit PDE3 and Ro to inhibit PDE4, as well as the broad spectrum PDE inhibitor (IBMX), we explored the role of these three PDEs in three different sets of experimental conditions (at basal, upon β-AR stimulation, and upon dual inhibition), and on nine different parameters: PKA phosphorylation level of TnI, MyBP-C, PLB, RyR2 and Cav1.2; amplitude and relaxation kinetics of sarcomere shortening and Ca 2+ transients. It is known that cAMP signals induced by -AR stimulation also activate the Ca 2+ /Calmodulin-dependent kinase II (CaMKII), which phosphorylates some of the main PKA substrates. [START_REF] Grimm | ß-Adrenergic receptor signaling in the heart: role of CaMKII[END_REF] Thus, it is conceivable that some of the effects of the PDE inhibitors are due to the activation of CaMKII. However, inhibition of this kinase by KN-93 (1 µM) did not alter the positive inotropic effects of IBMX (Supplemental Figure 2), suggesting that PKA is the main effector of the increased Ca 2+ transients and sarcomere shortening observed upon PDE inhibition.

Our results indicate that only PDE2 and PDE3 appear to regulate ECC under basal conditions. However, upon β-AR stimulation, individual inhibition either of PDE2, PDE3 or PDE4 potentiates the β-AR response of Ca 2+ transient and cell contraction to a similar extent.

This suggests that PDE2 and PDE3 are active in basal conditions, whereas PDE4 becomes activated upon β-AR stimulation or PDE3 inhibition.

In ARVMs, biochemical and functional measurements indicate that PDE3 and PDE4 are the major PDEs degrading cAMP. [START_REF] Rochais | A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gscoupled receptors in adult rat ventricular myocytes[END_REF][START_REF] Leroy | Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases[END_REF][START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF] Accordingly, experiments shown here indicate that PDE3 and PDE4 dominate. This is especially true for the control of the SR Ca 2+ load (Fig. 5), which determines Ca 2+ transient amplitude. [START_REF] Trafford | Stimulation of Ca-induced Ca release only transiently increases the systolic Ca transient: Measurements of Ca fluxes and sarcoplasmic reticulum Ca[END_REF] Interestingly, while PDE3 inhibition alone does not increase fractional release (probably because it affects only SR Ca 2+ load) the combination Cil + Ro have a tremendous effect on this parameter. This can be explained by the increased ICa,L amplitude [START_REF] Leroy | Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases[END_REF] and RyR2 open probability leading to an increase of the gain of ECC [START_REF] Ginsburg | Modulation of excitation-contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca 2+ load and Ca 2+ current trigger[END_REF] occurring only when both PDEs are inhibited. Hence, inhibition of these two PDEs induces a massive phosphorylation of ECC proteins, and a large increase in Ca 2+ transients and sarcomere shortening (Fig. 4,6). Other dual combinations of PDE inhibition had much smaller effects on protein phosphorylation and ECC, indicating that PDE3 and PDE4 can compensate for each other, and that their function is partially redundant.

Although PDE2 represents only a few percents of total hydrolytic activity in ARVMs, [START_REF] Rochais | A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gscoupled receptors in adult rat ventricular myocytes[END_REF] we show here that it regulates basal and β-AR-stimulated ECC. This is consistent with previous studies showing that PDE2 modulates cAMP levels and β-AR responses in rodent cardiac myocytes. [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF][START_REF] Mongillo | Compartmentalized phosphodiesterase-2 activity blunts ß-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway[END_REF] In particular, PDE2 modulates the β-AR regulation of the L-type Ca 2+ current (ICa,L) in cardiac myocytes [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF] thus providing a possible mechanism for the effects of PDE2 inhibition on Iso-stimulated Ca 2+ transients and contraction. In contrast, the mechanism by which PDE2 regulates basal ECC is less clear since PDE2 inhibition has no effect on basal ICa,L in ARVMs. [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF] MyBP-C could participate in the effects of PDE2 on basal ECC by increasing cross bridge cycling and myofilament Ca 2+ sensitivity. [START_REF] Cazorla | The cAMP binding protein cAMP PDEs and cardiac EC coupling -D. Mika et al. 22 Epac regulates cardiac myofilament function[END_REF] In contrast to PDE4, selective PDE3 inhibition increased the amplitude and relaxation kinetics of Ca 2+ transients and sarcomere shortening under basal conditions (Fig. 2), and this was associated with enhanced phosphorylation of PLB, TnI and MyBP-C but not Cav1.2 or RyR2 (Fig. 6). These data are in line with previous findings showing a subcellular localization of PDE3 in SR-enriched membrane fraction [START_REF] Lugnier | Characterization of indolidansensitive and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions[END_REF][START_REF] Movsesian | Beta-adrenergic receptor agonists and cyclic nucleotide phosphodiesterase inhibitors: Shifting the focus from inotropy to cyclic adenosine monophosphate[END_REF] and the positive effects of PDE3 inhibitors on SR Ca 2+ uptake and SR Ca 2+ content. [START_REF] Yano | Effect of milrinone on left ventricular relaxation and Ca 2+ uptake function of cardiac sarcoplasmic reticulum[END_REF][START_REF] Xiong | Diminished inotropic response to amrinone in ventricular myocytes from myopathic hamsters is linked to depression of high-gain Ca 2+induced Ca 2+ release[END_REF][START_REF] Beca | PDE3A regulates basal myocardial contractility through interacting with SERCA2a-signaling complexes in mouse heart[END_REF] This is also consistent with the lack of effect of selective PDE3 inhibition on basal ICa,L in ARVMs. [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF] In the present study, we established that PDE2, PDE3 and PDE4 have distinct roles in controlling ECC. In the absence of hormonal stimulation, when intracellular cAMP concentration is low, PDE2 and especially PDE3 are dominant to control cardiac contraction.

PDE3 contribution to the degradation of basal cAMP could be explained by its high affinity for cAMP (Km<1µM). [START_REF] Shakur | Regulation and function of the cyclic nucleotide phosphodiesterase (PDE3) gene family[END_REF] For PDE2, the scheme must be different because of its low affinity for cAMP. Nonetheless, under basal conditions, localized production of cGMP may occur to cAMP PDEs and cardiac EC coupling -D. Mika et al.

16

activate PDE2 by binding to its regulatory GAF-B domain. [START_REF] Martinez | The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding[END_REF] PDE2 activation mediated by cGMP could exacerbate its role in controlling cAMP levels regulating ECC as recently reported in cardiomyocytes. [START_REF] Stangherlin | cGMP Signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes[END_REF] When cAMP levels are increased, PDE4 turns on and becomes crucial to control global cAMP homeostasis and ECC. However, despite a modest effect on cAMP level, inhibition of PDE2 and PDE3 also enhances β-AR inotropic and lusitropic effects, demonstrating that PDE2, PDE3 and PDE4 act in concert to modulate β-AR stimulation of cardiac contraction. Interestingly, although cardiotonic drugs such as milrinone produce beneficial inotropic effects upon acute administration, [START_REF] Konstam | Short-term use of intravenous milrinone for heart failure[END_REF] these agents increase mortality in patients with dilated cardiomyopathy upon chronic treatment. [START_REF] Packer | Effect of oral milrinone on mortality in severe chronic heart failure. The PROMISE Study Research Group[END_REF] A challenge of future research will be to determine the molecular mechanisms that underlie long term detrimental effects of PDE inhibitors in heart failure in order to design new therapeutic approaches to enhance the beneficial effects and suppress detrimental long term effects of PDE inhibitors.

Limitations

The present study focused on the role of three different PDE families in controlling the ECC in isolated ARVMs. It is known that the expression level of these PDEs varies among different species and is dependent on the development stage and on the cardiac territory investigated. [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF] However, PDE expression is relatively conserved between murine models and human. [START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF] In mammalian heart, PDE3 and PDE4 remain the two major enzymes degrading cAMP and controlling cardiac ECC. Thus, even if our results might not be fully transposable to any cellular cardiac models or human heart, we unveil how cardiac function is finely tuned by these PDE families. Furthermore, their expression and distribution are altered under pathophysiological conditions. [START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF] Therefore, the relative contribution of PDE2, PDE3 and PDE4 to compartmentalize cAMP signals and maintain specific PKA phosphorylation of the key ECC proteins, is most probably differently affected in various cardiac diseases. Future studies investigating their participation in pathological conditions are necessary to design new therapeutic strategies targeting PDEs.

cAMP

  PDEs and cardiac EC coupling -D. Mika et al. 10 To investigate the functional consequences of individual PDE inhibition on basal and β-AR stimulated ECC in ARVMs, Ca 2+ transients and sarcomere shortening were simultaneously recorded in Fura-2-loaded ARVMs. In quiescent myocytes, the average diastolic sarcomere length (SL) was 1.68±0.01 µm (n=95) (Supplemental Table

Figure 1

 1 Figure 1 Effect of PDE2 inhibition on ECC in ARVMs. (A) Representative traces of Ca 2+ transients (top) and sarcomere shortening (bottom) recorded in ARVMs paced at 0.5 Hz and loaded with Fura-2 AM (5 µM) showing the effect of PDE2 inhibition by Bay (0.1 µM) (in black) on basal conditions (dashed lines) or upon β-adrenergic stimulation by 1 nM Iso (in gray). (B) Mean amplitude (± SEM) of Ca 2+ transients (upper graph, in % of diastolic ratio) and sarcomere shortening (lower graph, in % of resting sarcomere length, SL) in control (white bar, n=24 cells, 13 rats), Bay (black bar, n=24 cells, 13 rats), Iso (dark gray bar, n=11 cells, 4 rats) and Iso + Bay (light gray bar, n=11 cells, 4 rats). C Average time to 50% relaxation of Ca 2+ transients (upper graph) and sarcomere shortening (lower graph) in similar experimental conditions as in B. Statistical significance is indicated as: *, p<0.05; **, p<0.01; ***, p<0.001;and ### , p<0.001 (One-way ANOVA, Newman-Keuls).

Figure 2

 2 Figure 2 Effect of PDE3 inhibition on ECC in ARVMs. (A) Typical traces of Ca 2+ transients (top) and sarcomere shortening (bottom) in Fura-2 loaded ARVMs electrically stimulated (0.5 Hz) representing the effect of PDE3 inhibition by cilostamide (Cil, 1 µM) (in black) under basal conditions (dashed lines) or during isoprenaline stimulation (Iso, 1 nM) (in gray). (B) Mean amplitude (± SEM) of Ca 2+ transients (upper graph) and sarcomere shortening (lower graph) in control conditions (white bar, n=16 cells, 7 rats), Cil (black bar, n=16 cells, 7 rats), Iso (dark gray bar, n=9 cells, 3 rats) and Iso + Cil (light gray bar, n=9 cells, 3rats). (C) Average time to 50% relaxation for Ca 2+ transients (upper graph) and sarcomere shortening (lower graph) are presented for each conditions tested. Statistical significance is indicated as: *, p<0.05; ***, p<0.001; and # , p<0.05; ### , p<0.001 (One-way ANOVA, Newman-Keuls).

Figure 3

 3 Figure 3 Effect of PDE4 inhibition on ECC in ARVMs. (A) Individual traces of Ca 2+ transients (top) and sarcomere shortening (bottom) in Fura-2 loaded ARVMs paced at 0.5 hz representing the effect of PDE4 inhibition by Ro (10 µM) (in black) under basal conditions (dashed lines) or after isoprenaline stimulation (Iso, 1 nM) (in gray). (B) Mean amplitude (± SEM) of Ca 2+ transients (upper graph) and sarcomere shortening (lower graph) in control conditions (white bar, n=11 cells, 6 rats), Ro (black bar, n=11, 6 rats), Iso (dark gray bar, n=12 cells, 6 rats) and Iso + Ro (light gray bar, n=12 cells, 6 rats). (C) Average time to 50% relaxation for Ca 2+ transients (upper graph) and sarcomere shortening (lower graph) are presented for

Figure 4

 4 Figure 4 Effect of concomitant PDE inhibition on Ca 2+ transients and cell contraction in ARVMs under basal conditions. (A) Representative traces of Ca 2+ transients (top) and sarcomere shortening (bottom) obtained in Fura-2 loaded ARVMs paced at 0.5 Hz showing the effect of simultaneous PDE2 and PDE4 inhibition by Bay (0.1 µM) and Ro (10 µM) (Bay + Ro); PDE2 and PDE3 inhibition by Bay and cilostamide (1 µM) (Bay + Cil); PDE3 and PDE4 inhibition by Cil and Ro (Cil + Ro); and global PDE inhibition by IBMX (100 µM). (B) Mean amplitude (± SEM) of Ca 2+ transients (upper graph) and sarcomere shortening (lower graph) in control conditions (white bar), or after concomitant PDE inhibition by Bay + Ro (light gray bar, n=14 cells, 5 rats), Bay + Cil (dark gray bar, n=11 cells, 4 Cil + Ro (black bar, n=9 cells, 4 rats) and IBMX (hatched bar, n=10 cells, 3 rats). (C) Mean (± SEM) time to 50% relaxation for Ca 2+ decay (upper graph) and sarcomere shortening (lower graph) the same conditions as in B. Statistical significance is indicated as: *, p<0.05; **, p<0.01; ***, p<0.001; and # , p<0.05; ### , p<0.001 (One-way ANOVA, Newman-Keuls).

Figure 5

 5 Figure 5 Effect of concomitant PDE3 and PDE4 inhibition on sarcoplasmic reticulum Ca 2+ load and fractional release in ARVMs. (A) Representative traces of Ca 2+ transients obtained with caffeine (10 mM) recorded in Fura-2 loaded ARVMs after pacing at 0.5 Hz, under basal conditions (Ctrl), upon PDE3 inhibition by cilostamide (Cil, 1 µM) or PDE3 and PDE4 inhibition by Cil and Ro (10 µM) (Ro + Cil). (B) Mean amplitude (± SEM) of calcium transients induced by caffeine (10 mM) estimating sarcoplasmic reticulum Ca 2+ content in Ctrl (white bar, n=8 cells, 3 rats), Cil (grey bar, n=8 cells, 3 rats), and Cil + Ro (black bar, n=9 cells, 3 rats). (C) Average fractional release (± SEM) in Ctrl (white bar, n=8 cells, 3 rats), Cil (grey bar, n=8 cells, 3 rats), and Cil + Ro (black bar, n=9 cells, 3 rats). Statistical significance is indicated as: *, p<0.05; ***, p<0.001 (versus control cells) and ### , p<0.001 (versus Cil treated cells) (Oneway ANOVA, Newman-Keuls).

Figure 6

 6 Figure 6Effect of PDE inhibitors on phosphorylation of key ECC proteins in basal conditions. Unstimulated ARVMs were treated or not with Ro (10 µM), cilostamide (Cil, 1 µM), Ro + Cil, Bay (0.1 µM), Bay + Cil, Bay + Ro and IBMX (100 µM) as indicated for 15 min. Whole proteins were extracted and 50 µg of proteins were analysed by Western blot using antibodies for phospho-troponin I (P-TnI) (A), phospho-myosine-binding protein C (P-MyBP-

Figure 7

 7 Figure 7Effect of PDE inhibition on the phosphorylation of key ECC proteins under -AR stimulation. ARVMs were treated for 10 min with isoprenaline (1 nM, Iso) followed by 10 min treatment with PDE inhibitors Bay (0.1 µM), Cil (1 µM), Ro (10 µM) or IBMX (100 µM).Representative Western blots of cardiac myocyte lysates probed for phospho-troponin I (P-TnI) (A), phospho-myosine-binding protein C (P-MyBP-C) (B), phospho-phospholamban (P-PLB) (C) phospho-ryanodine receptor (P-RyR2) (D) and phospho-Cav1.2 (E). The membranes were stripped and reprobed with calsequestrin (CSQ) antibodies used as a loading control. A representative blot is shown, and at least 5 separate experiments were performed (n=5 for P-TnI, n=7 for P-MyBP-C, n=6 for P-PLB, n=6 for P-RyR and n=5 for P-CaV1.2), giving similar results. Phosphorylated proteins/CSQ ratios were quantified, expressed as means ± SEM and normalized to untreated ARVMs (Ctrl). Statistical significance is indicated as: *, p<0.05; **, p<0.01; ***, p<0.001; and # , p<0.05; ## , p<0.01; ### , p<0.001 (One-way ANOVA, Newman-Keuls).

Figure 1 * 2

 12 Figure 1

3 Measurements of Ca 2+ transients and sarcomere shortening

	cAMP PDEs and cardiac EC coupling -D. Mika et al. cAMP PDEs and cardiac EC coupling -D. Mika et al.	7 8
	2.Isolated cardiomyocytes were loaded with 5 µM Fura-2 AM (Invitrogen) at room temperature 2.
	for 15 min and then washed with Ringer solution containing (in mM) NaCl 121.6, KCl 5.4,
	NaHCO3 4.013, NaH2PO4 0.8, HEPES 10, glucose 5, Na pyruvate 5, MgCl2 1.8, CaCl2 1.8, pH
	7.4. The loaded cells were field stimulated (5 V, 4 ms) at a frequency of 0.5 Hz. Sarcomere
	length and Fura-2 ratio (measured at 512 nm upon excitation at 340 nm and 380 nm) were
	simultaneously recorded using an IonOptix System (IonOptix, Milton, MA, USA). Briefly,
	myocytes were imaged at 240 Hz using an IonOptix Myocam-S CCD camera. Images were
	displayed within the IonWizard acquisition software (IonOptix). The myocyte of interest was
	aligned horizontally, parallel to the field of view. The sarcomere length was measured from a
	user-defined region of interest that included at least 7 sarcomeres to maximize the accuracy of
	the measurements. The IonWizard software determined a density trace corresponding to the
	4 mL/min for 1 h with the same solution alternating dark and light A-and I-bands of the cardiomyocyte, and analyzed the periodicity in
	containing 1 mg/mL collagenase A (Boehringer, Mannhein, Germany) and 300 µM EGTA and the density trace by calculation of a fast Fourier transform. This analysis allowed a direct real-
	CaCl2 to adjust free Ca 2+ concentration to 20 µM. The ventricles were then separated from atria, time measurement of sarcomere length.
	finely chopped and gently agitated to dissociate individual cells. The resulting cell suspension The measurements of Ca 2+ transients and sarcomere shortening were performed on
	was filtered on a gauze and the cells were allowed to settle down. The supernatant was discarded individual myocytes superfused for few minutes with control Ringer solution and then
	and cells resuspended four more times with Ringer solution at increasing [Ca 2+ ] from 20 µM to challenged with PDE inhibitors (single: Bay, 100 nM; Cil, 1 µM; Ro, 10 µM or IBMX, 100
	300 µM. Freshly isolated cells were suspended in minimal essential medium (MEM: M 4780; µM; or combinations: Bay + Ro, Bay + Cil or Cil + Ro) during 15 minutes or with 1 nM Iso
	Sigma, St Louis, MO USA) containing 1.2 mM [Ca 2+ ] supplemented with 2.5% fetal bovine during 10 min followed by 10 min treatment with PDE inhibitors (Bay, Cil or Ro). Sarcoplasmic
	serum (FBS, Invitrogen, Cergy-Pontoise, France), 1% penicillin-streptomycin, 20 mM HEPES reticulum (SR) Ca 2+ load was measured by rapid application of caffeine (10 mM) to ARVMs
	(pH 7.6) and plated on 35 mm, laminin-coated culture dishes (10 μg/mL) at a density of 10 4 after pacing at 0.5 Hz in control Ringer solution or after Cil or Cil+Ro application. Fractional
	cells per dish. After 1 h the medium was replaced by 300 μL of FBS-free MEM. All experiments Ca 2+ release was calculated as the ratio between the Ca 2+ transient amplitude and the caffeine
	were performed on primary cell cultures 24h after plating. transient amplitude (SR Ca 2+ load) measured in the same ARVM.

6 mL/min at 37°C with a Ca 2+ -free Ringer solution containing (in mM) NaCl 117, KCl 5, NaHCO3 4.4, KH2PO4 1.5, MgCl2 1.7, D-glucose 11.7, Na-phosphocreatine 10, taurine 20 and HEPES 21, pH 7.1 during 5 min, followed by a perfusion at

4 Cell extracts and western blot analysis

  Iso followed by 10 min treatment with PDE inhibitors (Bay, Cil or Ro). To prepare whole ARVM cell lysates, cells were lysed in cold HNTG lysis buffer containing (in mM): Hepes 50 (pH 7.5), NaCl 400, NaF 100, Na-pyrophosphate 10, MgCl2 1.5, EGTA 1, 10% Glycerol, 1%

	Prior to cell homogenization and biochemical analysis, intact ARVMs were treated at room
	temperature for 15 min with PDE inhibitors (single: Bay, 100 nM; Cil, 1 µM; Ro, 10 µM or
	IBMX, 100 µM; or combinations: Bay + Ro, Bay + Cil or Cil + Ro), or for 10 min with 1 nM

  Supplemental Table1). The relaxation kinetics of both parameters, as estimated by the time to 50% decay (t1/2 off), were on average 0.43±0.01 s for Ca 2+ transients and 0.49±0.02 s for cell contraction (n=95). On average, application of 1 nM Iso increased the amplitude of Ca 2+ transients by 257±20.3% and sarcomere shortening by 1033.8±61.2%, (n=62, p<0.001 vs.

control). Iso also significantly accelerated the relaxation phases of both signals, decreasing t1/2 off for Ca 2+ decay by 38.4±1.6% and t1/2 off for contraction by 56.3±1.3% (n=62, p<0.001 vs.

Acknowledgements

The authors wish to thank Florence Lefebvre and Aurore Germain for technical assistance with cardiomyocyte isolation, and Dr Andrew R. Marks (Columbia University, USA) for kindly providing the RyR2 phosphospecific antibody.

Funding

This work was supported by Leducq cycAMP grant 06CVD02 (to RF), by European Union contract LSHM-CT-2005-018833/EUGeneHeart (to RF), the Fondation de France (to GV), the Agence Nationale de la Recherche 2010 BLAN 1139-01 (to GV), the Fondation pour la Recherche Médicale (to RF), the Société Française de Cardiologie (to JL), the Investment for the Future program ANR-11-IDEX-0003-01 within the LABEX ANR-10-LABX-0033 (to RF) and a PhD grant from Ile de France CORDDIM (to PB).

Supplementary material

Supplementary material is available at Cardiovascular Research online. Supplemental Individual cardiac myocytes were superfused for few minutes with control Ringer solution and then challenged with PDE inhibitors (single: Bay 60-7550, Bay, 100 nM; cilostamide, Cil, 1 µM; Ro 20-1724, Ro, 10 µM or IBMX, 100 µM; or combinations: Bay + Ro, Bay + Cil or Cil + Ro) during 15 min or with Isoproterenol (Iso, 1 nM) during 10 min followed by 10 min treatment with PDE inhibitors (Bay, Cil or Ro). Diastolic and systolic Fura-2 ratio, ΔR (systolic Fura-2 ratio-diastolic Fura-2 ratio), and Fura-2 ratio (%) (ΔR/diastolic Fura-2 ratio*100) are expressed as mean ± SEM for each stimulation.

Conflict of interest