PhD Cristina E Molina

Hind Mehel, Roel L H M Daniel M Johnson

G Spätjens

PhD Delphine Mika

MD Vincent Algalarrondo

Haj Zeineb

Patrick Slimane

Najah Lechêne

Henk J Abi-Gerges

Van Der Linde

PhD Hind Mehel

BS Roel L H M G Spätjens

PhD Zeineb Haj Slimane

BS Patrick Lechêne

PhD Najah Abi-Gerges

BS Henk J Van Der Linde

PhD Jérôme Leroy

MD Paul G A Volders

PhD Rodolphe Fischmeister
email: rodolphe.fischmeister@inserm.fr

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

Interventricular Differences in β-Adrenergic Responses in the Canine Heart: Role of Phosphodiesterases

Keywords: heart ventricles, dog, β-adrenergic stimulation, cAMP, phosphodiesterases

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The sympathetic nervous system is responsible for adaptation of cardiac output to stress and physical exercise. This "fight-or-flight" response is mediated primarily by noradrenaline and adrenaline acting on β-adrenergic receptors (β-ARs) at the surface of cardiac myocytes. β-ARs are coupled through Gαs to adenylyl cyclases (AC) and the generation of cAMP, which in turn activates the cAMP-dependent protein kinase (PKA).

PKA then phosphorylates key proteins involved in excitation-contraction coupling (ECC) including sarcolemmal L-type Ca 2+ channels (ICa,L), ryanodine receptors (RyR2), phospholamban and troponin I. [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF] In addition, PKA phosphorylates slowly-activating delayed rectifier K + (IKs) channels to control cardiac repolarization. [START_REF] Marx | Requirement of a macromolecular signaling complex for â adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel[END_REF] The levels of cAMP and thus the degree of PKA activation are finely regulated by cyclic nucleotide phosphodiesterases (PDEs) that degrade the second messenger into 5'-AMP. Cardiac PDEs degrading cAMP belong to 5 families (PDE1-4 and PDE8) which can be distinguished by distinct enzymatic properties and pharmacology. [START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF] In rodents, PDE3 and PDE4 are the major contributors to the total cAMP-hydrolytic activity [START_REF] Rochais | A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes[END_REF][START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF] and PDE4 is dominant to modulate β-AR regulation of cAMP levels. [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 | 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] 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] Multiple PDE4 variants associate with β-ARs, [START_REF] Baillie | â-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates âadrenoceptor switching from Gs to Gi[END_REF][11][START_REF] De Arcangelis | Differential association of phosphodiesterase 4D isoforms with ß2-adrenoceptor in cardiac myocytes[END_REF] RyR2, 13 SERCA2, [START_REF] Kerfant | PI3KY is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes[END_REF][START_REF] Beca | Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca 2+ release and cardiac contractility, independently of L-type Ca 2+ current[END_REF] ICa,L, [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] and IKs [START_REF] Terrenoire | The cardiac IKs potassium channel macromolecular complex includes the phosphodiesterase PDE4D3[END_REF] to exert local control of ECC. In larger mammals, PDE3 activity is dominant in microsomal fractions 18- 20 and PDE3 inhibitors exert a potent positive inotropic effect. [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF] Selective inhibition of PDE3 with milrinone has been shown to improve cardiac contractility in patients with congestive heart failure. [START_REF] Monrad | Assessment of long-term therapy with milrinone and the effects of milrinone withdrawal[END_REF] The role of PDE4 is less well defined but evidence is emerging that PDE4 may also play an important role in these species. In the canine heart, a large PDE4 activity is found in the cytoplasm [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF] but PDE4 is also present in microsomal fractions, where it accounts for ~20% of the activity. [START_REF] Lugnier | Characterization of indolidan-sensitive and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions[END_REF] Recent studies have indicated that

Molina et al.

Interventricular Differences in β-AR Signaling 4 PDE4 is expressed in human ventricle where, similar to rodents, it associates with β-ARs, RyR2 and phospholamban. [START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF][START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine receptor complex promotes heart failure and arrhythmias[END_REF] Moreover, PDE4 controls ECC and arrhythmias in human atrium. [START_REF] Molina | Cyclic AMP phosphodiesterase type 4 protects against atrial arrhythmias[END_REF] The right (RV) and left (LV) ventricles originate from different progenitor cells [START_REF] Zaffran | Right ventricular myocardium derives from the anterior heart field[END_REF][START_REF] Verzi | The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field[END_REF] and differ in several important ways. Both ventricles have different mass (under normal conditions LV mass is ~6-fold RV mass), volume, morphologies and pressures. [START_REF] Ho | Anatomy, echocardiography, and normal right ventricular dimensions[END_REF][START_REF] Haddad | Right ventricular function in cardiovascular disease, part I: Anatomy, physiology, aging, and functional assessment of the right ventricle[END_REF] Electrical heterogeneity has been well characterized between ventricular epicardial, endocardial and midmyocardial layers. [START_REF] Sicouri | A subpopulation of cells with unique electrophysiological properties in the deep subepicardium of the canine ventricle. The M cell[END_REF][START_REF] Antzelevitch | Heterogeneity within the ventricular wall -Electrophysiology and pharmacology of epicardial, endocardial, and M-Cells[END_REF][START_REF] Zygmunt | INaCa contributes to electrical heterogeneity within the canine ventricle[END_REF][START_REF] Gaborit | Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart[END_REF] Although less studied, different electrophysiological properties were also reported between the two ventricles. In rats and dog, the action potential (AP) is shorter in the RV than in the LV. [START_REF] Watanabe | Heterogeneity of the action potential in isolated rat ventricular myocytes and tissue[END_REF][START_REF] Volders | Repolarizing K + currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium[END_REF] In dog, the notch in phase 1 of the AP is deeper in the RV than the LV. [START_REF] Volders | Repolarizing K + currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium[END_REF][START_REF] Diego | Ito and action potential notch are smaller in left vs. right canine ventricular epicardium[END_REF] These differences are related to larger repolarizing K + currents, Ito and IKs in dog RV. [START_REF] Volders | Repolarizing K + currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium[END_REF][START_REF] Diego | Ito and action potential notch are smaller in left vs. right canine ventricular epicardium[END_REF] In this species, a larger RV IKs correlates with a higher expression of KCNQ1 and KCNE1, the principal and auxiliary subunit of the IKs channel, respectively. [START_REF] Ramakers | Coordinated downregulation of KCNQ1 and KCNE1 expression contributes to reduction of IKs in canine hypertrophied hearts[END_REF] Regional heterogeneity between the LV and the RV may also exist for ATP-activated K + current, IKATP. [START_REF] Pandit | Left-to-right ventricular differences in IKATP underlie epicardial repolarization gradient during global ischemia[END_REF][START_REF] Choi | Increased expression of ATP-sensitive K + channels improves the right ventricular tolerance to hypoxia in rabbit hearts[END_REF] In contrast to these electrophysiological studies, a recent proteomic study reported no difference in the expression level of more than 600 proteins between the RV and the LV from pig and rabbit. Most of these were contractile/structural proteins, oxidative phosphorylation components, and enzymes from intermediary metabolism. No plasma membrane voltage-gated ion channels were analyzed, but a few major Ca 2+ handling proteins (SERCA2, RyR2) and signal transduction components (Ca 2+ /Calmodulin kinase IIδ, PKA type I and II) showed no variation in expression level. [START_REF] Phillips | Homogenous protein programming in the mammalian left and right ventricle free walls[END_REF] These studies raise the question of whether functional differences between the LV and the RV are limited to electrophysiological features or extend to other aspects of ventricular function. Surprisingly, only limited information is available concerning ECC and its neurohumoral regulation in RV versus LV in the healthy heart. However, a previous report in rat has shown that, despite similar interventricular expression levels, a higher proportion of the total SERCA2a pool is associated with phospholamban in RV myocytes compared with LV myocytes, resulting in lower Ca 2+ reuptake rate and prolonged Ca 2+ transients. [START_REF] Sathish | Mechanistic basis of differences in Ca 2+ -handling properties of sarcoplasmic reticulum in right and left ventricles of normal rat myocardium[END_REF] In mouse, opposing inotropic responses to α1-AR stimulation have been reported, and were attributed to different effects on myofilament Ca 2+ sensitivity. [START_REF] Wang | Contrasting inotropic responses to alpha1-adrenergic receptor stimulation in left versus right ventricular myocardium[END_REF] In human, recent clinical studies indicate that ventricular load increases more for the RV than the LV during exercise [START_REF] Gerche | Disproportionate exercise load and remodeling of the athlete's right ventricle[END_REF] and this may reflect, at least in part, relative differences in maximal pulmonary-arterial versus aortic pressure. It is currently unclear whether interventricular differences exist in the sympathetic responsiveness of the human heart.

In canine, while the absolute dP/dt values are far greater in LV than in RV, stimulation of the cardiac sympathetic nerves induces greater relative changes in contractile force in the RV than in the LV. [START_REF] Norris | Responses of the canine myocardium to stimulation of thoracic cardiac nerves[END_REF] These changes are insensitive to α1-AR blockade by phentolamine, implicating differences in β-AR response. [START_REF] Abe | The effect of cardiac sympathetic nerve stimulation on the right ventricle in canine heart[END_REF] However, several studies reported no difference in β-AR density, AC activity and its activation by catecholamines between RV and LV under normal conditions. [START_REF] Vatner | Effects of pressure overload, left ventricular hypertrophy on ß-adrenergic receptors, and responsiveness to catecholamines[END_REF][START_REF] Bristow | Beta 1-and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1receptor down-regulation in heart failure[END_REF][START_REF] Fan | Alterations in cardiac betaadrenoceptor responsiveness and adenylate cyclase system by congestive heart failure in dogs[END_REF][START_REF] Calderone | Dysfunction of the ß-and alpha-adrenergic systems in a model of congestive heart failure. The pacing-overdrive dog[END_REF][START_REF] Liang | Chronic beta-adrenoceptor blockade prevents the development of beta-adrenergic subsensitivity in experimental right-sided congestive heart failure in dogs[END_REF][START_REF] White | Age-related changes in ßadrenergic neuroeffector systems in the human heart[END_REF] Intrigued by these apparent differences, we examined the β-AR regulation of cardiac contractility in dog RV and LV in isolated myocytes and in vivo. Our data reveal enhanced sensitivity of the RV to β-AR stimulation, and provide evidence that PDE3 and PDE4 shape distinct compartmentalized cAMP signals that underlie interventricular dispersion in β-AR stimulation. The study was conducted in accordance with the Declaration of Helsinki principles, and approved by the Ethical Committees of our institutions.

Methods

In vivo Experiments

General anesthesia was induced in 4 beagle dogs (3 F/1 M; average weight 111 kg) by lofentanil (0.075 mg/kg body weight i.v.), scopolamine (0.015 mg/kg), succinylcholine (1.0 mg/kg), and subsequent hourly slow injections of fentanyl (0.025 mg/kg i.v.) and continuous infusion of etomidate (1.5 mg/kg/h). Dogs were ventilated with 30% oxygen in pressurized air to normocapnia. The body temperature was kept at 37°C with a heated water mattress. ECG standard lead II was continuously recorded. Under closed-chest conditions, LV and RV intracavitary pressures were recorded simultaneously with highfidelity catheter-tip micromanometers introduced via the femoral artery and vein (Gaeltec Ltd, Dunvegan, UK and Millar Instruments Inc, Houston, TX, USA). In each animal a bolus injection of isoproterenol (2.5 μg/kg) was administered and repeated twice, with each next infusion given after baseline values had been stably reestablished.

Cell-Isolation Procedure

Adult female beagle dogs were used for myocyte isolations. A total of 12 dogs were included in the study. Anesthesia was induced with 45 mg/kg pentobarbital. Once full anesthesia was reached, the chest was opened via a left thoracotomy and the heart was excised and placed in an O2-gassed, Ca 2+ -free standard buffer solution at approximately 4°C. The cell-isolation procedure was the same as previously described. [START_REF] Volders | Repolarizing K + currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium[END_REF] Briefly, both the left-anterior-descending and right coronary arteries were cannulated and perfused simultaneously. After ~20 min of collagenase perfusion and subsequent washout of the enzyme, the epicardial surface layer was removed from wedges of both the LV and RV until a depth of ≥3 mm was reached. Softened tissue samples were collected from the midmyocardial layer underneath, while contamination with the endocardium was avoided. Samples were gently agitated, filtered and washed. Midmyocytes were stored at room temperature in standard buffer solution (vide infra) and only quiescent rod-shaped cells with clear cross-striations and without granulation were used for the experiments.

Cells were used within 48 h of isolation.

Adenoviral Infection of Dog Ventricular Myocytes

Isolated cells were suspended in minimal essential medium (MEM: M 4780; Sigma, St Louis, Missouri, USA) containing 1.2 mmol/L Ca 2+ , 2.5% fetal bovine serum (FBS, Invitrogen, Cergy-Pontoise, France), 1% penicillin-streptomycin, 2% HEPES (pH 7.6) and plated on 35 mm, laminin-coated culture dishes (10 μg/mL laminin, 2 h) at a density of 10 4 cells per dish. Dishes were kept in an incubator (95% O2, 5% CO2, 37ºC) for 2 h.

Then the medium was replaced by 400 μL of FBS-free MEM containing adenoviruses encoding for the cytoplasmic cAMP sensor Epac2-camps 50 at a multiplicity of infection (MOI) of 1000 pfu/cell, the plasma-membrane targeted pmEpac2-camps [START_REF] Wachten | Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells[END_REF] during 400 ms. The use of -50 mV as holding potential allowed the inactivation of voltage-dependent Na + currents. K + currents were blocked by replacing all K + ions with external and internal Cs + . Amplifiers RK-400 (Bio-Logic, Claix, France) or Axopatch 200B (Axon Instruments, Inc., Union City, CA, USA) were used for voltage clamping and acquisition. Currents were analogue filtered at 3 kHz and digitally sampled at 10 kHz using a 12-bit analogue-to-digital converter (DT2827; Data translation, Marlboro, Massachusetts, USA) connected to a compatible PC or a Digidata 1440A interface connected to a computer equipped with pClamp 10 software (Axon Instruments, Inc., Union City, CA, USA). The maximal amplitude of whole-cell ICa,L was measured 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] Currents were not compensated for capacitance and leak currents.

These experiments were performed at room temperature.

IKs Recordings

The whole-cell configuration of the patch-clamp technique was also used to record IKs. with NaOH at 37°C. During the recordings, KCl was omitted from the external solution to increase IKs amplitude, which occurs through changes in the concentration gradient, while leaving the kinetics of IKs activation and deactivation uninfluenced. 54 ICa,L was blocked with nifedipine (5 μM) and IKr with dofetilide (1 μM). The myocytes were depolarized from a holding potential of -50 mV to +50 mV for 3 s, with the tail currents on repolarization to 0 mV quantified as IKs. Ten baseline pulses were recorded followed by 3 pulses during brief (15 s) stimulation with Iso (plus or minus PDE inhibitor) and this was followed by a further 50 pulses during perfusion with 0 mmol/L K + ( PDE inhibitor).

Fast solution changes were achieved with the complete VC-6 fast-step perfusion system (Harvard Apparatus, Holliston, MA, USA) together with a multibarrel glass pipette that was positioned ~50 μm from the cell during drug application.

Recordings of Ca 2+ Transients and Sarcomere Shortening

Myocytes were loaded with 5 μM Fura-2 AM (Invitrogen) during 15 min at room temperature and then washed with Ringer solution containing (in mmol/L): NaCl 121.6, KCl 5.4, MgCl2 1.8, CaCl2 1.8, NaHCO3 4, NaH2PO4 0.8, D-glucose 5, Na-pyruvate 5, HEPES 10, adjusted to pH 7.4 with NaOH. The loaded cells were field-stimulated (5 V, 4 ms) at a frequency of 0.3 Hz. Then, 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). Cell contraction was assessed as the percentage of sarcomere shortening, which is the ratio of twitch amplitude (difference of end-diastolic and peak systolic sarcomere lengths) to end-diastolic sarcomere length. 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 10 end-diastolic ratio. All parameters were calculated offline with dedicated software (IonWizard 6x, IonOptix). These experiments were performed at room temperature.

FRET Measurements

The same Ringer solution as described above for Ca 2+ transients and cell shortening measurements was used in these experiments. Images were captured every 5 s using the 40x oil immersion objective of a Nikon TE 300 inverted microscope connected to a software-controlled (Metafluor, Molecular Devices, Sunnyvale, California, USA) cooled charge coupled (CCD) camera (Sensicam PE; PCO, Kelheim, Germany). CFP was excited during 150-300 ms by a Xenon lamp (100 W, Nikon, Champigny-sur-Marne, France) using a 440/20BP filter and a 455LP dichroic mirror. Dual-emission imaging of CFP and YFP was performed using an Optosplit II emission splitter (Cairn Research, Faversham, UK) equipped with a 495LP dichroic mirror and BP filters 470/30 and 535/30, respectively. A region of interest including the entire cell was used for measurement of average CFP and YFP intensity. CFP and YFP intensities were background corrected and the YFP emission was corrected for CFP bleed through. The ratio of CFP over corrected YFP was used as an index of cAMP concentration.

Reagents

Cilostamide was from Tocris Bioscience (Bristol, UK) and Ro 20-1724 was from Calbiochem (Darmstadt, Germany). Unless specified, all other drugs were from Sigma-Aldrich (Saint Quentin, France). Isoproterenol (Iso) was first dissolved in distilled water containing 30 μM ascorbic acid and then kept in the dark at 4°C until use.

for comparison of multiple effects and to check for the absence of correlation within animals. Sidak Adjustment Multiple Comparisons was used to evaluate the significance of measurements over time.

Results

Sarcomere Shortening and Ca 2+ -Transient Measurements in Response to β-AR

Stimulation in RV and LV Midmyocytes

Sarcomere shortening and Ca 2+ transients were simultaneously measured in Fura-2loaded RVMs and LVMs obtained from the same hearts and paced at 0.3 Hz. There was no significant difference in basal sarcomere length, basal sarcomere shortening, basal Fura-2 ratio and basal Ca 2+ -transient amplitude (Table 1). As shown by the individual traces of Figure 1A using the cytoplasmic sensor Epac2-camps. [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF] Figure 2A shows pseudocolor images illustrating the percent increase of the CFP/YFP ratio 1 min and 3 min after application of Iso (100 nmol/L, 15 s) in a LVM and a RVM expressing Epac2-camps. As shown in the inset graph of Figure 2B, there was no difference in the basal FRET ratio between LVMs and RVMs. At 1 min after application of Iso, the CFP/YFP ratio increased to a greater extent in RVMs than in LVMs. On average, Iso led to a transient increase in the CFP/YFP ratio that was ~2-fold higher in RVMs versus LVMs (p<0.001) reflecting enhanced cytoplasmic cAMP accumulation in RVMs (Figure 2B). Because PKA is the primary target of cAMP for short-term regulation of ECC, cytoplasmic PKA activity was monitored using the PKA FRET sensor AKAR3-NES. [START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters[END_REF] As with Epac2-camps, there was no significant difference in the average basal FRET ratio (inset graph of Figure 2D). As shown in the pseudocolor images illustrating the relative effect of Iso (100 nmol/L, 15 s) in Figure 2C and on the average time course of Figure 2D, although the maximal increase in YFP/CFP ratio was identical at 1 min, the signal decayed faster in LVMs than RVMs and became significantly higher in RVMs than LVMs after 2 min. This indicates that cytoplasmic PKA activity was higher in RVMs than in LVMs in response to β-AR stimulation. The lack of difference in the YFP/CFP ratio at the beginning of the stimulation was most likely due to saturation of the AKAR3-NES sensor at this

Midmyocytes

Because subsarcolemmal cAMP plays an important role in ECC, we next used a plasmamembrane targeted version of Epac2-camps (pmEpac2-camps [START_REF] Wachten | Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells[END_REF] ) to monitor cAMP specifically in this compartment. To our surprise, comparison of LVMs and RVMs revealed no difference in subsarcolemmal cAMP ([cAMP]pm) generated by β-AR stimulation with an Iso pulse. Indeed, neither the maximal cAMP elevation nor its decay kinetics was different between LVMs and RVMs (Figure 3A). We next wondered whether the same was true for the major sarcolemmal targets of the β-AR/cAMP/PKA pathway, ICa,L and IKs. As shown in Table 1, there was no difference in basal ICa,L density between RVMs and LVMs. A pulse stimulation with Iso (100 nmol/L, 15 s) resulted in a similar transient increase of ICa,L between LVMs and RVMs, both in terms of amplitude and duration (Figure 3B). In contrast to ICa,L, basal IKs density was higher in RVMs than LVMs (Table 1), which is consistent with earlier findings. [START_REF] Volders | Repolarizing K + currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium[END_REF] However, pulse β-AR stimulation increased IKs to similar degrees in LVMs and RVMs (Figure 3C). These results indicate that i) differences in β-AR cAMP signals between LVMs and RVMs are compartment-specific and ii) the increased stimulation of ECC by Iso in RVMs does not involve up-regulation of ICa,L or down-regulation of IKs.

PDE3 and PDE4 Shape Distinct β-AR cAMP Signals in RVMs and LVMs

We next investigated whether phosphodiesterases (PDEs) are involved in the differences in cytoplasmic cAMP accumulation observed between RVMs and LVMs upon β-AR stimulation. We first tested the implication of PDE3, because of its critical importance for the control of cardiac contractility in dog. [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF] Thus, myocytes expressing Epac2-camps were challenged with Iso (100 nmol/L, 15 s) in the presence of the specific PDE3 inhibitor cilostamide (Cil, 1 µmol/L), and the inhibitor was maintained during Iso washout. Figure 4A shows that Cil doubled the maximal amplitude of the Iso-induced cAMP transient in LVMs, while having little effect in RVMs. Cil also profoundly impaired cAMP recovery in both LVMs and RVMs, as indicated by the ~3-fold increase in time to half maximal decay (t1/2off) values (Figure 4A). We next tested the implication of PDE4, which represents the major soluble cAMP-PDE activity in dog ventricle [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF] by using the specific PDE4 inhibitor Ro-201724 (Ro, 10 µmol/L). Figure 4B shows that Ro induced a 3-fold increase in the amplitude of the cAMP transient elicited by Iso in LVMs and a 2-fold increase in RVMs. PDE4 inhibition also significantly delayed cAMP recovery, as demonstrated by a ~2.5-fold increase in time-to-half-maximal decay (t1/2off) values. Thus, PDE3 and PDE4 regulate cytoplasmic cAMP accumulation upon β-AR stimulation in canine ventricular midmyocytes. However, this control is less stringent in RVMs compared to LVMs, resulting in higher cytoplasmic cAMP ([cAMP]cyt) upon β-AR stimulation.

Regulation of Membrane cAMP, ICa,L and IKs by PDE3 and PDE4 in RVMs and

LVMs

Given the large contribution of PDE4 to cytoplasmic cAMP hydrolysis upon β-AR stimulation, we compared the respective roles of PDE3 and PDE4 in the β-AR regulation of subsarcolemmal cAMP in canine ventricular myocytes. As shown in Figure 5A, inhibition of PDE3 with 1 µmol/L Cil had no effect on the maximal cAMP elevation at the plasma membrane but delayed cAMP recovery in LVMs and RVMs. Interestingly, the difference in average t1/2off values between Iso and Iso+Cil reached statistical significance only in LVMs (t1/2off was 107±17 s for Iso, and 232±50 s for Iso+Cil, p<0.05 whereas in RVMs, t1/2off was 112±17 s for Iso alone and 171±39 s for Iso+Cil, p=0.36) suggesting a stronger contribution of membrane-bound PDE3 in LVMs versus RVMs to cAMP hydrolysis upon β-AR stimulation. In sharp contrast to what was observed in the cytoplasm, PDE4 inhibition with Ro at 10 µmol/L had no effect, neither on the amplitude nor on the recovery kinetics of the cAMP transient generated by Iso pulse stimulation at the plasma membrane (Figure 5B). We next investigated the respective contribution of PDE3 and PDE4 to the regulation of ICa,L and IKs. As shown in Figure 6A, PDE3 inhibition had no effect on the maximal ICa,L stimulation induced by Iso (100 nmol/L, 15 s), but significantly delayed ICa,L recovery in LVMs and RVMs (in LVMs, t1/2off was 91±7 s for Iso, and 203±22 s for Iso+Cil, p<0.001; in RVMs, t1/2off was 113±11 s for Iso alone and 176±19 s for Iso+Cil, p<0.05). Interestingly, PDE3 inhibition also slowed IKs recovery in RVMs and LVMs (data not shown). In contrast, inhibition of PDE4 with Ro at 10 µmol/L did not modify ICa,L (Figure 6B) nor IKs recovery. However, when PDE3 was inhibited by Cil, concomitant inhibition of PDE4 drastically prolonged the β-AR stimulation of the current (Figure 6C), with t1/2off values reaching 381±66 s in RVMs (p<0.05 versus Iso+Cil) and 381±51 s in LVMs (p<0.01 versus Iso+Cil). These results indicate that although PDE3 is dominant for β-AR regulation of ICa,L in canine myocytes, PDE4 becomes important when PDE3 is inhibited.

In vivo Inotropic Response to Isoproterenol in RV and LV

Molina et al.

Interventricular Differences in β-AR Signaling 16 Altogether, the above results indicate that β-AR stimulation of ECC is more efficient in RVMs versus LVMs due to a stronger contribution of compartmentalized PDE3 and PDE4 to hydrolyze cAMP in the cytoplasm. Since these results were obtained in isolated cells, it seemed important to test whether enhanced β-AR responsiveness could be observed at a more integrated level. For this, pressure recordings were obtained simultaneously from the RV and LV at baseline and during infusion of Iso in anesthetized beagle dogs. Representative examples relative to the ECG are shown in Figure 7A. At baseline, systolic pressures and maximal dP/dt were significantly higher in the LV than in the RV (Figure 7B). For example, maximal dP/dt was 2582±194 mm Hg/s in the LV versus 534±45 mm Hg/s in the RV (p<0.001). Upon Iso, maximal dP/dt increased ~3.4fold in the LV compared to almost 5-fold in the RV (p<0.05; Figure 7B, left panel).

Accordingly, the relative increase in systolic pressure by β-AR stimulation was significantly higher in the RV than LV, reaching on average 66±19 mm Hg (+61±6%) and 172±31 mm Hg (+20±4%), respectively (p<0.01) during Iso (Figure 7B, right panel).

In addition, the relative increase in maximal dP/dt and systolic pressure was more sustained in the RV than the LV after the Iso bolus infusions (Figure 7C). Diastolic pressures did not show discernible interventricular differences from baseline to Iso.

Discussion

This study provides evidence that the RV is more sensitive than the LV to β-AR stimulation, in part due to intrinsic postsynaptic differences in myocyte β-AR signaling.

The enhanced efficiency of Iso to stimulate Ca 2+ transients and sarcomere shortening in RVMs compared to LVMs isolated from the same hearts is correlated with an increased positive inotropic effect of Iso observed in the RV in vivo. Enhanced β-AR responsiveness in RVMs is not associated with modifications of β-AR signaling at the plasma membrane, since β-AR stimulation of subsarcolemmal [cAMP]i and of membrane currents ICa,L and IKs are similar in RVMs and LVMs, even though basal IKs density showed interventricular differences, in agreement with our previous study. [START_REF] Volders | Repolarizing K + currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium[END_REF] However, RVMs display a higher cAMP accumulation and exacerbated PKA response in the cytoplasm compared to LVMs, and the difference in cAMP is abolished by inhibition of PDE3 and PDE4. Thus, PDE3 and PDE4 shape distinct compartmentalized cAMP signals that underlie regionally-specific differences in the cardiac ventricles upon β-AR stimulation.

Recent clinical studies indicate that during intense physical exercise, the afterload of the RV increases more than that of the LV. [START_REF] Gerche | Disproportionate exercise load and remodeling of the athlete's right ventricle[END_REF][START_REF] Gerche | Exercise-induced right ventricular dysfunction and structural remodelling in endurance athletes[END_REF] Because the sympathetic nervous system is strongly activated during exercise, these observations suggest that the RV could be more sensitive to sympathetic stimulation than the LV in humans. Earlier studies that compared the responsiveness of the LV versus RV to sympathetic nerve stimulation in dog are consistent with this notion: Norris and Randall showed that stimulation of the left and right ansae subclavia has a stronger inotropic effect in the RV than in the LV. [START_REF] Norris | Responses of the canine myocardium to stimulation of thoracic cardiac nerves[END_REF] Similarly, Abe et al. [START_REF] Abe | The effect of cardiac sympathetic nerve stimulation on the right ventricle in canine heart[END_REF] reported stronger effects of sympathetic nerve stimulation on RV systolic pressure, which were not modified by α1-AR blockade. Thus, previous data suggested the existence of differential β-AR stimulation in RV versus LV, but until now its clear demonstration has been lacking and the potential mechanisms involved remain elusive. Our experiments show that Iso infusion in vivo exerts a stronger relative increase in RV contractility compared to that of the LV (Figure 7). By themselves, these in vivo data do not allow the exclusion of a differential effect of Iso on innervations or loading conditions of the two ventricles. However, consistent with our hypothesis, Piao et al., using RV and LV Langendorff preparations in rat, have shown recently that the inotropic effect of dobutamine is more efficient in the RV than in the LV. [START_REF] Piao | GRK2-mediated inhibition of adrenergic and dopaminergic signaling in right ventricular hypertrophy: therapeutic implications in pulmonary hypertension[END_REF] Importantly, myocytes isolated from the midmyocardial layer of the RV showed enhanced and prolonged stimulation of Ca 2+ transients and contraction by Iso compared to their LV counterparts (Figure 1). These results demonstrate that the high sensitivity of the RV to β-AR stimulation relies at least partly on an intrinsic property of RV myocytes, and raise the question of which mechanisms are involved.

β-AR control of cardiac ECC operates mainly through activation of cAMP and PKA. Consistent with increased functional effects of β-ARs, cytoplasmic cAMP and phosphorylation of a specific PKA sensor were found to be increased in RVMs upon Iso pulse stimulation (Figure 2). While it seems very likely that increased phosphorylation of the PKA probe was due to enhanced PKA activity in RVMs versus LVMs, the slower decay kinetics of AKAR-NES dephosphorylation could also result from a lower phosphatase activity in RVMs. To the best of our knowledge, differences in phosphatase activity have not been documented in LV versus RV. Because we focused on the role of phosphodiesterase and compartmentalization of cAMP, we did not pursue the investigation of phosphatases. However, this point would deserve further consideration in future studies.

Interestingly, this difference was compartment-specific since enhanced β-AR responses were not observed at the plasma membrane. Indeed neither β-AR regulation of subsarcolemmal cAMP signals nor that of ICa,L and IKs were modified in RV compared to LV (Figure 3). Since ICa,L was not involved in the enhanced inotropic effect of Iso in RV, other ECC proteins must be differentially regulated. Likely candidates are RyR2 and PLB, which control SR Ca 2+ release and reuptake by SERCA2a, respectively. Although differential SERCA2a-PLB association was observed in RV versus LV in rat, [START_REF] Sathish | Mechanistic basis of differences in Ca 2+ -handling properties of sarcoplasmic reticulum in right and left ventricles of normal rat myocardium[END_REF] we did not observe differences in the decay kinetics of Ca 2+ transients in RV versus LV dog myocytes (data not shown). To our knowledge, whether RyR2 and PLB are differentially phosphorylated in response to Iso stimulation in RV versus LV has not been investigated. Enhanced β-AR functional effects in RVMs could result from increased cAMP generation in RVMs. However, the lack of difference at the plasma membrane (Figure 3) does not support this idea. Moreover, previous studies in dog and humans provide compelling evidence that the density of β-ARs, AC activity and AC stimulation by β-ARs are similar between RV and LV. [START_REF] Vatner | Effects of pressure overload, left ventricular hypertrophy on ß-adrenergic receptors, and responsiveness to catecholamines[END_REF][START_REF] Bristow | Beta 1-and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1receptor down-regulation in heart failure[END_REF][START_REF] Fan | Alterations in cardiac betaadrenoceptor responsiveness and adenylate cyclase system by congestive heart failure in dogs[END_REF][START_REF] Calderone | Dysfunction of the ß-and alpha-adrenergic systems in a model of congestive heart failure. The pacing-overdrive dog[END_REF][START_REF] Liang | Chronic beta-adrenoceptor blockade prevents the development of beta-adrenergic subsensitivity in experimental right-sided congestive heart failure in dogs[END_REF][START_REF] White | Age-related changes in ßadrenergic neuroeffector systems in the human heart[END_REF] Because in rodents PDEs can generate cAMP gradients within cardiac myocytes, [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][START_REF] Jurevicius | cAMP compartmentation is responsible for a local activation of cardiac Ca 2+ channels by ß-adrenergic agonists[END_REF][START_REF] Zaccolo | Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes[END_REF] we tested the hypothesis that they could be involved in the interventricular differences in cytoplasmic cAMP accumulation in dog.

Our results indicate that PDE3 inhibition strongly increased maximal [cAMP]cyt in LVMs upon β-AR stimulation, while having a weak effect on maximal [cAMP]cyt in RVMs, thus abolishing the differences between RVMs and LVMs. PDE3 inhibition also significantly prolonged the effect of Iso on [cAMP]pm in LVMs but not in RVMs (Figure 6A). PDE4 inhibition potentiated the maximal [cAMP]cyt in both RVMs and LVMs upon β-AR stimulation, with a more pronounced effect in RVMs. As a consequence, the interventricular difference in [cAMP]cyt accumulation was attenuated upon PDE4 inhibition. We conclude that regionally-specific differences in subcellular cAMP compartmentation arise from a more stringent control of β-ARs cAMP signals by PDE3 and PDE4 in LVMs than in RVMs.

Upon β-AR stimulation, the relatively balanced contribution of PDE3 and PDE4 to cAMP hydrolysis in the cytoplasm (Figure 4) and the dominance of PDE3 at the sarcolemma (Figure 5) is at variance with previous results in rodents, where PDE4 is dominant in both compartments. [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 | 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] 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] Because in dog PDE3 inhibitors have strong inotropic effects in contrast to PDE4 inhibitors, [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF] our results raise the question of the functional role of PDE4 in dog. When the respective role of these two PDEs was evaluated on the stimulation of ICa,L by Iso, inhibition of PDE3 but not PDE4 significantly prolonged ICa,L upregulation (Figure 6). This is consistent with a predominance of PDE3 in T-tubular membranes, whereas only a fraction of PDE4 is sarcolemmal and most of the activity is soluble. [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF][START_REF] Lugnier | Characterization of indolidan-sensitive and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions[END_REF] However, when PDE3 was inhibited, PDE4 inhibition drastically prolonged ICa,L recovery from β-AR stimulation (Figure 6). Thus, PDE4 can regulate ICa,L, but under normal conditions it is masked by the predominant PDE3. Therefore, the hierarchy between PDE3 and PDE4 appears to be opposite in dog compared to rat. [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] In conclusion, the current study demonstrates that RV and LV differ in their sensitivity to β-AR stimulation both in vivo and in vitro, and reveals regionally-specific differences in β-AR coupling to PDE3 and PDE4 and subcellular cAMP compartmentation in the heart. The fact that these results were obtained in a large mammal increases the likelihood, at least to some extent, that they represent the human situation. If this is the case, such enhanced sensitivity to β-AR stimulation may be an important factor for RV dysfunction not only in the context of sports medicine, but also in RV failure consequent to left-sided heart failure or pulmonary hypertension (PH) where activation of the sympathetic nervous system, although beneficial on the short term, becomes detrimental for cardiac function in the long run. In this context, our results suggest that β-blockers could be particularly effective in right heart failure. Consistent with this hypothesis, β-adrenoceptor blockade was reported to prevent β-AR down-regulation and to attenuate desensitization in experimental right heart failure induced by progressive pulmonary artery banding and tricuspid avulsion in dog. [START_REF] Liang | Chronic beta-adrenoceptor blockade prevents the development of beta-adrenergic subsensitivity in experimental right-sided congestive heart failure in dogs[END_REF] Similarly, β-blockers were shown to have beneficial effects on RV function and morphology in rat models of PH (reviewed in [START_REF] De Man | Neurohormonal axis in patients with pulmonary arterial hypertension: friend or foe?[END_REF] ). Interestingly, although β-blockers are not recommended in humans with PH because of their acute negative chronotropic and inotropic effects, low doses of bisoprolol were shown to

Molina et al.

Interventricular Differences in β-AR Signaling 21 improve RV contractility and filling and to delay the progression of right heart failure in the monocrotaline rat model of PH. [START_REF] De Man | Bisoprolol delays progression towards right heart failure in experimental pulmonary hypertension[END_REF] Our study also shows that the coupling of β-AR to PDE3 and PDE4 differs in the normal LV versus RV. It will be important in future studies to determine whether this is related to a differential expression and/or localization of PDE3 and PDE4 in the two ventricles, and how this coupling is modified during hypertrophy and heart failure. Indeed, whereas a decrease in β1-AR expression is known to occur in both the failing LV and RV, [START_REF] Bristow | Beta 1-and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1receptor down-regulation in heart failure[END_REF] much less is known concerning PDE remodeling in right heart failure. A deeper characterization of interventricular differences in cAMP signaling compartmentation may provide new indications for therapies that preferentially target the failing RV.

Sources of Funding

This study was supported in part by a Postdoctoral Lefoulon Delalande grant from the Institut de France and a Marie Curie Intra-European Fellowship for Career Development

  This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animal handling was in accordance with the European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609/EU).

  at a MOI of ICa,L Recordings The whole-cell configuration of the patch-clamp technique was used to record ICa,L. Patch-electrode resistance was between 1-2 MΩ when filled with internal solution containing (in mmol/L): CsCl 118, EGTA 5, MgCl2 4, Na2phosphocreatine 5, Na2ATP 3.1, Na2GTP 0.42, CaCl2 0.062 (pCa 8.5), HEPES 10, adjusted to pH 7.3 with CsOH. Extracellular Cs + -Ringer solution contained (in mmol/L): CaCl2 1.8, MgCl2 1.8, NaCl 107.1, CsCl 20, NaHCO3 4, NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10, adjusted to pH 7.4 with NaOH. The cells were depolarized every 8 s from -50 to 0 mV

9 (

 9 Patch electrodes had a resistance between 0.5-2 MΩ when filled with internal solution containing (in mmol/L): K-aspartate 125, KCl 20, MgCl2 1.0, MgATP 5, HEPES 5 and EGTA 10, pH 7.2 with KOH. The external solution had the following composition Molina et al. Interventricular Differences in β-AR Signaling mmol/L): NaCl 145, KCl 4.0, CaCl2 1.8, MgCl2 1.0, glucose 11 and HEPES 10, pH 7.4

  and 1B, a pulse application of Iso (100 nmol/L, 15 s) increased the amplitude of sarcomere shortening and Ca 2+ transient in both LVMs and RVMs, but these effects were exacerbated in RVMs. Indeed, on average Iso increased sarcomere shortening by ~25-fold in RVMs versus ~10-fold in LVMs and increased Ca 2+ transients ~3-fold in RVMs versus 2-fold in LVMs (Figure 1C and 1D). The effect of Iso also lasted longer in RVMs than in LVMs, as indicated by the time to half-maximal recovery (t1/2off) of sarcomere shortening (79±11 s in RVMs versus 54±3 s in LVMs) and Ca 2+ transient Molina et al. Interventricular Differences in β-AR Signaling 12 (82±7 s in RVMs versus 49±6 s in LVMs) (Figure 1E and 1F). Thus, β-AR stimulation of ECC was more efficient in dog RVMs versus LVMs. β-AR Regulation of Cytoplasmic cAMP Signals and PKA Activity in RV and LV Midmyocytes Since β-AR stimulation of ECC involves cAMP mobilization, this second messenger was measured in intact LVMs and RVMs by fluorescence resonance energy transfer (FRET)

Molina

  et al. Interventricular Differences in β-AR Signaling 13 concentration of Iso, as indicated by the lack of a sharp peak in RVMs and by previous experiments in rat ventricular myocytes showing that a ~40% change in YFP/CFP represents the maximal FRET change that can be obtained with this sensor. 55 β-AR Regulation of Subsarcolemmal cAMP, ICa,L and IKs in RV and LV

[

  PIEF-GA-331241] (to CEM), an ANR grant 2010 BLAN 1139-01 (to GV) and the Fondation Leducq for the Transatlantic Network of Excellence cycAMP grant 06CVD02 (to RF). PGAV is supported by The Netherlands Heart Foundation (NHS2010B216) and a Vidi grant from the Netherlands Organization for Scientific Research (ZonMw 91710365).

Figure 1 .

 1 Figure 1. β-AR regulation of ECC in LV and RV midmyocytes. A and B, Raw traces of sarcomere shortening (A) and Ca 2+ transients (B) recorded in Fura-2 loaded LVMs and

Figure 2 .

 2 Figure 2. RV midmyocytes display enhanced cAMP accumulation and PKA activity in

Figure 3 .

 3 Figure 3. β-AR regulation of subsarcolemmal cAMP, ICa,L and IKs in LV and RV

Figure 4 .

 4 Figure 4. Regulation of β-AR cytoplasmic cAMP signals by PDE3 and PDE4 in LVMs

Figure 5 .

 5 Figure 5. Regulation of subsarcolemmal cAMP by PDE3 and PDE4 after brief β-AR

Figure 6 .

 6 Figure 6. Regulation of ICa,L by PDE3 and PDE4 after brief β-AR stimulation. Average

Figure 7 .

 7 Figure 7. Differential response to β-adrenergic stimulation in the LV versus RV in

Figure 7

 7 Figure1

Table 1 : Basal parameters measured in RVMs and LVMs

 1

		RVMs	n	LVMs	n
	Contraction
	SL (µm)	1.74 ± 0.01	20	1.72 ± 0.01	18
	ΔL (%)	1.30 ± 0.27	20	1.23 ± 0.20	18
	Calcium
	Fura-2 ratio	4.93 ± 0.28	20	4.66 ± 0.27	18
	ΔR (%)	8.31 ± 0.84	20	9.00 ± 1.01	18
	Electrophysiology
	Cell capacitance (pF)	138.7 ± 3.8	107	132.9 ± 4.03	105
	ICa,L density (pA/pF)	2.9 ± 0.2	89	3.3 ± 0.2	92
	IKs density (pA/pF)	1.1 ± 0.2	18	0.8 ± 0.1 (p<0.05)	13

Acknowledgments

We thank Florence Lefebvre for superb technical assistance, Dr. Viacheslav Nikolaev for providing the Epac2-camps and pmEpac2-camps, Dr. Jin Zhang for providing the AKAR3 sensor and Dr. Bertrand Crozatier for helpful discussions.

Data Analysis and Statistics

All results are expressed as mean ± SEM. For statistical evaluation the paired and unpaired Student's t-test were used, and a difference was considered statistically significant when p was <0.05. Levene and Shapiro-Wilke tests were carried out in all the experiments in order to confirm homogeneity and normal distribution. Higher-order ANOVA without the assumption of sphericity (Greenhouse-Geisser correction) was used

Disclosures

NAG is employed by AstraZeneca and HJVDL is employed by Janssen. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.


S Al Bacha

M Zakhour

M Nakhl

J.-L Bobet

Effect of ball milling in presence of additives (Graphite, AlCl 3 , MgCl 2 and NaCl) on the hydrolysis performances of Mg 17 Al 12

Keywords: Mg17Al12, hydrogen production, ball milling, hydrolysis, Mg-Al alloys

In most of the Mg-Al alloys, Al forms with Mg the intermetallic compound Mg17Al12. In order to understand the hydrogen production from the Mg-Al alloys waste by the hydrolysis reaction in "model" seawater (i.e. 3.5 wt. % NaCl), hydrolysis with Mg17Al12 was investigated. The effect of ball milling time, the nature of the additives (graphite, NaCl, MgCl2 and AlCl3) and the synergetic effects of both graphite and AlCl3 were investigated. It has been established that increasing ball milling time up to 5h is necessary to activate the intermetallic and to decrease sufficiently its crystallites and particles size. On one hand, the presence of AlCl3 provides the best hydrolysis performance (14 % of the theoretical hydrogen volume in 1h). On the other hand, the mixture obtained by simultaneous addition of graphite and AlCl3 shows the best hydrolysis performances with 16% of the theoretical H2 volume reached in 1h.

Introduction

Magnesium alloys are used as structural materials in various industries where the intended application requires weight reduction (aeronautics, automotive, ...) [START_REF] Friedrich | Research for a "new age of magnesium" in the automotive industry[END_REF][START_REF] Kurihara | The role of aluminum in automotive weight reduction-Part II[END_REF][3]. However, the problem that limits the extension of the use of Mg-based alloys is related to their corrosion. In recent years, many commercial Mg alloys (with Al, Zn, RE,…) have been developed [START_REF] You | Recent research and developments on wrought magnesium alloys[END_REF].

Nowadays, only magnesium-based alloy wastes class 1 and 2 (i.e. high purity) are recycled to secondary ingots [START_REF] Hanko | Recycling automotive magnesium scrap[END_REF]6]. It has been shown that other magnesium-based alloy wastes can be used to generate H2 by the hydrolysis reaction [START_REF] Uan | Generation of hydrogen from magnesium alloy scraps catalyzed by platinum-coated titanium net in NaCl aqueous solution[END_REF][START_REF] Uan | Producing hydrogen in an aqueous NaCl solution by the hydrolysis of metallic couples of low-grade magnesium scrap and noble metal net[END_REF][START_REF] Uan | Evolution of hydrogen from magnesium alloy scraps in citric acid-added seawater without catalyst[END_REF][START_REF] Yu | Effects of concentrations of NaCl and organic acid on generation of hydrogen from magnesium metal scrap[END_REF][START_REF] Figen | Hydrogen generation from waste Mg based material in various saline solutions (NiCl2, CoCl2, CuCl2, FeCl3, MnCl2)[END_REF][START_REF] Figen | Hydrogen production by the hydrolysis of milled waste magnesium scraps in nickel chloride solutions and nickel chloride added in Marmara Sea and Aegean Sea Water[END_REF][START_REF] Bacha | Hydrogen generation via hydrolysis of ball milled WE43 magnesium waste[END_REF]. Hydrolysis with magnesium-based materials is an electrochemical reaction where Mg is oxidized to Mg 2+ (i.e. Mg corrosion) and water is reduced to produce H2 gas [START_REF] Uan | Evolution of hydrogen from magnesium alloy scraps in citric acid-added seawater without catalyst[END_REF].

AZ91 is one of the most used magnesium alloys. Our objective is to produce hydrogen by the hydrolysis reaction with AZ91 alloy waste in a solution similar to seawater (i.e. 3.5 wt.% of NaCl) without the use of external catalyst. Previously, Uan et al. reported that AZ91 can generate H2 by hydrolysis and the reaction performance was improved using a Pt/Ti catalyst due to the galvanic coupling between Mg and Pt [START_REF] Uan | Generation of hydrogen from magnesium alloy scraps catalyzed by platinum-coated titanium net in NaCl aqueous solution[END_REF].

The as-cast AZ91 microstructure has typically a primary α-phase matrix (Mg matrix) and a βphase precipitate (Mg17Al12 intermetallic phase). As a galvanic coupling exists between the βphase and the magnesium, depending on the amount and the specific surface of the β-phase (and so the surface contact between both phases), two opposite behavior can be observed: acceleration of the corrosion rate or either creation of a corrosion barrier hindering corrosion [START_REF] Song | Influence of microstructure on the corrosion of diecast AZ91D[END_REF][START_REF] Zhao | Influence of the β-phase morphology on the corrosion of the Mg alloy AZ91[END_REF][START_REF] Song | Understanding Magnesium Corrosion-A Framework for Improved Alloy Performance[END_REF][START_REF] Zhao | Corrosion of AZ91 -Influence of the β-Phase Morphology[END_REF].

In order to better understand and then to enhance the hydrolysis performance of AZ alloys, the behavior of the two mains components (i.e. Mg and Mg17Al12) has to be studied separately. The hydrolysis of Mg have been widely reported [START_REF] Grosjean | Hydrogen production from highly corroding Mg-based materials elaborated by ball milling[END_REF][START_REF] Grosjean | Hydrogen production via hydrolysis reaction from ball-milled Mg-based materials[END_REF][START_REF] Zou | The preparation of Mg-based hydro-reactive materials and their reactive properties in seawater[END_REF][START_REF] Kravchenko | Formation of hydrogen from oxidation of Mg, Mg alloys and mixture with Ni, Co, Cu and Fe in aqueous salt solutions[END_REF][START_REF] Oh | Fabrication of Mg-Ni-Sn alloys for fast hydrogen generation in seawater[END_REF][START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF][START_REF] Wang | Hydrolysis reaction of ball-milled Mg-metal chlorides composite for hydrogen generation for fuel cells[END_REF][START_REF] Sun | A study of hydrogen generation by reaction of an activated Mg-CoCl2 (magnesium-cobalt chloride) composite with pure water for portable applications[END_REF][START_REF] Liu | Hydrogen generation from the hydrolysis of Mg powder ball-milled with AlCl3[END_REF][START_REF] Grosjean | Hydrolysis of Mg-salt and MgH2-salt mixtures prepared by ball milling for hydrogen production[END_REF][START_REF] Alasmar | Hydrogen generation from Nd-Ni-Mg system by hydrolysis reaction[END_REF] whereas the "Mg17Al12 -H2" subject is not sufficiently investigated. Previous studies have shown that Mg17Al12 can be used for hydrogen storage [START_REF] Lee | Effects of Ni addition on hydrogen storage properties of Mg17Al12 alloy[END_REF][START_REF] Peng | Investigation on preparation and hydrogen storage performance of Mg17Al12 alloy[END_REF][START_REF] Crivello | Hydrogen absorption properties of the γ-Mg17Al12 phase and its Al-richer domain[END_REF][START_REF] Xiao | Hydrogen generation by CaH2-induced hydrolysis of Mg17Al12 hydride[END_REF] while the production of hydrogen by the hydrolysis reaction has never been reported.

Magnesium hydrolysis can be improved by ball milling with additives. Considering the similarity between Mg and Mg17Al12, the effect of ball milling on the hydrolysis performance (i.e. yield and kinetics) of Mg17Al12 using relatively cheap additives such as graphite, NaCl, MgCl2 and AlCl3 is relevant. Graphite improved the hydrolysis performance of Mg and WE43 due to the formation of a thin layer which limits the oxidation and prevents the adhesion of Mg(OH)2 to Mg surface [START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF].

Ball milling with 3 mol.% of NaCl was optimum to improve the hydrolysis performance of MgH2 where NaCl facilitates the reduction of the particles size and the creation of surface defects [START_REF] Li | Influence of chloride salts on hydrogen generation via hydrolysis of MgH2 prepared by hydriding combustion synthesis and mechanical milling[END_REF].

Total hydrolysis reaction (i.e. yield = 100%) was reported for Mg in the ball milled mixture Mg with 10 mol. % of MgCl2 [START_REF] Grosjean | Hydrolysis of Mg-salt and MgH2-salt mixtures prepared by ball milling for hydrogen production[END_REF]. The enhancement was attributed to the extra heat generated by the exothermic dissolution of MgCl2. Similarly, the exothermic dissolution of AlCl3 increases the temperature and generates HCl in the reaction medium which dissolve the formed Mg(OH)2 and improve the hydrolysis reaction [START_REF] Liu | Hydrogen generation from the hydrolysis of Mg powder ball-milled with AlCl3[END_REF].

The effects of ball milling time, heteromaterials (Graphite, NaCl, MgCl2 and AlCl3) addition during the mechanical process and the synergetic effect of both graphite and AlCl3 were also studied. To our knowledge, no previous work has investigated the hydrolysis of Mg17Al12; in order to better understand the hydrolysis of AZ magnesium alloy. Experimental results show that ball milling increases the hydrolysis performance of the intermetallic in a 3.5 wt.% NaCl solution (i.e. similar to seawater) especially when milled in presence of graphite and AlCl3 (e.g. simultaneous addition) leading to the best result with a hydrogen production of 136 mL.(g of powder) -1 which corresponds to a yield of 16%. The results are explained in terms of microstructural (XRD and SEM-EDS analysis) and morphological (SEM-Granulometry) effects.

Experimental Procedure

Mg17Al12 synthesis

Based on the binary phase diagram of Mg and Al, commercial magnesium and aluminum powders were mixed with 17:12 atomic ratio by an ultrasonic homogenizer. This homogeneous mixture was put into stainless-steel container and closed in a glovebox to get fully rid of air contamination.

Afterwards, the container was heated with a heating rate of 2 K/min up to 813K (higher than the congruent melting temperature of Mg17Al12 because the appearance of the liquid phase is beneficial for the diffusion process, which is the key for the alloying of Mg17Al12), and kept at this temperature for 2h. Then the temperature was decreased to 723K (melting temperature of Mg17Al12) with a rate of 4.5 K/h. Finally, cooling down to room temperature was done by turning off the furnace.

Ball milling

Graphite (G) (Fisher Scientific), sodium chloride NaCl (Fisher Scientific, 99.5%), anhydrous magnesium chloride MgCl2 (Aldrich, 98%) and anhydrous aluminum chloride AlCl3 (Acros organics, 98.5%) powders were used as milling additives. The chlorides were stored in a glove box to avoid hydration. Based on our previous studies [START_REF] Bacha | Hydrogen generation via hydrolysis of ball milled WE43 magnesium waste[END_REF][START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF], we have adopted the addition of 5 wt. % of additives (i.e. G, NaCl, MgCl2 or AlCl3) during ball milling. The Mg17Al12 -X (X = G, NaCl, MgCl2 or AlCl3) mixtures were prepared by ball milling (using a Fritsch Pulverisette ® 5) in a stainless-steel vial under argon with the sequence: 15 min of continuous milling followed by 2 min of rest (to allow the system to cool down). The ball to powder weight ratio was 17:1 corresponding to 1.18 g of powder and 5 stainless steel balls (10 mm of diameter) and the rotation speed was fixed to 250 rpm. The milling vial was handled in a glove box filled with purified argon.

Characterization

The hydrolysis reaction was carried out in a salted water solution (3.5 wt % NaCl) at room temperature (more details can be found in reference [START_REF] Tayeh | Production of hydrogen from magnesium hydrides hydrolysis[END_REF]). Hydrogen production is presented as the conversion yield (%), which is defined as the volume of produced hydrogen over the theoretical volume of hydrogen if Mg17Al12 powder is completely consumed.

The samples were characterized by X-ray diffraction (XRD) using a Philips PANalyticalX'Pert (PW1820) diffractometer with Cu Kα1 radiation (λ=1.5405Å). Crystallites sizes were estimated from XRD refinement using EVA software (according to the weighted Scherrer formula) and the cell parameters were evaluated from Rietveld Refinement using FullProf software. Scanning Electron Microscopy (SEM) coupled with Energy Dispersive Spectrometer (EDS) observations were made using a TESCAN VEGA3 SB microscope. Particles size distribution was evaluated by laser granulometry using a MASTERSIZER2000 from Malvern®.

Results and discussion

Hydrolysis reaction

Pure Mg17Al12 was synthesized as shown from X-ray diffraction pattern [START_REF] Bacha | Experimental evidence of H2 spillover during the corrosion of Mg17Al12[END_REF]. The hydrolysis reaction of Mg17Al12 was performed in 0.6M HCl until the complete dissolution of the intermetallic is achieved to evaluate the maximum generation of H2.

The X-ray diffractogram of the solid product obtained after neutralization with KOH and drying (Supplementary material figure 1) revealed the presence of Mg(OH)2, MgCl2.6H2O and Al(OH)3 and the absence of peaks related to Mg17Al12 justifying the total reaction. Mg17Al12 generates 85% of the theoretical H2 volume considering that both Mg and Al react (Supplementary material figure 2). During the hydrolysis reaction, OH -ions are generated in the solution increasing the medium's pH. According to Pourbaix diagram of magnesium at the Open Current Potential of Mg17Al12 (E= -1.2 V/ECS), the corrosion zone extends to pH = 8.5 while for aluminum, the passivation zone extends from pH=1.5 [START_REF] Pourbaix | Atlas of Electrochemical Equilibria in Aqueous Solutions[END_REF].

Through the hydrolysis reaction, Mg(OH)2 and Al(OH)3 protective films are formed preventing the diffusion of water to the unreacted Mg and Al atoms. Since Mg is more reactive than Al and Al(OH)3 is more protective than Mg(OH)2, we consider that all Mg atoms and 2/3 of Al atoms are oxidized (i.e. the number of reactive aluminum atoms is calculated based on the hydrolysis yield results).

Then, the equation for the hydrolysis reaction with Mg17Al12 can be written as:

Mg17Al12 + 58 H2O  17 Mg(OH)2 + 8 Al(OH)3 + 4 Al + 29 H2 (1)
The use of acids increases the corrosive effect but for practical application, handling with relatively neutral solutions such as seawater (i.e. aqueous NaCl solution) is preferable.

Ball milling time

The As-Cast Mg17Al12 barely reacts in 3.5 wt.% aqueous NaCl solution.

Figure 1: Hydrogen generation by: as-cast Mg17Al12, Mg17Al12 ball milled for 1h, 3h and 5h, and Mg17Al12 ball milled with graphite (denoted Mg17Al12 + G) for 1h, 3h and 5h.

Only 6% of the theoretical hydrogen volume are generated after 1 hour of reaction (figure 1). Ball milling of magnesium under inert atmosphere (i.e. Argon atmosphere) was proved beneficial to increase the hydrolysis reactivity of the materials. The effect of ball milling duration on the reactivity of Mg17Al12 was studied by varying the milling time (e.g. 1h, 3h and 5h).

Mg17Al12 has a cubic unit cell. The cell parameter of the synthesized Mg17Al12 is 10.53 Å and stay approximately constant after ball milling (maximum variation of 0.15%, cf figure 3).

7 Crystallites size, denoted "L", decrease from 74 nm to 23 nm after 1 hour of ball milling and barely decrease after 3h and 5h of ball milling (15 nm and 14 nm respectively) (figure 2). The medium (close to the average) particles size, denoted "d0.5", decreased from 500 μm to 40 μm after 5h of ball milling (figure 2). Considering that defects may act as hydrolysis initiators sites [START_REF] Li | Production of an Mg/Mg2Ni lamellar composite for generating H2 and the recycling of the post-H2 generation residue to nickel powder[END_REF], the average number of bulk defects in the milled material can be roughly estimated by dividing the particles volume by crystallites volume (denoted Vp/VL). From this estimation, we consider that all defects are equivalent while actually, when particle size decreases, more surface defects are formed which are more efficient than bulk defects. Therefore, Vp/VL value is better revealing when d0.5 and L varies slightly (and or when d0.5 is almost constant).

It is well known that ball milling is an effective way to create defects. Nevertheless, after 1h milling of Mg17Al12 the ratio Vp/VL decreases (from 321x10 9 to 0.5x10 9 ) which could indicate that the number of defects is decreasing. This is mainly due to particles size reduction. Figure 2 shows this ratio increases after 3h of milling to 1.2x10 9 indicating that more bulk defects are formed in addition to surface defects.

Figure 1 shows that increasing ball milling time enhances the hydrolysis performance of Mg17Al12. This is due to the destruction of the oxide layer formed on Mg17Al12 and to the decrease of crystallinity.

Ball milling reduces particles size and, therefore, increases the reactive surface area. The particle size decreases and the reactive area increases which improve the hydrolysis performance by favoring the dissolution of Mg(OH)2. This phenomenon is described by the Noyes-Whitney equation [START_REF] Hattori | Dissolution process analysis using model-free Noyes-Whitney integral equation[END_REF] where the rate of dissolution of a solid is proportional to its surface area (considering the diffusion layer thickness, the diffusion coefficient, the concentration of Mg(OH)2 in the bulk and in the diffusion layer as constants because the hydrolysis experiment is realized in the same conditions with the same NaCl solution).

Graphite addition

Ball milling with 5 wt. % of graphite improves the hydrolysis performance of Mg [START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF] and WE43 magnesium waste [START_REF] Bacha | Hydrogen generation via hydrolysis of ball milled WE43 magnesium waste[END_REF]. The effect of ball milling with graphite on the hydrolysis performance of Mg17Al12 was investigated by varying ball milling time (i.e. 1h, 3h and 5h) and graphite content (5 wt.% and 10 wt.%). As previously reported without G, the variation of the unit cell parameter (figure 3) is not significant since the maximum variation is 0.2%. Contrariwise, ball milling with graphite (5 wt.% and 10 wt.%) prevents the crystallite size reduction (figure 3). This phenomenon may be attributed to the lubricant effect of graphite [START_REF] Bacha | Hydrogen generation via hydrolysis of ball milled WE43 magnesium waste[END_REF][START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF]. It is worth pointing out that increasing graphite content up to 10 wt.% does not change the crystallite size variation and consequently the materials reactivity. Based on these results, the addition of 5 wt.% of graphite was adopted while varying the ball milling time. Figure 2 shows that adding graphite during ball milling of Mg17Al12 promotes the reduction of particle size due to the anti-sticking effect of graphite [START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF] and subsequently decreases the crystallinity.

Mg17Al12 milled with graphite for 1h, 3h and 5h presents the same hydrolysis performance with a generation of 7% of the theoretical H2 volume within 1 hour of reaction (Figure 1, e.g. Mg17Al12 milled for 5h produces 11% of the theoretical H2 volume). That means that addition of graphite is not beneficial for the hydrolysis performance of Mg17Al12. Ball milling with AlCl3 decreases particles size and crystallinity (figure 4). Increasing ball milling time with AlCl3 from 1h to 5h reduces the particle size from 22 μm to 11 μm and the crystallites size from 40 nm to 25 nm respectively. Considering Vp/VL ratio; the number of defects slightly decreases but it has to be considered that as the particles size decrease a lot, the number of surface defects will increase. Figure 5 shows that adding AlCl3 to Mg17Al12 enhances its hydrolysis performancea maximum yield of 14% was obtained with Mg17Al12 + AlCl3 5h. As seen from SEM analysis (figure 6), the presence of chlorides during ball milling creates cracks. It is known that the microstructural defects (e.g. cracks and crystallite boundaries) may act as hydrolysis initiators sites, withstand the continuous formation of the passive layer of Mg(OH)2 [START_REF] Li | Production of an Mg/Mg2Ni lamellar composite for generating H2 and the recycling of the post-H2 generation residue to nickel powder[END_REF], and enhance the pitting corrosion by the localized enrichment in chlorides [START_REF] Grosjean | Hydrogen production from highly corroding Mg-based materials elaborated by ball milling[END_REF]. Additionally, AlCl3 is a strong water-absorbing agent and almost completely undergoes hydrolysis in water to generate chloride acid, which favor the dissolution of the formed Mg(OH)2 [START_REF] Liu | Hydrogen generation from the hydrolysis of Mg powder ball-milled with AlCl3[END_REF][START_REF] Buryakovskaya | Hydrogen production properties of magnesium and magnesium-based materials at low temperatures in reaction with aqueous solutions[END_REF]. Indeed, the pH of the solution increases in the first 5 As observed from SEM analysis (figure 6 and supplementary material figure 3), ball milling in the presence of AlCl3, MgCl2 and NaCl for 5h slightly decreases the particles size favoring more fresh reactive Mg17Al12 surfaces (compared to Mg17Al12 5h) . Cracks were also clearly observed in Mg17Al12 + AlCl3 5h (e.g. red circle on figure 6 down left and supplementary material figure 3). Addition of MgCl2 leads to better hydrolysis performance of Mg17Al12 than addition of NaCl (Figure 7). These results may be attributed to the exothermic dissolution of MgCl2 in the hydrolysis medium (ΔH = -155 kJ.mol -1 ) [START_REF] Grosjean | Hydrolysis of Mg-salt and MgH2-salt mixtures prepared by ball milling for hydrogen production[END_REF]. On the other hand, adding NaCl to Mg17Al12 does not favor its hydrolysis performance as NaCl dissolution is endothermic [START_REF] Grosjean | Hydrolysis of Mg-salt and MgH2-salt mixtures prepared by ball milling for hydrogen production[END_REF].

Chlorides addition

The effect of pH overrides the effect of the exothermicity of the dissolution of salts.

Synergetic effect of Graphite and AlCl3

The addition of 5 wt.% of 2 additives (Graphite and Nickel) was found to enhance Mg [START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF] and WE43 [START_REF] Bacha | Hydrogen generation via hydrolysis of ball milled WE43 magnesium waste[END_REF] hydrolysis rate. Previously, we demonstrated that ball milling with AlCl3 gives the best hydrolysis performance for Mg17Al12. Considering that the role of graphite and AlCl3 are different in term of enhancing the hydrolysis reaction, the synergetic effect of graphite and AlCl3 was studied.

Increasing ball milling time from 1h to 5h with addition of 5 wt.% of graphite and 5 wt.% of AlCl3 improve the hydrolysis performance. Mg17Al12 + G + AlCl3 BM 1h, 3h and 5 h generates 10%, 12% and 16% of the theoretical H2 volume respectively (figure 7). Particles size and crystallinity of Mg17Al12 decrease when increasing ball milling time from 1h to 5h. Figure 8 shows that graphite particles tend to agglomerate after prolonged ball milling (i.e. 5h).

The same phenomenon was observed in a previous work [START_REF] Bacha | Hydrogen generation via hydrolysis of ball milled WE43 magnesium waste[END_REF]. Adding AlCl3 reduces particles size and favor cracks formation (section 3.4) and graphite may form a fine protective layer on the fresh surface [START_REF] Bacha | Hydrogen generation via hydrolysis of ball milled WE43 magnesium waste[END_REF][START_REF] Awad | Effect of carbons (G and CFs), TM (Ni, Fe and Al) and oxides (Nb2O5 and V2O5) on hydrogen generation from ball milled Mg-based hydrolysis reaction for fuel cell[END_REF].

It is worth pointing out that milling Mg17Al12 with graphite and AlCl3 gives the best hydrolysis performance with a generation of 16% of the theoretical H2 volume (which is almost triple that obtained for as cast Mg17Al12) within 1 hour of reaction.

Conclusion

In this work, the hydrolysis reaction of Mg17Al12 was investigated. The as-cast intermetallic produces 6% of the theoretical hydrogen volume. Ball milling was performed in order to enhance the hydrolysis performance. Increasing ball milling time to 5h improves the hydrolysis performance with 11% reached in 1 hour. Upon all the tested additives (i.e. graphite, NaCl, MgCl2 and AlCl3), AlCl3 showed the best performance with a generation of 14% of the theoretical hydrogen volume. This is attributed to the dissolution of AlCl3, by generating HCl in the hydrolysis medium, which favor the dissolution of the formed Mg(OH)2. Moreover, the extra heat released through the exothermic salt dissolution (i.e. MgCl2 and AlCl3) would also help to increase the yield and kinetics of the hydrolysis reaction. The addition of graphite and AlCl3 simultaneously during ball milling of Mg17Al12 further enhances its hydrolysis with a generation of 16% of the theoretical H2 volume. Based on our findings, a total hydrolysis of AZ alloys can not be achieved since, in the best case, 85% of Mg17Al12 does not react in 3.5 wt.% NaCl solution.

It is then worth pointing out that the hydrolysis of Mg17Al12 can be enhanced by ball milling with cheap additives such as graphite and AlCl3. Subsequently, by following the same procedure (i.e.

ball milling with additives), the hydrolysis of AZ magnesium alloys will be further improved.

Therefore, this process can enable the low-cost production of pure hydrogen for fuel cells from magnesium alloys waste.

Figure 2 :

 2 Figure 2: Variation of the crystallite size (L), the medium particles size (d0.5) and the number of crystallite boundaries per particle (Vp/VL) of as-cast Mg17Al12, Mg17Al12 after ball milling for 1h, 3h and 5h and Mg17Al12 after ball milling with graphite (denoted Mg17Al12 + G) for 1h, 3h and 5h.

Figure 3 :

 3 Figure 3: Variation of the unit cell parameter "a" (relative variation) symbolized by • and the crystallite size "L" symbolized by ■ of the as-cast Mg17Al12, Mg17Al12 after ball milling without graphite (0 wt.% G), with 5 wt.% G and with 10 wt.% G for 1h, 3h and 5h.

Figure 4 :

 4 Figure 4: Variation of the crystallite size (L), the medium particles size (d0.5) and the number of crystallite boundaries per particle (Vp/VL) of as-cast Mg17Al12, Mg17Al12 after ball milling for 1h, 3h and 5h and Mg17Al12 after ball milling with AlCl3 (denoted Mg17Al12 + AlCl3) for 1h, 3h and 5h.

Figure 5 :

 5 Figure 5: Hydrogen generation for hydrolysis with Mg17Al12 after ball milling for 1h, 3h and 5h and Mg17Al12 after ball milling with AlCl3 (denoted Mg17Al12 + AlCl3) for 1h, 3h and 5h and pH monitoring during the hydrolysis of Mg17Al12 + AlCl3 5h.

  min of the hydrolysis reaction and reaches a value of 10 indicating the rapid formation of Mg(OH)2. The exothermic dissolution of AlCl3 increases the local temperature (ΔH = -223 kJ.mol - 1 )[START_REF] Liu | Hydrogen generation from the hydrolysis of Mg powder ball-milled with AlCl3[END_REF][START_REF] Grosjean | Hydrolysis of Mg-salt and MgH2-salt mixtures prepared by ball milling for hydrogen production[END_REF] at the interface Mg17Al12/H2O and generates HCl through the following reaction[START_REF] Liu | Hydrogen generation from the hydrolysis of Mg powder ball-milled with AlCl3[END_REF]:AlCl3 + 3 H2O  Al(OH)3 + 3 HCl (2)and shifts the final theoretical pH value from 10.8 (pH at the Mg 2+ /Mg(OH)2 equilibrium) to 9.7.In fact, the pH of the solution can be calculated following the Henderson-Hasselbalch equation for a weak base: pH = pKa + log( the molar solubility of the undissociated Mg(OH)2, S is the molar concentration of the dissociated Mg(OH)2 and pKa is that of Mg 2+ /Mg(OH)2. When pH shifts to lower values, the solubility of Mg(OH)2 in the medium increases.Based on our previous results, the reactivity of Mg17Al12 ball milled with MgCl2 and NaCl for 5hwas compared to that of Mg17Al12 ball milled 5h with AlCl3. It is well known that the presence of chlorides additives improves the efficiency of the milling process by creating microstructural defects and enabling the reduction of particles size thus increasing the surface area[START_REF] Li | Influence of chloride salts on hydrogen generation via hydrolysis of MgH2 prepared by hydriding combustion synthesis and mechanical milling[END_REF].

Figure 6 :

 6 Figure 6: SEM images of Mg17Al12 5h, Mg17Al12 + NaCl 5h, Mg17Al12 + AlCl3 5h and Mg17Al12 + MgCl2 5h.

Figure 7 :

 7 Figure 7: Hydrogen generation for hydrolysis with as-cast Mg17Al12, Mg17Al12 5h, Mg17Al12 + G 5h, Mg17Al12 + AlCl3 5h, Mg17Al12 + MgCl2 5h, Mg17Al12 + NaCl 5h and Mg17Al12 + G + AlCl3 5h.

Figure 8 :

 8 Figure 8 : SEM and EDX mapping of Mg17Al12 + AlCl3 5h.

Acknowledgments

This work was financially supported by the AZM & SAADE Association and the Lebanese University through a scholarship to S. Al Bacha. Authors also thank Lisea Carbone foundation for financial support and also Fondation Bordeaux Université (Mr Adrien Le Léon and Mrs Iryna Danylyshyna) for saluable help on administratives duty.