Alexandre Besson

Cécile Formosa-Dague

Pascal Guiraud
email: pguiraud@insa-toulouse.fr

Prof Pascal Guiraud

Flocculation-flotation harvesting mechanism of Dunaliella salina: from nanoscale interpretation to industrial optimization

Keywords: Dunaliella salina, Harvesting, Flocculation, Flotation, Atomic force microscopy Besson et

Dunaliella salina is a green microalgae species industrially exploited for its capacity to produce important amounts of carotenoid pigments. However in low nitrogen conditions in which they produce these pigments, their concentration is low, which results in harvesting difficulties and high costs. In this work, we propose a new solution to efficiently harvest D. salina at the pre-industrial scale, using flocculation/flotation harvesting induced by NaOH addition in the medium. We first show, using numerical simulations and nanoscale atomic force spectroscopy experiments, that sweeping mechanism in formed magnesium hydroxide precipitate is only responsible for D. salina flocculation in hypersaline culture medium upon NaOH addition. Based on this understanding of the flocculation mechanism, we then evaluate the influence of several parameters related to NaOH mixing and magnesium hydroxide precipitation and show that NaOH concentration, mixing, and salinity of the medium can be optimized to achieve high flocculation/flotation harvesting efficiencies in laboratory-scale experiments. We finally successfully scale-up the data obtained at lab-scale to a continuous pre-industrial flotation pilot, and achieve up to 80% of cell recovery. This interdisciplinary study thus provides original results, from the nano-to the pre-industrial scale, which allow the successful development of an efficient large-scale D. salina harvesting process. We thus anticipate our results to be the starting point for further optimization and industrial use of this flocculation/flotation harvesting technique.

Introduction

Microalgae are receiving increasing attention worldwide as an alternative and renewable source of energy because of their eminent oil producing capacity [START_REF] Pragya | A review on harvesting, oil extraction and biofuels production technologies from microalgae[END_REF]. But the potential of microalgae is in fact even greater and they also represent an important source of biomass and of molecules of interest for the fields of food, feed or health. Indeed, microalgae are unique microorganisms which convert light energy, water and inorganic nutrients into biomass resource rich in value-added products such as lipids, carbohydrates, proteins and pigments [START_REF] Minhas | A Review on the Assessment of Stress Conditions for Simultaneous Production of Microalgal Lipids and Carotenoids[END_REF][START_REF] Pragya | A review on harvesting, oil extraction and biofuels production technologies from microalgae[END_REF]. Among the wide diversity of microalgae [START_REF] Metting | Biodiversity and application of microalgae[END_REF], Dunaliella salina, a halotolerant green microalgae, is among the species currently industrially exploited, as it is one of the most important producer of natural β-carotene pigments [START_REF] Pirwitz | Comparison of flocculation methods for harvesting Dunaliella[END_REF][START_REF] Ambati | Industrial potential of carotenoid pigments from microalgae: Current trends and future prospects[END_REF]. Because of this specificity, D. salina has been the subject of many studies dedicated to the optimization of its culture conditions [START_REF] Prieto | Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes[END_REF][START_REF] Kim | Optimization of culture conditions and comparison of biomass productivity of three green algae[END_REF][START_REF] Khadim | Mass cultivation of Dunaliella salina in a flat plate photobioreactor and its effective harvesting[END_REF][START_REF] Béchet | Full-scale validation of an algal productivity model including nitrogen limitation[END_REF] and of its harvesting [START_REF] Besson | High-pH-induced flocculation-flotation of the hypersaline microalga Dunaliella salina[END_REF][START_REF] Pirwitz | Comparison of flocculation methods for harvesting Dunaliella[END_REF][START_REF] Xiong | Facile sand enhanced electro-flocculation for cost-efficient harvesting of Dunaliella salina[END_REF].

Moreover, other studies have showed the utility of this microalgae species in genetic and metabolic engineering [START_REF] Feng | Dunaliella salina as a novel host for the production of recombinant proteins[END_REF][START_REF] Anila | Metabolic engineering of Dunaliella salina for production of ketocarotenoids[END_REF], and also recently in concrete biomedical applications, for example to reduce tumor growth in mice [START_REF] Srinivasan | Oral administration of lyophilized Dunaliella salina, a carotenoid-rich marine alga, reduces tumor progression in mammary cancer induced rats[END_REF].

However, under β-carotene production culture conditions (nitrogen deficiency conditions), D. salina reaches low cell concentrations which thus has consequences on the energy input and overall cost of their separation from water in industrial processes. In a general manner, several methods can be used for microalgae harvesting, including centrifugation, filtration, flocculation and flotation [START_REF] Garg | Flotation of marine microalgae: effect of algal hydrophobicity[END_REF]. However, most of these methods are synonymous with high costs and energy consumption, often for low efficiency rates. For example, centrifugation, the most commonly used method, consumes a large amount of energy and can cause damages to the cells because of high shear forces [START_REF] Pragya | A review on harvesting, oil extraction and biofuels production technologies from microalgae[END_REF]. Filtration involves using membranes, which, in the case of microalgae separation, can get clogged because of the small size of the cells and/or of the exopolysaccharides they secrete, resulting in high operating costs (Uduman et al., 2010). As for flocculation, it seems to be a promising low-cost approach for large-scale harvesting; however, contamination is a major issue in this technique, as the chemical flocculants classically used to induce flocculation end up in the harvested biomass, and can interfere with the final application of the biomass (food or feed) (Vandamme et al., 2013).

In this context, flotation is believed to be a promising harvesting technique that takes advantage of microalgae's natural low density and self-floating tendency [START_REF] Garg | Flotation of marine microalgae: effect of algal hydrophobicity[END_REF]. Assisted flotation consists in air or gas transformed into bubbles rising through a microalgae suspension. As a result, microalgae cells get attached to bubbles and are carried out and accumulated on the surface.

Thus flotation allows for low-cost cell harvesting, without necessarily using flocculants that could damage the cells. In addition, it is a relatively rapid operation, which needs low space and has moderate operational costs. However, as both the surface of microalgae and bubbles present a negative charge in aqueous medium [START_REF] Yang | Measurement of the Zeta Potential of Gas Bubbles in Aqueous Solutions by Microelectrophoresis Method[END_REF][START_REF] Garg | Flotation separation of marine microalgae from aqueous medium[END_REF], and given the low hydrophobicity of microalgae cells, the interactions between cells and bubbles are repulsive, which prevents adhesion, and thus capture and flotation, resulting in the poor efficiency of this harvesting technique.

Among the possible strategies to enhance flotation efficiency, natural auto-flocculation is an interesting alternative. There are several known auto-flocculation mechanisms, among which one is based on the precipitation of magnesium ions into magnesium hydroxide at high pH (Sukenik and Shelef, 1984). Such increased pH in the culture medium can result either from the photosynthesis activity of the cells or from the direct addition of OH -. Then the flocculation of the cells can occur through charge neutralization, i.e. through the interaction between the positively charged formed magnesium hydroxide precipitates and cells, through sweeping where cells are entrapped in the massive precipitation of the magnesium hydroxide in the medium, or both (Vandamme et al., 2013).

Auto-flocculation of cells at high pH using NaOH addition has already been described for different microalgae species, such as Chlorella vulgaris [START_REF] Wu | Evaluation of flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium[END_REF], or Phaeodactylum tricornutum [START_REF] Wu | Evaluation of flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium[END_REF][START_REF] Vandamme | Alkaline flocculation of Phaeodactylum tricornutum induced by brucite and calcite[END_REF][START_REF] Formosa-Dague | Towards a better understanding of the flocculation/flotation mechanism of the marine microalgae Phaeodactylum tricornutum under increased pH using atomic force microscopy[END_REF] and has been showed to occur by both sweeping flocculation and charge neutralization. In the case of D. salina, a previous study that we conducted in 2013 showed that addition of NaOH was necessary to increase the pH in the medium, and that further flocculation was caused by sweeping of the cells by forming magnesium hydroxide precipitate [START_REF] Besson | High-pH-induced flocculation-flotation of the hypersaline microalga Dunaliella salina[END_REF]. In the experimental conditions used in this study, the more NaOH was added, the more magnesium hydroxide was precipitated and the more efficient the flocculation was; a cell recovery up to 100% could be achieved using further flotation. The results obtained also showed that the NaOH flow rate had no influence on the flocculation and further flotation recovery; however they suggested that the type of injection of NaOH, and thus it's mixing in the medium and the speed of magnesium hydroxide precipitation, could have an influence in the final harvesting efficiency.

In the work presented here, we will first evidence the only contribution of sweeping mechanism in the flocculation of cells induced by the precipitation of magnesium hydroxide at high pH, and exclude the role of charge neutralization. For that we will first simulate the precipitation of magnesium hydroxide in the medium as a function of the NaOH concentration to understand the behavior of the culture medium during flocculation. Then, we will use recent developments made in the team that consist in using atomic force microscopy (AFM) [START_REF] Binnig | Atomic Force Microscope[END_REF] to probe the interactions between microalgae cells and particles [START_REF] Formosa-Dague | Towards a better understanding of the flocculation/flotation mechanism of the marine microalgae Phaeodactylum tricornutum under increased pH using atomic force microscopy[END_REF]. For that, AFM tips functionalized with magnesium hydroxides particles will be used in force spectroscopy experiments to measure directly, at the piconewton scale, the interactions between these particles and the cells.

Then, knowing precisely the flocculation mechanism at play, we will be able to optimize parameters to harvest, in the most effective possible way, D. salina cells at laboratory scale but also at preindustrial scales (suspension flow rate 0.6 m 3 /h). For that, we will complete our previous study and determine the role of NaOH mixing in the medium by testing different injection types, agitation and medium salinity, and by evaluating their effects on the flocculation/flotation recovery of the cells at laboratory scale. Based on these results and their interpretation, we will adapt and optimize an injection system to efficiently flocculate and harvest cells by flotation in a pre-industrial continuous cultivation system. This way we will provide a cost-effective solution to efficiently harvest lowconcentrated D. salina cells at industrial scales, thus contributing to the industrial valorization of this β-carotene rich biomass.

Material and methods

Strain and culture conditions

Dunaliella salina strain CCAP19/25 (Culture Collection of Algae and Protozoa) was cultivated in dechlorinated tap water containing the following: NaCl (107 g/L); MgSO 4 .7H 2 O (4.8 g/L); MgCl 2 .6H 2 O (4.0 g/L); CaCl 2 (1.1 g/L); KCl (0.1 g/L). A complete nutritive Conway medium (without silicates) [START_REF] Walne | Studies on the food value of nineteen genera of algae to juvenile bivavlves of the genera Ostrea, Crassostrea, Mercenaria and Mytillus[END_REF] was also added. The salinity of this synthetic water is of 10%, the ionic strength of 2.3. This medium was used for all the experiments performed in laboratory. It mimics the medium resulting from the evaporation of see water in which D. salina can grow with a limited competition from other microorganisms, used in pre-industrial scale cultures. A D. salina strain isolated from saline ponds in Gruissan, Occitanie, France, was also used to evaluate the influence of the strain on the harvesting efficiencies.

Laboratory-scale cultures: For AFM experiments, cells were cultivated at 20°C, under agitation (120 rpm), in 75 mL non-coated culture flasks (15 mL of culture), 500 mL non-coated culture flasks (150 mL of culture). The incubator was equipped with white neon light tubes providing illumination of approximately 40 µmol photons m -2 s -1 with a photoperiod of 12h light : 12h dark. For flotation experiments, D. salina culture was achieved in 10 L glass photobioreactors. The culture was continuously agitated by the gentle bubbling of 0.2 µm filtered atmospheric air (2 L/min).

Illumination was provided by daylight fluorescent tubes (OSRAM FQ 965 Biolux) with a photoperiod of 16h light : 8h dark. The temperature of the culture was regulated by air conditioning at 21 ± 3°C.

Pre-industrial scale cultures: The pre-industrial experimental site of the Salinalgue research project (2010)(2011)(2012)(2013)(2014) was located in a salt marsh on the Mediterranean Sea coast, at Gruissan, Occitanie, France (Latitude: 43.094 N; Longitude: 3.084 E), as fully described in Béchet et al. [START_REF] Béchet | Full-scale validation of an algal productivity model including nitrogen limitation[END_REF]. Briefly, cultures were achieved in 4 outdoor raceways of 250 m², containing between 30 and 100 m 3 . See water concentrated by sun evaporation (salinity of 12%) and disinfected with bleach fed the raceways before being inoculated. The raceways were mixed by paddle wheels and by suspension pumping which was also used to limit the pH to 7.4 by a monitored addition of dissolved CO 2 in the recirculated suspension.

Precipitation model for hydroxide magnesium in the culture medium used

The concentration of magnesium hydroxide, calcium carbonate and dolomite were modeled as a function of the added NaOH concentration in the culture medium. To do so, we simulated the NaOH addition effects and developed a numerical model, using the software Phreeqc, based on the Pitzer equations to simulate the precipitate formation in our culture medium. Indeed, in order to assess the equilibrium state of hypersaline solutions in which D. salina is grown and understand the precipitation phenomena involved during NaOH addition, it is necessary to evaluate the activity of species in solution. This requires the evaluation of coefficients of activity, which take into account the various ionic interactions in these complex environments. The models for calculating these activity coefficients are generally based on the coupling of the Debye-Hückel [START_REF] Debye | The theory of electrolytes. I. Lowering of freezing point and related phenomena[END_REF] theory translating electrostatic ionic interactions, and of the theory of the ionic association that takes into account ionic interactions at short distances. These models are well suited for solutions with ionic strength that does not exceed the one of regular seawater (ionic strength of 0.7). But since the culture of D. salina is carried out in a hypersaline environment, the ionic forces encountered in its culture medium exceed the areas of validity of the different ion association models (for a 10% salinity culture medium, the ionic strength is of 2.3). Therefore, the models obtained with these theoretical descriptions of saltwater thermodynamics are not satisfactory for concentrated saline water. In the case of D. salina, it was thus necessary to implement other types of models, known as specific ion interaction models, such as the one proposed by Pitzer. The Pitzer model is based on a different thermodynamic approach, and is well suited to evaluate the thermodynamic properties of hypersaline solutions. The model used is based on the Pitzer equations, and allows to simulate the ionic equilibria during the injection of NaOH in our culture medium. The models developed by Pitzer [START_REF] Pitzer | Thermodynamics of electrolytes. I. Theoretical basis and general equations[END_REF]Pitzer andMayorga, 1973, 1974;[START_REF] Pitzer | Thermodynamics of electrolytes. IV. Activity and osmotic coefficients for mixed electrolytes[END_REF][START_REF] Pitzer | Thermodynamics of electrolytes. V. Effects of higher-order electrostatic terms[END_REF] describes the specific ionic interactions of diverse species in complex media, at high ionic strength. For pure components, this model uses the parameters given by (Pitzer andMayorga, 1973, 1974). The asymmetric mixing parameters come from Pitzer et and Kim [START_REF] Pitzer | Thermodynamics of electrolytes. IV. Activity and osmotic coefficients for mixed electrolytes[END_REF][START_REF] Pitzer | Thermodynamics of electrolytes. V. Effects of higher-order electrostatic terms[END_REF]. The whole set of parameters were validated and listed by [START_REF] Harvie | The prediction of mineral solubilities in natural waters: The Na-K-Mg-Ca-H-Cl-SO4-OH-HCO3-CO3-CO2-H2O system to high ionic strengths at 25°C[END_REF], the relevant database being "pitzer.dat" This database accounts for the significant elements for Mediterranean see water (Na-K-Mg-Ca-H-Cl-S-O-C-Fe-Mn-Ba-Sr-B-Li-Br) with a large spectrum of possible precipitates. Before its application for the simulations presented here, the model has been successfully [START_REF] Besson | Etude multi-échelle de la récolte de Dunaliella salina-Développement d'un procédé d'autofloculation-flottation de microalgues[END_REF] compared to the experimental results of sea water evaporation proposed by Baseggio [START_REF] Baseggio | The composition of sea water and its concentrates[END_REF]. The composition of the culture medium used for the simulations (Table 1) was entered using a temperature of 25°C and a total dissolved carbon concentration of 0.00021 mol/kg_water.

Flotation experiments

Due to the small size of the microalgae, Dissolved Air Flotation (DAF) was used to harvest D. salina after NaOH-induced flocculation. Figure 1 presents the DAF devices used in this study.

Laboratory scale: A detailed presentation of the laboratory scale flotation experiments can be found in [START_REF] Besson | High-pH-induced flocculation-flotation of the hypersaline microalga Dunaliella salina[END_REF]. DAF experiments were performed in a Multiplace Orchidis™ FTH3 Flottatest. Three flotation-test beakers were run simultaneously, in which 600 mL samples were collected from the algal culture and added to each beaker. Then NaOH was added: in the case of direct NaOH injection, x mL of NaOH at a concentration of 1M was added to the microalgae suspension, as well as 100 -x mL of distilled water. In the case of the diluted NaOH injection, a unique solution containing x mL of NaOH at a concentration of 1M and 100 -x mL of distilled water was added to the microalgae suspension. The volume of NaOH (x) added was calculated depending on the final concentration wanted. This procedure is presented in figure 4a.

The depressurization at atmospheric pressure of 200 mL culture medium free of algae and saturated by air at 6 bars for 15 min induced the formation of microbubbles. The recycle ratio (pressurized culture medium volume/initial sample volume) was of 33%. For flocculation, the concentration of the added NaOH solution was calculated taking into account the volume of the culture and the volume of the added white water, so that the pH does not change upon addition of the white waters.

Pre-industrial scale: The pre-industrial DAF system was adapted from the CY1 flotation unit proposed by Sérinol (Bram, Occitanie, France), and is presented in produced in these saline waters have a diameter smaller than 60 µm [START_REF] Besson | Effects of pH adjustment on Dissolved Air Flotation harvesting of the hypersaline microalga Dunaliella salina[END_REF].

NaOH solutions at different concentrations in distilled water were added at different injection places on the supply line at a controlled flow injection using a peristaltic pump. Given that the microalgae suspension flow rate is of 300 L/h, and the final NaOH concentration is of 0.02 mol/L, if a NaOH solution of 0.1 mol/L is injected, the flowrate is of 66 L/h, and if a NaOH solution of 0.2 mol/L is injected, the flowrate is of 0.33 L/h. Flowrates were measured thanks to Khrone Optif lux -4100 electromagnetic flowmeters. For the operating conditions, microalgae suspensions were extracted from the external raceways using a peristaltic pump, into the flotation unit. Simultaneously, the pressurization system was launched and the NaOH solutions were injected on the supply line. The scrapping of the microalgae started as soon as the pressurization system was launched. After one hour at constant operating parameters (three times the time of the continuous state settling), samples required to quantify the separation efficiency (treated suspension) were harvested: the harvesting efficiency was then measured as described below.

Harvesting efficiency quantification

Harvesting efficiency quantification is based on optical density measurements. The harvesting efficiency represents the quantity of algae floated compared to the quantity in the initial suspension: it was evaluated using the following equation:

E % = 1 - OD V OD V × 100
Where ODi and Vi are the initial optical density at 800 nm and the volume of algal suspension before NaOH addition and flotation, ODa and Va are the optical density at 800 nm of the aqueous phase and the volume of the aqueous phase after injection of pressurized water.

Zeta potential measurements

The global electrical properties of D. salina cell surface were assessed by measuring the electrophoretic mobility which corresponds to the velocity of suspended cells exposed to an electric field. To this end, microalgae were harvested by centrifugation (1500 rpm, 3 min), washed two times in sorbitol buffer 375 mM at pH = 10 and resuspended in the same buffer at a final concentration of 1.5 × 10 6 cell/mL. Using this procedure, electrolytes present in the culture medium do not interfere and only the surface charge of the cells is measured. The electrophoretic mobility was then measured using an automated laser zetameter (Zetasizer NanoZS, Malvern Instruments). Cell suspensions coming from 2 independent cultures were analyzed.

AFM tip functionalization with hydroxides

To prepare functionalized AFM with Mg(OH) 2 , MLCT AUWH tips were first dipped into a thin layer of UV-curable glue (NOA63, Norland Edmund Optics), then into a thin layer of Mg(OH) 2 particles (Sigma-Aldrich) deposited on a glass slide. Functionalized tips were then put under UV-light for 10 min to allow the glue to cure.

AFM imaging and force spectroscopy experiments

Before AFM experiments, cells were harvested by centrifugation (3000 rpm, 10 min) and washed two times in sorbitol buffer 375 mM at pH = 10. This salt-free buffer is used for force spectroscopy experiments as it allows to accurately measure interactions between the cells and magnesium hydroxide particles functionalized on the AFM tips without introducing a bias from electrolytes present in D. salina culture medium. Moreover, the sorbitol present in this buffer keeps the cells from exploding because of the osmotic pressure. Finally, the pH of 10 allows to reproduce the conditions in which the cells are during flocculation induced by addition of NaOH. Cells were then immobilized on polyethylenimine (PEI, Sigma P3143) coated glass slide prepared as previously described [START_REF] Francius | Nanostructure and nanomechanics of live Phaeodactylum tricornutum morphotypes[END_REF]. Briefly, freshly oxygen activated glass slides were covered by a 0.2% PEI solution in deionized water and left for incubation overnight. Then the glass slides were rinsed with deionized water and dried under nitrogen. A total of 1 mL of cell suspension was then deposited on the PEI slides, allowed to stand for 30 min at room temperature, and rinsed with sorbitol buffer 375 mM at pH = 10. For force spectroscopy experiments, MLCT AUWH cantilevers with a nominal spring constant of 0.01 N/m, functionalized with hydroxides or not, were used at a constant applied force of 0.25 nN. The cantilevers spring constants were determined using the thermal noise method [START_REF] Hutter | Calibration of atomic-force microscope tips[END_REF]) before each experiment.

Results and discussion

Precipitation modeling and AFM force spectroscopy confirm that D. salina is flocculated by sweeping in magnesium hydroxide precipitate at high pH

In our previous study in 2013 [START_REF] Besson | High-pH-induced flocculation-flotation of the hypersaline microalga Dunaliella salina[END_REF], our work evidenced the positive effect of NaOH addition on the flocculation of D. salina. Our main results showed indeed an increase in the harvesting efficiency with the addition of NaOH into the medium, up to 80% of cell recovery.

While the hypothesis that the flocculation occurred through sweeping in magnesium hydroxide precipitate could be formulated, notably thanks to ions chromatography experiments performed on the suspension before and after flocculation/flotation, no proof of this mechanism were brought.

The first part of this study thus focus on providing a full understanding of the NaOH-induced flocculation mechanism. For that we first assessed the equilibrium state of the hypersaline solutions in which D. salina is grown to understand the precipitation phenomena involved when flocculating this microalgae using NaOH addition. For that, we used the model described in section 2.2, elaborated from the Pitzer model, to successfully simulate the influence of the addition of NaOH on the ionic equilibria in our culture medium described in Table 1. Results are presented in Figure 2: on this graph, the precipitation of magnesium hydroxide Mg(OH) 2 is plotted as a function of the added NaOH concentration. The resulting pH as well as two other precipitate candidate concentrations are also presented. Even if the measure of pH in hypersaline solutions with a high ionic strength presents some problems, the simulated pH profile is similar to experimental measurements, as the one showed in our previous study [START_REF] Besson | High-pH-induced flocculation-flotation of the hypersaline microalga Dunaliella salina[END_REF]. The pH sharply increases until the beginning of the precipitation of the magnesium hydroxide. Then, it continues to slightly increase until the end of the Mg(OH) 2 precipitation, that is to say when all the Mg present in the solution is used. Thus this shows that upon addition of NaOH, magnesium hydroxide precipitation increases until reaching a maximum for a NaOH concentration between 0.08 and 0.1 mol/kg of water, while calcium carbonates precipitation remains non-significant. Thus we can conclude from this simulation that the hypothesis made in our previous study, stating that flocculation of D. salina at increased pH, under hypersaline conditions at least, results from the precipitation of only magnesium hydroxide at high pH, among a great number of other possible salts, is correct. While this precipitation creates a gel that entraps the cells and flocculate them through sweeping, the question is now to know if charge neutralization is also involved in this flocculation mechanism, thus to have a full understanding of it.

To answer this question we used AFM and performed force spectroscopy experiments using functionalized tips with magnesium hydroxides particles. In this type of experiments, the AFM tip is moved towards the surface until touching it, and then retracted. If an interaction takes place between the tip and the sample, upon retraction, the tip will bend until the force applied is higher than the force of the interaction and the interaction breaks. The tip then goes back to its initial position, which is materialized on AFM retract force curves as a peak, referred to as retract adhesion.

The results of these experiments are presented in figure 3, they show in the case of bare AFM tips no adhesion between the tip and the cells, as seen on the retract force curves which present no retract adhesions (Figure 3a and b, n=2400 curves recorded on 6 cells from 2 independent cultures). In the case of tips functionalized by magnesium hydroxides (Figure 3C andd), force curves also show no retract adhesions, thus demonstrating that hydroxide particles do not interact with cells (n=3200 curves recorded on 8 cells coming from 2 independent cultures, with 7 different Mg(OH) 2 tips). Note that in order to avoid any interaction between electrolytes in the medium and the AFM tips, experiments were performed in a salt-free buffer at pH=10 to make sure only the interactions between the cells and the tips are probed. As no interactions were probed in this buffer, it is then evident that in the hypersaline waters in which D. salina grows, these interactions do not occur neither given the high quantity of charged ions that can screen both cells and Mg(OH) 2 particles charges. To give an explanation to this absence of interactions between the cells and Mg(OH) 2 particles, we then performed zeta potential measurements of D. salina cells in the same salt-free buffer at high pH (pH = 10), and measured a surface charge of -15.4 ± 0.9 mV (n=8 measurements on cell suspensions coming from 2 independent cultures). Thus the cells do not present a sufficient negative surface charge in our conditions, and do not interact with the positive surface of magnesium hydroxide [START_REF] Lin | Adsorption of dyes using magnesium hydroxide-modified diatomite[END_REF].

Therefore thanks to these simulations and AFM experiments, we are able to confirm the hypothesis we made in our previous study, and show that addition of NaOH in the culture medium precipitates only magnesium hydroxide. This precipitation results in the formation of a gel that is only responsible for entrapping the cells and flocculating them, as AFM experiments proved that charge neutralization is not involved in the case of D. salina. Moreover, in further experiments that we performed, Mg(OH) 2 already formed was directly added to D. salina cultures, which resulted in no flocculation of the cells, thus reinforcing our nanoscale conclusions. This is an interesting point as for all the microalgae species for which pH-induced flocculation has been described, charge neutralization mechanism is always involved (Sukenik and Shelef, 1984;[START_REF] Wu | Evaluation of flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium[END_REF]Vandamme et al., 2012;[START_REF] Nguyen | Harvesting Chlorella vulgaris by natural increase in pH: effect of medium composition[END_REF][START_REF] Vandamme | Alkaline flocculation of Phaeodactylum tricornutum induced by brucite and calcite[END_REF][START_REF] Formosa-Dague | Towards a better understanding of the flocculation/flotation mechanism of the marine microalgae Phaeodactylum tricornutum under increased pH using atomic force microscopy[END_REF][START_REF] Branyikova | Physicochemical approach to alkaline flocculation of Chlorella vulgaris induced by calcium phosphate Besson et al. precipitates[END_REF]. Indeed, microalgae often have a cell wall, composed of lipids, proteins and polysaccharides. These last ones have a pK a of 11-12; when the pH increases in the medium, the hydroxyl functions of these polysaccharides are deprotonated, which can give the surface a more important negative charge. As microalgae are usually grown in waters containing calcium or magnesium ions, which precipitate into positively charged particles at high pH, then flocculation occur also thanks to the interaction between these particles and the cells. However in our case, D. salina cells do not interact with positively charged magnesium hydroxide. Indeed, Dunaliella genus is unique in the absence of a rigid polysaccharidic cell wall; cells only present a thin plasma membrane [START_REF] Oren | A hundred years of Dunaliella research: 1905-2005[END_REF][START_REF] Chen | Osmotic responses of Dunaliella to the changes of salinity[END_REF]. This thus explains why its surface charge is not more negative at high pH, and thus why charge neutralization is not involved in its flocculation mechanisms in presence of magnesium hydroxide. Thus our results give a full understanding of the mechanism of D.

salina flocculation at high pH; based on these information, we can now evaluate the influence of different parameters, such as NaOH concentration, agitation and salinity in order to determine the best possible separation conditions of the cells from the water, and provide a solution for high-scale harvesting.

Magnesium hydroxide precipitation phase is determinant for flocculating the cells

Because the flocculation of the cells occur only through sweeping, the precipitation of magnesium hydroxide is then determinant for the successful flocculation/flotation of the cells.

Indeed, the precipitation phase must be carried out as homogeneously as possible, so that as much of the sample volume as possible is affected by the trapping of microalgae in the precipitate in formation. Therefore, the conditions for adding NaOH to the suspension should be adapted to optimize its fast mixing. To address this point, we performed flocculation/flotation experiments using two different injection ways (Figure 4a), reaching the same final NaOH concentration. In the first way (direct injection), NaOH (1 mol/L) and water are directly injected in a flotation unit containing the microalgae suspension. In the second way, called here diluted injection, water and NaOH (1 mol/L) are first mixed into one solution, which is further added to the microalgae suspension. Then, in the first case, concentrated NaOH is delivered locally into the medium and in the second case, the local instant concentration of the NaOH solution is reduced. Using these two injection scenarios, flocculation/flotation harvesting was performed: the results, presented in Figure 4b show a clear increase of the harvesting efficiency in the case of diluted injection, for the same final NaOH concentration in the microalgae suspension (black-filled symbols). For example at 0.02 mol/L of final NaOH concentration, the harvesting efficiency using direct injection is of almost 30% whereas using the diluted injection system, it reaches up to 90%. Indeed, in the case of direct injection, a high quantity of concentrated NaOH is locally delivered in the solution, thus the speed of precipitation is increased but only concerns a local area of the suspension. Therefore in this case, only the cells present in this area can be entrapped during the precipitation of magnesium hydroxide. In the case of the diluted injection, the low local concentration allows for a slower precipitation, which then has time to occur in the entire, or at least a larger volume of the microalgae suspension, leading to the entrapment of more cells. This phenomena is further illustrated in Figure 4c; for a final NaOH concentration in the microalgae suspension of 0.02 mol/L, the more diluted the added NaOH solution is, the more efficient the harvesting is. Indeed, for an added NaOH solution of 0.1 mol/L, the flocculation/flotation cell recovery reaches 80% while for NaOH solutions higher than 0.5 mol/L, the harvesting efficiency is reduced to 20%.

The results of these experiments then prove that the local concentration of NaOH and the phase of the magnesium hydroxide precipitation is determinant for the harvesting efficiency. While diluted injection allows lowering this local NaOH concentration, to optimize the conditions to reach the best cell separation possible, other parameters such as the agitation and the salinity (quantity of magnesium ions available) of the medium should be taken into account as they will thus also influence the harvesting efficiency.

Agitation and salinity are parameters that influence harvesting efficiency

While diluted injection allows the magnesium hydroxide precipitate to form slowly in the medium, agitation of the medium allows for the precipitation phase to sweep the entire microalgae suspension. In Figure 5a, the effects of different agitation speed on the flocculation/flotation harvesting efficiency were measured. It is clear on this figure that an increase from 20 to 40 rpm allows increasing by approximately a two-fold the cell separation whatever the final NaOH concentration in the microalgae suspension. However, increasing again the agitation speed from 40 to 80 rpm do not show the same effect, as the harvesting efficiency in the case of 80 rpm agitation is slightly lower than in the case of 40 rpm. This can be explained by the fact that a too fast agitation can change the structure of the flocs formed and thus have consequences on their capture by the bubbles during flotation. As for the salinity of the medium, to assess its effects on the harvesting efficiency, we performed experiments with D. salina cells grown in natural seawaters presenting salinities of 7.5, 12 and 15.2%, using a dilute injection system and an agitation of 40 rpm. The results obtained, presented in Figure 5b, show that the more saline the medium is, the less efficient the harvesting is, whatever the NaOH final concentration. Indeed, while high magnesium concentrations found in highly saline media can be thought, in the case of sweeping flocculation, to enhance harvesting efficiency, it is the opposite effect. In high saline media, the NaOH added directly meets high concentrations of magnesium ions, which induces a fast precipitation of magnesium hydroxide.

OH -has thus no time to reach the entire volume of the suspension. Then a high salinity has similar effects than injection of concentrated NaOH, where only the cells present in the area where fast precipitation of Mg(OH) 2 occurs can be entrapped in the precipitate, leading to lower separation rates.

Therefore thanks to these experiments, the parameters to reach the best separation rate possible can be optimized: while a dilute injection is needed, the agitation of the medium must be optimized to allow an efficient mixing of NaOH with cells and a Mg(OH) 2 precipitation that reaches the entire volume, without having negative effects on the flocs formed. As for the salinity of the culture medium, it needs to be low enough to avoid supersaturation of magnesium hydroxide precipitation, the adequate NaOH concentration depending on the salinity. In order to make sure that only parameters related to the magnesium hydroxide precipitation have an influence on the harvesting efficiency, we also evaluated the influence of the calcium concentration, the D. salina and Shelef, 1984). The Conway medium used here to cultivate the cells containing phosphate, we thus used waters of different calcium concentrations to evaluate the potential effect of calcium phosphate precipitate on the flocculation of D. salina. Our results (Supplementary Data 1) showed no difference in the harvesting efficiencies, thus reinforcing our conclusions on the role of only magnesium hydroxide precipitate in sweeping the cells. The influence of the D. salina strain and of its nutritive conditions have then also been evaluated, and showed that for the two strains we tested (the CCAP 19/25 and one other D. salina strains isolated from saline ponds in France), the same flocculation/flotation conditions resulted in the same harvesting efficiencies (Supplementary Data 2).

Regarding the nutritive conditions in which cells are grown, we chose to focus on nitrogen deficiency conditions. Indeed, the overproduction of β-carotene after nitrogen starvation (i.e. in conditions of unbalanced growth in response to lack of nitrogen) is a well-documented biological process in D.

salina [START_REF] Lamers | Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga[END_REF][START_REF] Bonnefond | Coupling and uncoupling of triglyceride and beta-carotene production by Dunaliella salina under nitrogen limitation and starvation[END_REF]. We thus chose to address this question as the harvesting method we propose here is intended to be used for industrial use. Our results, with cells grown in nitrogen deficient conditions, showed no difference in the harvesting efficiencies obtained.

Thus these two last points show that the flocculation/flotation method that we focus on in this study, when used with the good injection system, agitation and salinity of the medium, is efficient for different D. salina strains, in different relevant culture conditions. This demonstrates the robustness of this harvesting method, and thus the parameters identified (NaOH injection, agitation and salinity) can now further be used to develop and adapt a NaOH injection system to efficiently flocculate and harvest cells by flotation in a pre-industrial continuous cultivation/harvesting system.

Development of a pre-industrial harvesting process of D. salina by flocculation/flotation.

In this part of the work, the knowledge previously acquired at the laboratory scale is used to harvest D. salina cells at high-scales. For that, raceways of 250 m² were built in Gruissan (Occitanie, France) and used to cultivate D. salina; an industrial continuous flotation unit (600 L) was adapted and installed on the raceway site to harvest cells using NaOH-induced flocculation/flotation. A network of pumps with controlled flow rates were used to collect the cells from the raceway and bring pressurized water into the flotation unit. For adapting the flotation unit to the specific conditions of D. salina, we first faced a technical challenge regarding the size of the bubbles produced in the pilot. Indeed, using a method based on laser light diffraction, we evaluated the size of the bubbles produced by DAF in D. salina culture medium (Supplementary Data 3a). These experiments show that in this medium (NaCl concentration of 107 g/L), bubbles have a size of approximately 40 µm, compared to 60-100 µm in freshwater [START_REF] Edzwald | Principles and applications of dissolved air flotation[END_REF]. Thus the salinity reduces the size of the bubbles, which results in the reduction of their ascending velocity. While this is not a problem at small-scale, because the flotation units consist of one small cylinder (1 L of maximum volume) with only one entry for the pressurized water at the bottom (Figure 1a), at highscale in a high-dimensioned flotation unit, presenting descending flow zones, it is a problem. For instance, this flotation unit presents holes at the base of the Clifford in order to accentuate the airlift effect and obtain, in nominal functioning conditions, descending speeds of 4 m/h (Figure 1b). We thus roughly calculated the speed of our bubbles, considering them as rigid particles obeying the Stokes law corrected by Oseen [START_REF] Oseen | Stokes' Formula and a Related Theorem in Hydrodynamics[END_REF][START_REF] Clift | Bubbles, drops, and particles[END_REF] and found in our conditions an average bubble ascending velocity comprised between 3.6 and 5.4 m/h depending on the contamination degree of their surface (1 and 1.5 mm/s, Supplementary Data 3b). Thus some of the bubbles may not reach the surface and can be aspirated in the flow descending zones of the flotation unit. Thus adaptations directly on the flotation unit were realized, and consisted in decreasing the surface of the holes present at the base of the Clifford to decrease the airlift effect and decrease the velocity in these descending flow zones. This way the rising velocity of the microbubbles is higher than the velocity in the descending flow zones, and thus a functioning similar to what is obtained at the laboratory-scale can be provided.

These adaptations made, we then scaled-up the NaOH-induced flocculation/flotation process optimized in batch mode at the laboratory scale, to a continuous pre-industrial scale mode. For that, given the specific flocculation mechanism of D. salina by sweeping, it is needed to adapt an injection system that will ensure a good mixing between the cells and the added NaOH, as it is determinant to efficiently flocculate the cells. Given the previous results concerning the salinity of the medium, these experiments were performed in natural seawater at a salinity of 12% that ensured the growth of the cells in the raceway without presenting precipitation supersaturation problems. For the experiments, microalgae cultures were injected into the industrial flotation unit, as well as pressurized waters, through supply lines following the injection system represented in Figure 6a.

Note that in this system, the waters used for pressurization are recycled from the flotation unit after microalgae recovery, and may contain also some microalgae. NaOH mixing with microalgae and microbubbles in this system takes place directly in the injection system, and is different depending on its location on this injection system. For instance, in positions 3 and 6 (Figure 6a), mixing will be more efficient than in position 1 and 4 because there the flow rate is the most important and the fluid is already a tri-phase fluid (culture medium, cells and bubbles). Thus to find the best mixing conditions, we chose to inject NaOH at of 0.2 mol/L (final concentration of 0.02 mol/L in the microalgae suspension) at the different places in the injection system represented in red on the schematic representation in Figure 6a. After flocculation/flotation, the harvesting efficiencies were measured: results are presented in Figure 6b. They show that the best cell separation rates, of approximately 60%, are reached when NaOH is injected in the locations 3 and 6, where mixing is the most efficient. If NaOH is injected into the microalgae supply line, the harvesting performance is slightly higher if this injection is made at the outlet of the elbow (position 1), where recirculation phenomena occur because of the 90° bend located just upstream of position 1. However, these performances do not reach those achieved for a NaOH injection in the positions 3 and 6. On the pressurized water supply line, the further away the injection is from the confluence with the suspension feed, the lower the harvesting efficiency is. For instance, the harvesting efficiency is significantly reduced when NaOH is injected in the position 4; this can be explained by the low presence of microalgae in this pipe. The magnesium precipitate needs to be formed in the presence of the cells to best entrap them, thus explaining the lower separation efficiencies reached in the pressurized water supply line.

However in these experiments, the maximum harvesting efficiency obtained is of 60%. They were realized with a NaOH solution at a concentration of 0.2 mol/L: as showed before, best separation rates are obtained for diluted NaOH solutions, the best efficiencies being achieved in the case of NaOH solutions of 0.1 mol/L (Figure 4c, 80% of cell separated from water). Thus, in order to optimize the NaOH concentration, using a NaOH injection in the position 3 in the injection system, the experiments were repeated with injected NaOH solutions of different concentrations (final NaOH concentration of 0.02 mol/L). The results are showed in Figure 7, where both the harvesting efficiencies obtained in batch-mode at laboratory scale and in continuous mode at pre-industrial scale are represented. It is clear on this graph that indeed, for a solution of NaOH of 0.1 mol/L, the separation rate reaches 80%, and decreases as the NaOH concentration increases. The interesting point is that for both harvesting modes (batch or continuous), the harvesting efficiencies are the same, thus showing the successful scale-up of our NaOH-induced flocculation/flotation process.

Therefore, we could adapt an efficient injection system, allowing to separate in a single pass up to 80% of the cells from their culture medium at high scale, and concentrate them by a factor of 230, compared to approximately 20 in laboratory-scale experiments. This difference in the concentration factor is explained by the fact that in the continuous DAF system, the rotation velocity of the scrapper, which removes floated microalgae, and its vertical position in the tank can be tuned in order to adapt the residence time of the foam containing the microalgae at the tank surface. During this residence, the microalgae concentration increases in the foam, due to the liquid drainage by gravity, resulting in higher concentration factors compared to lab-scaled experiments.

Conclusions

We provide here an interdisciplinary multi-scale study to propose an efficient harvesting method for D. salina cells using flocculation/flotation, which can be used at industrial scales.

Experiments at the nanoscale as well as simulations allowed first to precisely understand the complete mechanism of flocculation by addition of NaOH in the complex hypersaline medium in which D. salina grows. We thus brought strong scientific arguments proving that addition of NaOH in the medium creates a magnesium hydroxide precipitate that entraps the cell and flocculate them through sweeping. Because no other mechanism is involved, the formation of this precipitate, at low speed, in the entire microalgae suspension, is determinant for the harvesting efficiency.

Understanding this then led us to evaluate the influence of pertinent parameters to achieve highefficiency harvesting, all related to the precipitation of Mg(OH) 2 , that are the NaOH concentration in the medium, the agitation and the salinity of the medium. Our results, in laboratory-scale flocculation/flotation experiments, allowed us to show that the added NaOH solution had to be diluted, the agitation had to be optimal to bring the NaOH in the entire volume without breaking the forming flocs, and that too high salinities were resulting in magnesium hydroxide supersaturation phenomena. It is based on this understanding of the separation mechanisms and on the identification of the influence of different parameters on the harvesting performances that the transition of the process to the pre-industrial scale could be addressed from in an efficient way. For that we focused on the mixing efficiency at the injection site and the NaOH concentration injected to provide optimal parameters and achieve efficient microalgae harvesting at high-scale. Now further studies needs to be done to evaluate with precision the cost and energy consumption of this process at high-scale, and optimize better flocculation conditions to achieve effective harvesting at lower costs. Experiments have already been performed in this way, using slaked lime as a base to induce the precipitation of magnesium hydroxide. Our results so far show promising harvesting efficiencies, achieved at lower cost as calcite is less expensive than NaOH.

Figure 1 .

 1 Built in 316L stainless steel to avoid corrosion by suspensions at high salinity, this continuous cylindrical (0.6 m of diameter) DAF separation equipment, with a conical bottom, works as an airlift. A cylindrical Clifford delimits the ascending contact zone where the suspension to be treated and the white waters, containing bubbles, mix. At the periphery, the descending annular separation zone is equipped with a vertical lamellar packing to keep the flow as quiet as possible. The nominal descending flow velocity is of 4.0 m/h. The flotation tank volume is of 600 L and its maximum flow capacity is of 1 m 3 /h. The floated microalgae are mechanically removed from the tank surface by a tunable rotating scrapper. Salted waters identical to the culture medium or recycled from the harvesting are continuously pressurized at 6 bars by a centrifugal pump and saturated with air within a pressurization tank. White waters are produced by passing this pressurized solution through a needle valve. The microbubble size distribution was measured by Laser Diffraction Sizer (Malvern Spraytec): most of the bubbles

  strain used, and the nutritive conditions in which the cells are grown. Regarding the influence of the calcium concentration in the medium, Sukenik et al. in 1984 have established that the precipitation of calcium and phosphate ions at high pH could induce the flocculation of microalgae cells (Sukenik

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Simulating the influence of the addition of NaOH on the ionic equilibria in D. salina

Figure 3 .

 3 Figure 3. Probing the interactions between D. salina cells and magnesium hydroxides. (a) Histogram

Figure 4 .

 4 Figure 4. Influence of the NaOH injection way on the harvesting efficiencies. (a) Scheme

Figure 5 .

 5 Figure 5. Influence of agitation and salinity on the harvesting efficiencies. (a) Influence of the

Figure 6 .

 6 Figure 6. Adaptation of the NaOH injection system for high-scale harvesting. (a) Schematic and

Figure 7 .

 7 Figure 7. Influence of the injected NaOH concentration on the harvesting efficiency.

Table 1 .

 1 Concentrations of the main ions in the culture medium used in simulations in g/L

	Na +	Cl -	SO 4	2-	Mg 2+	Ca 2+	K +
	42.12	67.08	1.87	0.95	0.39	0.04

Table 1 .

 1 Concentrations of the main ions in the culture medium used in g/L

	Na +	Cl -	SO 4	2-	Mg 2+	Ca 2+	K +
	42.12	67.08	1.87	0.95	0.39	0.04

Acknowledgements A. B. is a PhD student supported by the FUI Salinalgue. C. F.-D. is a postdoctoral researcher supported by the AgreenSkills fellowship programme, which has received funding from the EU's Seventh Framework Programme under grant agreement No. FP7-609398 (AgreenSkills+contract). Sukenik, A., Shelef, G., 1984. Algal autoflocculation--verification and proposed mechanism. Biotechnol. Bioeng. 26, 142-147. https://doi.org/10.1002/bit.260260206 Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., Hoadley, A., 2010. Dewatering of microalgal cultures: A major bottleneck to algae-based fuels. J. Renew. Sustain. Energy 2, 012701. https://doi.org/10.1063/1.3294480 Vandamme, D., Foubert, I., Fraeye, I., Meesschaert, B., Muylaert, K., 2012. Flocculation of Chlorella vulgaris induced by high pH: Role of magnesium and calcium and practical implications. Bioresour. Technol. 105, 114-119. https://doi.org/10.1016/j.biortech.2011.11.105 Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31, 233-239.


Xavier Loyer

Caroline Dubroca

Maxime Branchereau

Graziellia Griffith

Luis Garcia

Dr Christophe Heymes
email: christophe.heymes@inserm.fr

Loyer Xavier

; Maxime Branchereau

; Luis

Neuronal NO synthase mediates plenylephrine induced cardiomyocyte hypertrophy through facilitation of NFAT-dependent transcriptional activity

Keywords: Neuronal NO synthase, cardiomyocyte hypertrophy, gene silencing, Adenovirus, NFAT

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

Introduction

Cardiovascular disease continues to be the leading cause of morbidity and deaths in industrialized countries, and left ventricular hypertrophy is an independent risk factor for cardiovascular mortality and morbidity. At first, cardiac hypertrophy is a critical adaptive response to preserve cardiac function in response to various stresses. However, continued cardiac stress caused pathological cardiac hypertrophy leading to heart failure [START_REF] Olson | A decade of discoveries in cardiac biology[END_REF][START_REF] Tham | Pathophysiology of cardiac hypertrophy and heart failure: signaling pathways and novel therapeutic targets[END_REF].

Although the physiological importance of nitric oxide (NO)-dependent signalling in heart hypertrophy has been long discussed, the precise role of cardiomyocyte nitric oxide synthase isoforms (NOS) in the development of cardiac hypertrophy is still controversial [START_REF] Loyer | Constitutive nitric oxide synthases in the heart from hypertrophy to failure[END_REF][START_REF] Carnicer | Nitric oxide synthases in heart failure[END_REF]. However, it appears that the main source of NO that modulates cardiomyocyte growth is produced by the neuronal nitric oxide synthase (NOS1).

NOS1 expression and activity were consistently increased in the hypertrophic or failing rodent myocardium and in failing human hearts [START_REF] Bendall | Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat[END_REF][START_REF] Damy | Increased neuronal nitric oxide synthasederived NO production in the failing human heart[END_REF][START_REF] Watts | Anti-hypertrophic and anti-oxidant effect of beta3-adrenergic stimulation in myocytes requires differential neuronal NOS phosphorylation[END_REF]. NOS1 gene deletion was associated with more severe cardiac remodeling following myocardial infarction [START_REF] Saraiva | Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium[END_REF][START_REF] Dawson | nNOS gene deletion exacerbates pathological left ventricular remodeling and functional deterioration after myocardial infarction[END_REF]. In contrast, we reported that mice with cardiomyocyte specific overexpression of NOS1 exhibited a greater cardiac hypertrophic response in a model of pressure overload-induced cardiac hypertrophy, as compared to wild-type mice [START_REF] Loyer | Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling[END_REF].

Although these previous studies have demonstrated that NOS1 was associated with cardiac hypertrophic growth, the direct functional contribution of NOS1 in this process remains to be elucidated. Hence, in the present study we attempted to use silent RNA and adenovirus mediated silencing or overexpression to investigate the role of NOS1 during phenylephrine (PE)-induced cardiac hypertrophy growth in neonatal rat ventricular cardiomyocytes (NRVMs). We demonstrate that NOS1 gene suppression clearly blunts NRVMs hypertrophy and the corresponding foetal gene program in response to PE. This inhibition of NRVMs growth was, at least, mediated through inhibition of the calcineurin signalling pathway. In contrast, exposing the NOS1 overexpressed NRVMs to PE-treatment markedly increased the hypertrophic growth and marker gene expression. Together, the results of the present study suggest that NOS1 is directly involved in controlling the development of cardiomyocyte hypertrophy.

Materials and methods

The present study was conducted in accordance with institutional guidelines and those formulated by the European Community for experimental animal use (L358-86/609/EEC). We used Wistar rat pups on post-natal day 1 to isolate neonatal rat ventricular myocytes (NRVMs).

NRVMs culture, transfections and manipulation

Monolayer cultures of neonatal rat cardiac cells were prepared as previously described. Briefly, hearts were removed and ventricular cells were dispersed by digestion with collagenase A (0.45 mg/ml) (Boehringer Mannheim) and pancreatin (0.05 mg/ml) (GIBCO) in Ads buffer (116 mM NaCl, 20 mM HEPES, 1 mM NaH2PO4, 5.4 mM KCl, 5.5 mM glucose, 0.8 mM MgSO4, pH 7.35).

Cardiomyocytes were purified on a discontinuous Percoll gradient (1.059/1.085) collected by centrifugation at 3000xg and resuspended (0.3x106 cells/ml) in Dulbecco's modified Eagle medium (DMEM, 1.8 mM Ca 2+ ), 17% Medium 199 (GIBCO), 10% horse serum, 5% newborn calf serum, 1% penicillin and 1% streptomycin. human NOS1. siRNA duplex for NOS1 (si-NOS1) and a siRNA containing the same nucleotides in a scrambled order (si-Scramb) were designed and provided from Quiagen France SAS. The sense and antisense oligonucleotides of NOS1 siRNA were, respectively, r(CAA UAC UAC UCA UCC AUU A)dTdT and r(UAA UGG AUG AGU AGU AUU G)dGdT. All the siRNAs were transfected using Lipofectamine ® Reagent (Thermo Fisher) according to the manufacturer's protocol. The human NOS1 (hNOS1, α isoform) coding sequence was a kind gift of Dr. Bernd Mayer (Department of Pharmacology and Toxicology, Karl Franzens Universität Graz, Austria). Adenovirus coding for NOS1 (Ad.NOS1) was constructed and amplified by Dr. Graziella Griffith. For all experiments using adenoviruses, control cells were infected with a control empty adenovirus and so called "Ad.Empty". After 24h, NRVMs were starved and hypertrophy was induced by phenylephrine stimulation (PE, Sigma aldrich, 10 -4 mol/L) for 48 hours. For experiments employing adenoviruses, the day after seeding, cells were infected overnight with adenoviruses. The day after, excess of adenoviruses is removed and cells were replaced in SFM and stimulated with PE. Viruses were used at a multiplicity of infection (MOI) of 100. Identical procedure was used when cells were transfected with siRNAs.

Incorporation of amino acids and cell size measurement

To quantify the incorporation of amino acids, NRVMs were incubated with [ 3 H]leucine for the last 24h with (1.0 µCi/ml, Perkin Elmer, USA). After three washes with PBS 1X, cells were treated for 2hours at 4°C with trichloroacetic acid (10% w/v) followed by solubilization in 0.1N NaOH (0.2 M) for 2 hours under agitation at 37°C. The radioactivity was determined by scintillation counting and expressed as percentage of control cells.

To perform cell size measurements, cells were fixed in 4% PFA and then stained with anti-alpha actinin antibody (Sigma Aldrich) and Hoescht staining for nucleus.

Pictures were recorded using a fluorescent microscope (Zeiss Imager Z1, France).

A minimun of 10 fields per condition was recorded at X20. Cardiomyocytes areas were determined using Image J Software.

Hypertrophic program determination using reporter assay

To determine the role of NOS1 on the hypertrophic program, cells were cotransfected with Lipofectamine 2000 (Invitrogen Life Technologies, France) in the presence of 1 µg of ANF-Luciferase plasmid construct (generously provided by Dr K. Knowlton). The day after transfection, cells were stimulated with PE for 48 hours and assayed for Luciferase activity. Results are expressed as percentage activation of control.

Superoxide measurement using Lucigenin-enhanced chemiluminescence

To determine if NOS1-derived superoxide anions production was involved in the hypertrophic response following PE stimulation, NOS1-derived superoxide production was measured in cardiomyocytes homogenates using Lucigeninenhanced chemiluminescence. Briefly, cells homogenates were placed into a 96well microplate luminometer together with dark-adapted lucigenin (5 μmol/L), and NADPH (300 μmol/L) was added before recording chemiluminescence. NOS1derived superoxide was assessed by pre-incubating cells homogenates with the selective NOS1 inhibitor LVNIO (1.10 -6 mol/L). Superoxide production was expressed as arbitrary light units over 20 min. Results are expressed as percentage activation of control.

Immunoblots

NRVMs were homogenized and subjected to Western Blot analysis. After migration on SDS-PAGE gels, proteins were transferred onto nitrocellulose membranes (Amersham, Chalfont St. Giles, UK). Afterwards, the membranes were incubated overnight at 4° C with the following primary antibodies in blocking solution: anti-NOS1 (Affinity Bioreagent), anti-NOS3 (Santa Cruz Biotechnology Inc.), anti-calcineurin (Becton Dickinson), anti-GSK-3β and anti-Phospho-GSK-3β (Cell signalling). After several washes, membranes were incubated with a secondary antibody coupled to horseraddish peroxidase (1:5000; Amersham, Chalfont St. Giles, UK). Protein detection was performed using ECL+ on a Fuji system and quantified by Image Gauge. Membranes were also blotted with an anti GAPDH antibody (Chemicon) to normalize.

Calcineurin NFAT activity

Calcineurin activity was quantified using the measurement of the NFAT-Luciferase reporter activity. The luciferase reporter plasmid driven by four NFAT consensus binding sites (NFAT-Luc) was obtained from Stratagene. NRVMs were cotransfected with Lipofectamine 2000 in the presence of 1 µg of the plasmid construct according to the manufacturers'instructions. The day after transfection, cells were stimulated by PE for 48 hours and were assayed for Luciferase activity.

Results were expressed as percentage activation of control.

Statistical analysis

Data are expressed as mean ± sem. Statistical analysis was performed with Prism. ANOVA followed by a Bonferroni-Dunn test was performed. P<0.05 after Bonferroni test is considered significant.

Results

NOS1 is upregulated and promotes the induction of cardiomyocyte hypertrophy induced by PE

Because we have previously demonstrated that increased expression and/or activity of NOS1 expression were associated to the progression of cardiac hypertrophy, we tested whether PE stimulation may regulate NOS1 protein expression and activity and whether blocking NOS1 activity would influence the development of cardiomyocyte hypertophy. As shown in Fig. 1A, PE treatment significantly increased NOS1 protein expression, as compared to non-stimulated cardiomyocytes (P<0.05 versus basal). As expected, PE-induced cardiomyocyte hypertrophy was demonstrated by a 36% increase in cell size (Fig. 1B) and a 74% induction in [ 3 H]-leucine incorporation (Fig. 1C). In line with our hypothesis, LVNIO treatment, the selective NOS1 inhibitor, significantly decreased PE-induced NRVMs hypertrophy and [ 3 H]-leucine incorporation (P<0.01 versus PE). Note that LVNIO treatment in absence of PE had no effect.

To determine if NOS1-derived superoxide anions production was involved in the hypertrophic response following PE stimulation, NOS1-derived superoxide production was measured in cardiomyocytes homogenates using Lucigeninenhanced chemiluminescence. As expected, PE induced a significant increase in superoxide production (+ 114% versus non-stimulated cells). However, LVNIO preincubation had no effect on superoxide production, suggesting that NOS1-derived superoxide was not involved in the hypertrophic response mediated by PE (Supplemental Fig. 1A).

NOS1 is involved in the induction of cardiomyocyte hypertrophy induced by PE

To further investigate the possible effects of NOS1 on cardiomyocyte hypertrophy, we used complementary strategies. To explore the role of native NOS1 in the hypertrophic effect of PE, we used a specific silent RNA targeting NOS1 (si-NOS1). As expected, NRVMs transfected with si-NOS1 showed a decreased level of NOS1 compared with silent RNA sequence control (si-Scramb, Fig. 2A). To mimic the results previously obtained in vivo and those obtained in vitro after PE stimulation, we constructed an adenovirus encoding the human NOS1 protein (Ad.NOS1). As expected, NRVMs infected with Ad.NOS1 showed an increased level of NOS1 compared with a control empty adenovirus (Ad.Empty, Fig. 2B). Both the transfection and infection efficiencies were maintained for at least 72 hours.

Then we investigated cardiomyocytes transfected with the si-NOS1 or Ad.NOS1 followed by PE treatment. As shown in Figure 3A and 3B, It can be observed that silencing of NOS1 significantly attenuated the increase in cell surface and in [ 3 H]leucine incorporation induced by PE stimulation. Similar findings were obtained on another marker of cardiomyocyte hypertrophy, ANF expression. Indeed, silencing NOS1 expression significantly inhibited PE-induced ANF-Luciferase gene transcriptional activity (Fig. 3C). Consistent with this finding, upregulation of NOS1 in NRVMs with the Ad.NOS1 further exacerbated the effect of PE on cell surface area compared with NRVMs infected with control adenovirus Ad.Empty (Fig. 3A).

The effect of Ad.NOS1 on cell growth was also confirmed by protein synthesis measurement (Fig. 3B). Finally, the ANF-Luciferase gene transcriptional activity was also further increased in NRVMs infected with Ad.NOS1 compared to cells treated with PE only. In absence of PE stimulation either silencing or overexpressing NOS1 has no effect on cardiomyocyte hypertrophy. In the same way, the si-Scramb and Ad.Empty had no effects on NRVMS hypertrophy with or without PE stimulation (data not shown). We finally examined if the effects on cardiomyocytes hypertrophy induced by NOS1 could be partially mediated through NOS3 protein expression Taken together, these date support the hypothesis that NOS1 participate in PEinduced cellular hypertrophy.

NOS1 mediates PE-induced cardiomyocyte hypertrophy in a NFAT-dependent signalling pathway

Several calcium-dependent signal transduction pathways have been implicated in cardiac hypertrophy. The calcium/calmodulin-dependent protein phosphatase calcineurin is an especially potent inducer of myocardial hypertrophy in vivo and in vitro [START_REF] Frey | Decoding calcium signals involved in cardiac growth and function[END_REF]. Calcineurin dephosphorylates a large number of cellular targets that could influence cardiac growth, including the nuclear factor of activated T cell (NFAT) proteins [START_REF] Crabtree | Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT[END_REF].

Since we have previously demonstrated that NOS1 cardiomyocyte specific overexpression in mice was associated with an increase in calcium content, we further characterize the NOS1 hypertrophic signaling pathway and investigate the effect of NOS1 protein modulation on calcineurin expression and activity (using the NFAT-luciferase reporter assay to monitor transcriptional activity of NFAT). Figure 4A and 4B shows that calcineurin protein expression and activity were both significantly increased in NRVMs treated with PE, as compared to non-treated cells.

In line with the above results on cells growth, these increases induced by PE were prevented by the selective si-NOS1. Interestingly, overexpressing NOS1 using Ad.NOS1 further increased calcineurin protein expression and activity in response to PE (Fig. 4B).

In contrary, glycogen synthase kinase-3β (GSK-3β) is a ubiquitous serine/threonine protein kinase that phosphorylates a series of sites of NFAT proteins, inhibiting its transcriptional activity. GSK-3β is highly active in unstimulated cells and becomes inactivated in response to hypertrophic stimulation. Conversely, dephosphorylation results in activation of the kinase [START_REF] Cohen | The renaissance of GSK3[END_REF]. To gain further insight on the mechanism of retarded cardiomyocyte hypertrophy induced by NOS1 gene silencing, we thus determined if GSK-3β activation (determined by the ratio phosphorylated form to non-phosphorylated form) is affected following PE stimulation. As shown in figure 4C, PE induced a significant increase in phospho/total ratio indicating that gsk3β is inactive and cannot repress hypertrophy.

Upon PE stimulation, silencing NOS1 leads to a decrease of the phospho/total ratio indicating that GSK-3β is active and can limit hypertrophy development.

All of these data demonstrate that NOS1 acts as a prohypertrophic factor by involving calcineurin pathway and limiting action of the GSK-3β one.

Discussion

The results of the present study reveal for the first time a key and direct contribution of NOS1 and NOS1-derived NO in PE-induced neonatal cardiomyocyte hypertrophy. Indeed, not only PE stimulation is associated with an increased expression of NOS1 but also a specific inhibitor of NOS1 prevents PE-induced NRVMs growth. In addition, silencing NOS1 expression blocked the hypertrophic effect of PE. Finally, overexpression of NOS1, using an adenovirus, further exacerbated NRVMs growth following PE stimulation.

Although we did not observe any effect of either silencing or overexpressing NOS1 on cardiomyocyte hypertrophy in the absence of PE stimulation, the present results confirmed our in vivo data where we did not observe any impact of NOS1 overexpression on adult cardiomyocyte hypertrophy without stressing the heart (following increased pressure-overload) [START_REF] Loyer | Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling[END_REF]. In fact, NOS1 binds calmodulin and NOS1 activity is regulated by Ca 2+ and calmodulin. In NOS1, calmodulin binding is brought about by an increase in intracellular Ca 2+ . Thus, to unmask any effect of NOS1 expression modulation, we need to promote its activation by increasing cardiomyocyte calcium level [START_REF] Förstermann | Nitric oxide synthases: regulation and function[END_REF]. Although we did not quantify Ca 2+ transients in response to PE, this hypertrophic stimulus has been shown to increase intracellular Ca 2+ influx in NRVMs via Gq protein activation. The activated Gq protein activates phospholipase C and in turn cleaves phosphatidyl inositol 4,5-biphosphate into the second messengers inositol 1,4,5-triphosphate and diacylglycerol leading to the increase of calcium in the cardiomyocyte [START_REF] Lee | microRNA-133a attenuates cardiomyocyte hypertrophy by targeting PKCδ and Gq[END_REF]. After PE stimulation, calciumcalmodulin complex levels increased resulting in increased NOS1 activity. In nonstimulated cardiomyocytes, calcium level are unchanged which may explain that silencing or overexpressing NOS1 have no impact on cardiomyocytes hypertrophy.

In the same way, ATP was shown to regulate cardiomyocyte NO production through modulation of calcium influx [START_REF] Sunggip | TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy[END_REF].

Experimental evidence has indicated that constitutive NO production may play an important part in regulating cardiac growth in response to stress [START_REF] Massion | Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice[END_REF][START_REF] Massion | Nitric oxide and cardiac function: ten years after, and continuing[END_REF]. However, selective gene deletion of the second NOS isoform present in the cardiomyocyte, NOS3, has not produced consistent results on cardiac remodeling, and NOS3 expression and activity have been found to be significantly suppressed or even absent in the myocytes of the hypertrophic heart [START_REF] Bendall | Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat[END_REF][START_REF] Damy | Increased neuronal nitric oxide synthasederived NO production in the failing human heart[END_REF][START_REF] Scherrer-Crosbie | Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice[END_REF][START_REF] Liu | Effect of ACE inhibitors and angiotensin II type 1 receptor antagonists on endothelial NO synthase knockout mice with heart failure[END_REF][START_REF] Damy | Up-regulation of cardiac nitric oxide synthase 1-derived nitric oxide after myocardial infarction in senescent rats[END_REF]. In contrast, the increase of NOS1 expression following PE stimulation is consistent with previous in vivo experimental evidence, demonstrating that NOS1 expression and activity were increased in the hypertrophic or failing rodent myocardium and in failing human hearts [START_REF] Bendall | Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat[END_REF][START_REF] Damy | Increased neuronal nitric oxide synthasederived NO production in the failing human heart[END_REF][START_REF] Loyer | Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling[END_REF]. Similarly, NOS1 expression and activity are significantly increased in the myocardium or isolated cardiomyocytes of hypertensive rats and in isolated LV myocytes following angiotensin II treatment, confirming the upregulation of cardiac NOS1 and/or activity with cardiomyocyte hypertrophy [START_REF] Umar | Activation of signaling molecules and matrix metalloproteinases in right ventricular myocardium of rats with pulmonary hypertension[END_REF][START_REF] Jin | Myofilament Ca2+ desensitization mediates positive lusitropic effect of neuronal nitric oxide synthase in left ventricular myocytes from murine hypertensive heart[END_REF][START_REF] Jang | ROS and endothelial nitric oxide synthase (eNOS)-dependant trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes[END_REF][START_REF] Jun | Neuronal nitric oxide synthase is up-regulated by angiotensin II and attenuates NADPH oxidase activity and facilitates relaxation in murine left ventricular myocytes[END_REF].

Emerging but conflicting data suggest that NOS1 may have important functions in the pathophysiology of adverse cardiac hypertrophy. Here, we provide the first in vitro evidence that both NOS1 and NOS1-derived NO act as positive regulators of cardiomyocyte hypertrophy. These results are in sharp contrast with previous in vitro studies, which demonstrated that non-selective inhibition of NOS isoforms potentiated cardiomyocyte hypertrophy [START_REF] Garcia | Factors and mechanisms involved in left ventricular hypertrophy and the anti-hypertrophic role of nitric oxide[END_REF][START_REF] Tang | Targeting NOS as a therapeutic approach for heart failure[END_REF][START_REF] Sunggip | TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy[END_REF]. However, these studies never used selective inhibitor of the different NOS isoforms neither manipulated NOS isoforms expression using specific silent RNA or adenovirus. In contrast, our results, demonstrating that NOS1 is a pro-hypertrophic factor in vitro, extend our data obtained in vivo previously. Although we didn't use the same experimental approach to overexpress NOS1, using a transgenic mouse with conditional cardiomyocyte specific NOS1 overexpression, we provided evidence that NOS1 overexpressing hearts undergo further hypertrophy retaining concentric left ventricular remodeling and full contractile function, whereas WT mice showed pronounced chamber dilation and impaired contractility in response to chronic pressure overload. In this study, we further demonstrated that isolated cardiomyocytes from NOS1 overexpressing mice had greater shortening, intracellular Ca 2+ transients and sarcoplasmic reticulum Ca 2+ load. The current results in NOS1 transgenic mice were consistent with the idea that NOS1 may prevent adverse remodeling and cardiac deterioration by preserving calcium cycling and contractile function even though the extent of hypertrophy is increased in response to pressure overload [START_REF] Loyer | Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling[END_REF]. Consistent with an adaptive and protective role for myocardial NOS1 overexpression, studies found that NOS1 in the left ventricular myocardium of chronically infarcted mice appears to attenuate adverse left ventricular remodelling and functional deterioration [START_REF] Dawson | nNOS gene deletion exacerbates pathological left ventricular remodeling and functional deterioration after myocardial infarction[END_REF]. More recently, Rosello-Lleti and collaborators demonstrated an increased NOS1 protein expression in ventricular tissue obtained from explanted human ischemic cardiomyopathy hearts. They also found a significant relationship between NOS1 protein levels and systolic ventricular function, indicating that an increased NOS1 level was associated with a better ventricular performing. Of note, the authors found no increase in NOS1-derived NO production in their experiments and suggested that the increased levels of NOS1 could be a mechanism to compensate the loss of its activity. The authors concluded that NOS1 may be important in the physiopathology of cardiac dysfunction in ischemic heart disease with a preservative role in maintaining myocardial homeostasis [START_REF] Rosello-Lleti | Human ischemic cardiomyopathy shows cardiac NOS1 translocation and its increased levels are related to left ventricular performance[END_REF]. Thus, increasing NOS1 protein expression within the cardiomyocyte may be a potential therapeutic target for heart disease. We now have to test our hypothesis in vivo using this adenovius-induced cardiomyocyte NOS1 protein expression (using AAV9 serotype) in different experimental models of heart failure induced by an increased pressure-overload or myocardial infarction.

In the same way, NOS1 and NOS1-derived NO were shown to be key triggers of skeletal muscle hypertrophy. Using a genetic model of Duchenne muscular dystrophy (MDX mouse) engineered to lack NOS1, the authors demonstrated that NOS1 depletion from MDX mice prevented compensatory skeletal muscle hypertrophy [START_REF] Froehner | Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice[END_REF]. In another model of overload-induced skeletal muscle hypertrophy, based on compensatory adaptation of the plantaris muscle after ablation of the tendons of the functionally synergistic muscles, NOS1 was found to modulate overload-induced fiber hypertrophy. Ito and collaborators [START_REF] Ito | Activation of calcium signaling through Trpv1 by nNOS and peroxynitrite as a key trigger of skeletal muscle hypertrophy[END_REF] demonstrated that, 7 days after ablation, NOS1-null mice showed a significant diminished increase in muscle weight, compared to in wild-type mice. Overload also increased the fiber size and total protein contents in the wild-type plantaris, whereas no such substantial increases were found in the NOS1-null plantaris. In this study, the authors also validated that the observed effects were mediated through an increase in NOS1-derived NO, using the specific inhibitor 7-nitroindazole. The latter results are similar to those we have obtained in our study demonstrating that LVNIO prevented PE-induced cardiomyocyte hypertrophy.

Cardiomyocyte growth is finely controlled by Ca 2+ -mediated signaling [START_REF] Dewenter | Calcium Signaling and Transcriptional Regulation in Cardiomyocytes[END_REF]; however, it was still unknown whether the action of NOS1 involved Ca 2+ -mediated pathways to induce positive regulation during hypertrophy. A major new paradigm in NO biology has emerged in the last few years to suggest that NOS1-derived NO plays an important role in the physiological regulation of myocyte calcium fluxes [START_REF] Tang | Targeting NOS as a therapeutic approach for heart failure[END_REF]. Although we did not quantify Ca 2+ transients in response to PE, this drug has been shown to increase intracellular Ca 2+ influx in NRVMs. In addition, we have previously demonstrated that cardiomyocytes overexpressing NOS1 higher Ca 2+ transients than control myocytes. Accordingly, we found that silencing NOS1 expression decreased both the expression and activity (measuring the NFAT transcriptional activity) of the Ca 2+ -dependent prohypertrophic protein calcineurin.

The opposite results were obtained when NOS1 was overexpressed. On the other hand, transcriptional activity of NFAT is also controlled by GSK-3β. As previously described, PE-induced cardiomyocyte hypertrophy induced an increase in phosphorylated GSK-3 β, the inactive form. In contrary, silencing NOS1 leads to a decrease of the phospho/total GSK-3β ratio indicating that GSK-3 β was more active

  modulation. As shown in Supplemental Fig 1B, silencing or overexpression NOS1 didn't induce any change in NOS3 protein expression after PE stimulation. Since the selective inhibitor LVNIO abolished PE-induced cardiomyocyte hypertrophy, NOS3 or potential NOS3-derived NO production seemed to be negligible in the development of NRVMs hypertrophy.

Figures legendFigure 1 .

 1 Figures legend

Figure 2 .

 2 Figure 2. Modulation of NOS1 expression by specific siRNA or adenovirus is

Figure 3 .

 3 Figure 3. NOS1 is involved in PE-induced NRVMs hypertrophy. A, Representative

Figure 4 .

 4 Figure 4. NOS1 effect on cardiomyocyte hypertrophy is mediated by the NFAT

mol/L), silent RNA (siRNA) against mouse NOS1 or adenovirus coding for the

and can limit the development of myocyte hypertrophy. Our results are in agreement with data from Drenning and colleagues [32] who demonstrated that NO could facilitate NFAT-dependent transcription in mouse myotubes through regulation of both pathways described in our study. More recently, NOS1 was shown to induce neuronal differentiation through inactivation of the GSK-3 β pathway [33].

In conclusion, we have provided in vitro evidence that NOS1 and NOS1-derived NO promote cardiac cells hypertrophy through a signaling pathway that includes calcineurin and GSK-3 β. These data improve our understanding of the regulation of myocyte hypertrophy by NOS1.

Source of fundings

This work was supported by INSERM, grants from INSERM Programme National de Recherche sur les Maladies Cardiovasculaires (DAPS-2007-02/MCPV/CC, C.H.), the Association Française contre les Myopathies (AFM 15305, C.H.), The French National Research Agency (ANR-08-GENOPAT-0032-01, C.H.).

Disclosures

None.

C.

Figure 4