Nelly Béchir

Émilie Pecchi

Karima Relizani

Christophe Vilmen

Yann Le Fur

Monique Bernard

Helge Amthor

David Bendahan

Benoit Giannesini
email: benoit.giannesini@univ-amu.fr

Benoît Giannesini

Benoît Giannesini

Mitochondrial impairment induced by postnatal ActRIIB blockade does not alter function and energy status in exercising mouse glycolytic muscle in vivo

Keywords: Skeletal muscle hypertrophy, Activin type IIB receptor, Myostatin, Force, Fatigue

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

INTRODUCTION

ActRIIB is a transmembrane serine-threonine kinase receptor highly expressed in mammalian skeletal muscle. It mediates signaling for myostatin (GDF8) and other members of the transforming growth factor β family that are involved in the negative regulation of skeletal muscle development [START_REF] Caestecker | The transforming growth factor-beta superfamily of receptors[END_REF][START_REF] Lee | Regulation of myostatin activity and muscle growth[END_REF]. Disruption of the ActRIIB signaling pathway leads to a rapid and massive increase in muscle mass resulting from fiber hypertrophy with increased myofiber protein synthesis [START_REF] Bogdanovich | Functional improvement of dystrophic muscle by myostatin blockade[END_REF][START_REF] Wang | Myostatin inhibition induces muscle fibre hypertrophy prior to satellite cell activation[END_REF]. In that context, several pharmacological approaches based on the postnatal ActRIIB signaling blockade have been considered for counteracting muscle wasting commonly associated to ageing, neuromuscular disorders and various catabolic diseases [START_REF] Dumonceaux | Current advances in the development of therapies for neuromuscular disorders based on myostatin signalling, 3rd International Institute of Myology Workshop[END_REF][START_REF] Lawlor | Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice[END_REF][START_REF] Lee | Regulation of muscle growth by multiple ligands signaling through activin type II receptors[END_REF]. These approaches mainly include the systemic delivery of neutralizing antibodies against ActRIIB or myostatin [START_REF] Lach-Trifilieff | An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy[END_REF][START_REF] Lebrasseur | Myostatin inhibition enhances the effects of exercise on performance and metabolic outcomes in aged mice[END_REF], and injection of a soluble recombinant form of ActRIIB (sActRIIB-Fc), which acts as a decoy receptor disrupting the interaction of endogenous ActRIIB receptor with its ligands [START_REF] Attie | A single ascending-dose study of muscle regulator ACE-031 in healthy volunteers[END_REF][START_REF] Lee | Regulation of muscle growth by multiple ligands signaling through activin type II receptors[END_REF][START_REF] Morrison | A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis[END_REF].

Although ActRIIB signaling blockade has been already tested in patients [START_REF] Krivickas | Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029[END_REF][START_REF] Wagner | A phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy[END_REF] and animal models [START_REF] Bogdanovich | Functional improvement of dystrophic muscle by myostatin blockade[END_REF][START_REF] Morrison | A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Pistilli | Targeting the activin type IIB receptor to improve muscle mass and function in the mdx mouse model of Duchenne muscular dystrophy[END_REF] suffering from dystrophic and neurological disorders, it is noteworthy that the impact of such a blockade has been very poorly documented in healthy muscle in vivo. This issue is however an important prerequisite for a better understanding of the effects of ActRIIB blockade in diseased muscle. It has been shown that postnatal disruption of the ActRIIB signaling in normal mice improves functional parameters such as grip strength [START_REF] Akpan | The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity[END_REF][START_REF] Lawlor | Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice[END_REF] and absolute force in isolated muscle [START_REF] Akpan | The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity[END_REF][START_REF] Chiu | Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents[END_REF], but on the other hand decreases the resistance to fatigue [START_REF] Chiu | Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents[END_REF][START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF]. These results are in all respects comparable to those obtained in myostatin-deficient mice in which the improved force has been documented [START_REF] Amthor | Lack of myostatin results in excessive muscle growth but impaired force generation[END_REF][START_REF] Giannesini | Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo[END_REF][START_REF] Mendias | Contractile properties of EDL and soleus muscles of myostatin-deficient mice[END_REF][START_REF] Mouisel | Myostatin is a key mediator between energy metabolism and endurance capacity of skeletal muscle[END_REF] and the increased fatigability has been assigned to a mitochondrial impairment evidenced on the basis of in vitro assays [START_REF] Mouisel | Myostatin is a key mediator between energy metabolism and endurance capacity of skeletal muscle[END_REF][START_REF] Ploquin | Lack of myostatin alters intermyofibrillar mitochondria activity, unbalances redox status, and impairs tolerance to chronic repetitive contractions in muscle[END_REF] and in vivo 31 P-MRS measurements [START_REF] Baligand | Functional assessment of skeletal muscle in intact mice lacking myostatin by concurrent NMR imaging and spectroscopy[END_REF][START_REF] Giannesini | Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo[END_REF]. Interestingly, long-term sActRIIB-Fc treatment in wild-type mice has been shown to reduce the expression of oxidative phosphorylation genes [START_REF] Rahimov | Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor[END_REF][START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF][START_REF] Zhao | Coordinated patterns of gene expressions for adult muscle build-up in transgenic mice expressing myostatin propeptide[END_REF] as well as the level of porin, an outer mitochondrial membrane protein involved in ATP transport between the cytosol and the mitochondrial matrix [START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF]. These findings suggest that ActRIIB blockade negatively affects the muscle mitochondrial function, which could have undesirable clinical consequences.

Nevertheless, this potent impairment has been so far assessed only though in vitro experiments, and the corresponding effects on energetics and mechanical performance in exercising muscle are then missing in vivo.

The aim of the current study was to investigate strictly noninvasively the impact of longterm ActRIIB signaling blockade on gastrocnemius muscle anatomy, energy metabolism and mechanical performance in wild-type mice. We intended especially to clarify (i) whether ActRIIB signaling blockade impairs mitochondrial function similarly to what has been reported in myostatin-deficient mice, and (ii) whether bioenergetics and force-generating capacity during a fatiguing bout of exercise are impaired under these conditions. Functional investigations were performed in electrostimulated muscle in vivo using MR imaging and 31 P-MR spectroscopy ( 31 P-MRS) while the electrophoretic separation of myosin heavy-chain (MHC) isoforms provided information about the fiber type composition. Gastrocnemius muscles were immediately removed, freeze-clamped with nitrogen-chilled metal tongs, and stored at -80°C for in vitro measurements. For each mouse, the delay between euthanasia and muscle freeze clamping was less than 20 s.

MATERIALS AND METHODS

Animal care and feeding

Sixteen

Experimental design

The postnatal blockade of ActRIIB signaling pathway was achieved using a soluble fusion protein (sActRIIB-Fc, also called RAP-031, Acceleron Pharma Inc., Cambridge, MA, USA) generated by fusing the ActRIIB extracellular domain to the mouse immunoglobulin Fc region [START_REF] Morrison | A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis[END_REF]. Mice were assigned to two groups (n = 8 per group) of comparable mean body weights. For a total of 8 weeks, animals were injected subcutaneously twice weekly with either sActRIIB-Fc (10 mg/kg body weight) or an equal volume of vehicle (10 mM PBS; control group).

Noninvasive investigation of gastrocnemius muscle function and energetics

Each animal was tested twice over a one-week period in order to evaluate strictly noninvasively bioenergetics, anatomy and force-generating capacity in electrostimulated gastrocnemius muscle. During the first testing session, MR imaging measurements were performed to get information about muscle volume, and metabolic changes associated to muscle activity were investigated using 31 P-MR spectroscopy throughout a standardized 6-min fatiguing exercise. During the second session, gastrocnemius muscle was electrostimulated with trains of incremental frequencies (1-150 Hz) and the corresponding force was measured.

Animal preparation. Mice were initially anesthetized in an induction chamber using 1.75% isoflurane in 33% O2 (0.2 L/min) and 66% N2O (0.4 L/min). The left hindlimb was shaved before an electrode cream was applied at the knee and heel regions to optimize transcutaneous electrostimulation. Anesthetized animal was placed supine in a home-built cradle which has been especially designed for the strictly noninvasive MR investigation of gastrocnemius muscle function [START_REF] Giannesini | A strictly noninvasive MR setup dedicated to longitudinal studies of mechanical performance, bioenergetics, anatomy, and muscle recruitment in contracting mouse skeletal muscle[END_REF]. This muscle was chosen because it is easily accessible for MR measurements and preferentially activated using our experimental methodology. The cradle integrates an ergometer consisting of 9 x 24 mm foot pedal coupled to a force transducer constructed by sticking a strain gauge (120-ohm internal resistance; ref 1-LY11-6/120A, HBM GmbH, Darmstadt, Germany) on a Bakelite slat (0.4 mm thickness) in a Wheatstone bridge design (3 × 120 ohm). The animal's foot was positioned on the pedal of the ergometer and the lower hindlimb was centered inside a 20-mm-diameter 1 H Helmholtz imaging coil while the belly of the gastrocnemius muscle was located above an elliptic (8 x 12 mm 2 ) 31 P-MRS surface coil. Corneas were protected from drying by applying ophthalmic cream, and animal's head was placed in a home-built facemask continuously supplied with 1.75% isoflurane in 33% O2 (0.2 L/min) and 66% N2O (0.4 L/min). Body temperature was controlled and maintained at a physiological level throughout the experiment using a feedback loop including an electrical heating blanket, a temperature control unit (ref. 507137, Harvard Apparatus, Holliston, MA, USA) and a home-built rectal thermometer.

Muscle electrostimulation procedure and force output measurement. Muscle contractions were achieved by transcutaneous electrostimulation using two rod-shaped 1.5mm-diameter electrodes integrated in the experimental cradle and connected to a constantcurrent stimulator (DS7A, Digitimer, Herthfordshire, United Kingdom). One electrode was placed at the heel level and the other one was located just above the knee joint. Electrical signal coming out from the force transducer was amplified (Operational amplifier AD620, Analog Devices, Norwood, MA, USA; 70-dB gain; 0-5 kHz bandwidth) and converted to a digital signal (PCI-6220, National Instrument, Austin, TX, USA) that was continuously monitored and recorded on a personal computer using the WinATS software version 6.5 (Sysma, Aix-en-Provence, France).

Preliminary adjustments. Before each experiment, muscle was passively stretched at rest by adjusting the angle between the foot and the lower hindlimb in order to produce maximal isometric twitch amplitude in response to supramaximal square wave pulses (1-ms duration). The individual maximal electrostimulation intensity was determined by progressively increasing the stimulus intensity until there was no further peak twitch force increase.

Fatigue exercise and force-frequency protocol. The fatiguing exercise was performed simultaneously to the dynamic 31 P-MRS acquisition. It consisted in 6 min of repeated maximal isometric contractions induced electrically at a frequency of 1.7 Hz.

Absolute force production (expressed as force-time integral, in N*s) was calculated by integrating the isometric force (in N) with respect to time. During the force-frequency protocol, gastrocnemius muscle was electrostimulated with trains (0.75 sec duration; rest interval of 30 sec) of incremental frequencies [START_REF] Akpan | The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity[END_REF][START_REF] Bogdanovich | Functional improvement of dystrophic muscle by myostatin blockade[END_REF][START_REF] Giannesini | A strictly noninvasive MR setup dedicated to longitudinal studies of mechanical performance, bioenergetics, anatomy, and muscle recruitment in contracting mouse skeletal muscle[END_REF][START_REF] Krivickas | Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029[END_REF][START_REF] Rahimov | Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor[END_REF]75, 100, and 150 Hz) and the corresponding force data were processed as followed: The isometric peak force was measured for each train and the overall dataset was fitted to the Hill equation for calculating f50, i.e., the electrostimulation frequency for which 50% of the maximal force was exerted. Specific force was calculated by normalizing the absolute force with the gastrocnemius muscle volume quantified from hindlimb MR images (see below). MR data were processed using custom-written analysis programs developed under the IDL (Interactive Data Language, Exelis Visual Information Solutions, Boulder, CO, USA) environment [START_REF] Fur | Grid-free interactive and automated data processing for MR chemical shift imaging data[END_REF][START_REF] Mattei | Segmentation of fascias, fat and muscle from magnetic resonance images in humans: the DISPIMAG software[END_REF]. For each MR image, region of interest was manually outlined so that the corresponding cross sectional area of the gastrocnemius muscle was measured. Muscle volume was calculated by summing the volumes between consecutive slices. Relative concentrations of phosphocreatine (PCr), inorganic phosphate (Pi) and ATP were obtained from 31 P-MR spectra by a time-domain fitting routine using the AMARES-MRUI Fortran code [START_REF] Vanhamme | Improved method for accurate and efficient quantification of MRS data with use of prior knowledge[END_REF] and appropriate prior knowledge for the ATP multiplets. Absolute amounts of phosphorylated compounds were expressed relative to a resting ATP concentration determined in vitro using a bioluminescence-based method (see below), and intracellular pH was calculated from the chemical shift of the Pi signal relative to PCr (3). ADP concentration was calculated from [PCr], [ATP] and pH considering the equilibrium constant (K = 1.67 10 9 M - 1 ) of the creatine kinase (CK) reaction [START_REF] Roth | Determination of cytosolic ADP and AMP concentrations and the free energy of ATP hydrolysis in human muscle and brain tissues with 31P NMR spectroscopy[END_REF]. VPCrstim (the initial rate of PCr degradation at the start of the fatiguing exercise, in mM/min) was calculated as the product of kstim (the pseudofirst-order rate-constant of PCr degradation) and [PCr]cons (the amount of PCr consumed at the end of the exercise). The constant kstim was determined by fitting with a least mean-squared algorithm the time-course of PCr degradation to a single exponential curve described by the oxidative ATP synthesis (Qmax, mM/min) was calculated using VPCrrec and the level of ADP measured at the end of the fatiguing exercise as previously described ( 28): Qmax = (1+Km/[ADP]end)VPCrrec where Km (the apparent Michaelis-Menten constant of mitochondrial respiration for ADP, i.e., the ADP concentration at half-maximal rate of oxidative ATP synthesis) is 30 μM in mouse gastrocnemius muscle [START_REF] Kemp | pH control in rat skeletal muscle during exercise, recovery from exercise, and acute respiratory acidosis[END_REF]. The rate of proton efflux from the muscle (in mM/min) was calculated in the early stage of the postexercise recovery period from the changes in pH and levels of high-energy phosphorylated contents as described previously [START_REF] Giannesini | Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo[END_REF][START_REF] Kemp | Quantitative interpretation of bioenergetic data from 31 P and 1 H magnetic resonance spectroscopic studies of skeletal muscle: an analytical review[END_REF]. PCr cost of contraction (PCrcost, in μmol/Ns) was calculated as the ratio between VPCrstim and the specific force generated at the start of the fatiguing exercise. Oxidative cost of contraction (Oxcost, in μmol/N*s) was calculated at the end of muscle activity as the ratio between VPCrrec and specific force produced [START_REF] Boska | Estimating the ATP cost of force production in the human gastrocnemius/soleus muscle group using 31P MRS and 1H MRI[END_REF] considering that, at this time, ATP production from oxidative phosphorylation is equal to VPCrrec [START_REF] Radda | The use of NMR spectroscopy for the understanding of disease[END_REF][START_REF] Taylor | Energetics of human muscle: exercise-induced ATP depletion[END_REF].

MR data acquisition and processing

Myosin heavy-chain isoforms analysis

Freeze-clamped muscles (50 mg) were homogenized in 600 µl of a solution containing (mM): NaCl 300, NaH2PO4 100, Na2HPO4 50, Na4P2O7 10, MgCl2 1, EDTA 10 and βmercaptoethanol 1.4; pH 6.5. After incubation for 24 h at 4°C, the homogenates were centrifuged (15 min, 13000 x g, 4°C). The supernatants were diluted 1:1 with glycerol and stored at -80°C. Total protein concentrations were measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, Illkirch, France) according to the manufacturer's instructions.

Two µg of total proteins were denatured and then subjected to electrophoresis as previously described (8) using a Mini Protean Tetra system (Bio-Rad, Marnes-la-Coquette, France). Gels were run at 72 V for 48 h, stained with IRDye® Blue Protein Stain (LI-COR Biosciences, Lincoln, NE, USA), and then scanned in the 700 nm channel of an infrared imaging system with a 42-µm spatial resolution (Odyssey®, LI-COR Biosciences, Lincoln, NE, USA). MHC isoforms were identified by comparing them with bands of myosin extracts from control soleus muscle [START_REF] Birot | Exercise-induced expression of vascular endothelial growth factor mRNA in rat skeletal muscle is dependent on fibre type[END_REF] and quantified with the ImageJ software (http://imagej.nih.gov/ij/).

Intramuscular ATP content

Freeze-clamped muscles (40-60 mg) were homogenized in 1.2 mL of ice-cold perchloric acid (0.6 M). After incubation for 15 min at 4°C, homogenates were centrifuged (15 min, 2000 x g, 4°C). The supernatants were neutralized with K2CO3, and placed for 30 min at 4°C. After being centrifuged (15 min, 2000 x g, 4°C) to remove precipitates, the supernatants were aliquoted and stored at -80°C. ATP concentrations were determined using the bioluminescence ATP determination kit (ref. A22066, Invitrogen, Eugene, Oregon, USA) according to the manufacturer's instructions. Luminescence was measured on a microplate reader (Victor X3, PerkinElmer, Waltham, MA, USA). All samples were run in duplicate.

Mitochondrial DNA content

Total genomic DNA was isolated from freeze-clamped gastrocnemius muscle using the Nucleospin Tissue extraction kit (Macherey-Nagel, Düren, Germany). Isolated gDNA was quantified using the Nanovue Plus Spectrophotometer (GE Healthcare, Life Technologies, France). A real time PCR was performed using the SYBR Green PCR Master Mix Protocol (Bio-Rad France, Marnes-la-Coquette, France) in triplicate on the CFX96 Real Time System (BioRad France). A 10 min denaturation step at 94°C was followed by 40 cycles of denaturation at 94°C for 10 s and annealing/extension at 60°C for 30 s. mtDNA content was determined with the mitochondrial cytochrome c oxidase II (COXII) gene expression (forward

5' GCCGACTAAATCAAGCAACA 3'
and reverse 5' CAATGGGCATAAAGCTATGG 3' ) normalized to the genomic NADH deshydrogenase uboquinone flavoprotein 1 (Ndufv1) gene (forward 5' CTTCCCCACTGGCCTCAAG 3' and reverse 5' CCAAAACCCAGTGATCCAGC 3' ).

Statistical analysis

Data are expressed as mean ± SE. Statistical analyses were performed using JMP software version 9 (SAS Institute Inc., Cary, NC, USA). Significant differences were determined by unpaired two-tailed Student's t-tests or by two-factor (group × time) analysis of variance (ANOVAs) with repeated measures on time followed, when appropriate, by Tukey-Kramer post-hoc multiple comparison tests for determining pairwise differences between groups. The significance level was set at P < 0.05.

RESULTS

Morphological changes

At the end of the 8-week treatment period, body weight and gastrocnemius muscle volume were larger (+29% and +58%, respectively) in animal treated with sActRIIB-Fc (Fig. 1A and1B). Furthermore, ActRIIB blockade did not alter the proportion of MHC isoforms in the gastrocnemius muscle (Fig. 1C) .

Mechanical performance

The force-frequency curve was constructed from data obtained during incremental frequency trains in electrostimulated gastrocnemius muscle (Fig. 2A). As shown in Fig. 2B and 2C, treatment with sActRIIB-Fc did not affect the maximal tetanic force but significantly reduced the maximal specific tetanic force. Besides, there was no difference between both groups for f50, i.e., the electrostimulation frequency for which 50% of the maximal force was exerted (Fig. 2D).

Changes in absolute force production and fatigue resistance during the 6-min in vivo fatiguing exercise are shown in Fig. 3A and3B. Absolute force transiently increased in the early stage of the exercise to reach a maximal value (Fmax); Fmax and force increase (Fmax-Fstart)

were larger (+26% and +33%, respectively) in animals receiving sActRIIB-Fc (Fig. 3C and3D).

Afterward, force progressively decreased until the end of the exercise as a sign of fatigue development (Fig. 3A). Interestingly, fatigue resistance became significantly lower in sActRIIB-Fc treated animals from the middle of the exercise (Fig. 3B). At the end of the exercise, the extent of force reduction was larger in animals treated with sActRIIB-Fc (68 ± 4 % vs. 50 ± 4 %; Fig. 3B). Overall, total force produced during the whole fatiguing exercise was 12% larger in the group receiving sActRIIB-Fc (Fig. 3E). Besides, the shape of specific force curve during the fatiguing exercise was similar between both groups (Fig. 4A). Nevertheless, maximal specific force (Fig. 4B) and total specific force produced during the whole fatiguing exercise (Fig. 4C) were significantly lower (-19% and -28%, respectively) in sActRIIB-Fc treated animals.

Bioenergetics

In resting gastrocnemius muscle, there were no differences between both groups for

[PCr]/[ATP] ratio, [Pi] and [ATP] (Table 1). On the contrary, [PCr], [ADP] and pH were significantly lower in animals treated with sActRIIB-Fc. Importantly, the mitochondrial DNA copy numbers remained largely unchanged following sActRIIB treatment (Fig. 5).

At the start of the fatiguing exercise, PCr was rapidly consumed (Fig. 6A) at a similar rate between both groups (Fig. 7A) and PCr cost of contraction was not affected by sActRIIB-Fc treatment (Fig. 7B). In the middle of the exercise, [PCr] reached a steady state that remained stable until the end of the contractile session (Fig. 6A), when the extent of PCr degradation (D[PCr]) did not differ between both groups (Table 1). For each animal, Pi accumulated throughout the fatiguing exercise with an initial phase of rapid and massive accumulation followed by a phase of steady state (Fig. 6B) whereas [ATP] remained close to its basal value during the whole exercise (Fig. 6C). At the end of the exercise, there were no differences between PBS and sActRIIB-Fc groups for [Pi] and D[ATP] (Table 1). The timecourse of intracellular pH was also strongly similar between both groups, with a rapid acidosis early in the exercise, followed by a phase of steady state that remained fairly constant until the cessation of electrostimulation (Fig. 6D). At that time, the extent of acidosis was not affected by sActRIIB-Fc treatment (Table 1). Besides, the oxidative cost of contraction did not differ between both groups (Fig. 7C) but the maximal rate of oxidative ATP synthesis (Qmax) was 44% lower in animals treated with sActRIIB-Fc (Fig. 7D).

During the recovery period following the fatiguing exercise, phosphorylated compound levels and pH progressively returned toward their respective basal values (Fig. 6A, 6B, 6C and 6D). Noteworthy, the initial rate of PCr resynthesis and proton efflux were lower (-34% and -92%, respectively) in animals treated with sActRIIB-Fc (Fig. 7E and7F).

DISCUSSION

This study is the first attempt for investigating in vivo the effect of long-term ActRIIB signaling blockade on muscle anatomy, energy metabolism and force-generating capacity. We mainly found that ActRIIB blockade (i) impairs oxidative mitochondrial function while (ii) absolute force generation is increased and (iii) specific force and fatigue resistance are both reduced. Despite these abnormalities, the bioenergetics status throughout a fatiguing bout of exercise was not altered.

One of the major findings of the current study is that long-term disruption of ActRIIB signaling impairs mitochondrial function in skeletal muscle, which is comparable to data obtained in myostatin-deficient animals [START_REF] Amthor | Lack of myostatin results in excessive muscle growth but impaired force generation[END_REF][START_REF] Baligand | Functional assessment of skeletal muscle in intact mice lacking myostatin by concurrent NMR imaging and spectroscopy[END_REF][START_REF] Giannesini | Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo[END_REF][START_REF] Mouisel | Myostatin is a key mediator between energy metabolism and endurance capacity of skeletal muscle[END_REF]. We found actually that sActRIIB-Fc treatment dramatically reduced the maximal rate of oxidative ATP synthesis (-42%), which is considered as a robust and highly reproducible in vivo index of oxidative mitochondrial capacity that has been widely used in research and clinical applications [START_REF] Arnold | Metabolic recovery after exercise and the assessment of mitochondrial function in vivo in human skeletal muscle by means of 31 P NMR[END_REF][START_REF] Bendahan | 31P NMR spectroscopy and ergometer exercise test as evidence for muscle oxidative performance improvement with coenzyme Q in mitochondrial myopathies[END_REF][START_REF] Kemp | Quantification of skeletal muscle mitochondrial function by 31P magnetic resonance spectroscopy techniques: a quantitative review[END_REF][START_REF] Lanza | Measurement of human skeletal muscle oxidative capacity by 31P-MR spectroscopy: a cross-validation with in vitro measurements[END_REF]. Our findings are in agreement with previous in vitro experiments conducted in wild-type mice showing that sActRIIB-Fc delivery reduces the expression of genes involved in mitochondrial oxidative phosphorylation [START_REF] Rahimov | Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor[END_REF][START_REF] Zhao | Coordinated patterns of gene expressions for adult muscle build-up in transgenic mice expressing myostatin propeptide[END_REF] and decreases the level of porin, a mitochondrial membrane protein involved in the transport of ATP between mitochondrial matrix and cytosol [START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF].

Nevertheless, in agreement with previous experiments in soleus and EDL muscles from healthy mice treated with sActRIIB-Fc ( 52), we measured that the mitochondrial DNA copy number in gastrocnemius muscle remained largely unchanged following sActRIIB treatment, hence indicating that the mitochondrial impairment was not caused by any decreased mitochondria content. Besides, it must be kept in mind that the ability of mitochondria to produce ATP critically depends on oxygen supply. Interestingly, we observed that pH and PCr level were both significantly lower in resting hypertrophied muscle. This indicates that sActRIIB-Fc limits muscle oxygen availability given that the combination of intracellular acidosis and PCr depletion is an indicator of basal muscle hypoxia [START_REF] Boutilier | Mechanisms of cell survival in hypoxia and hypothermia[END_REF]. Accordingly, we found that proton efflux at the start of the postexercise period was significantly slower in mice treated with soluble ActRIIB, hence leading to assume that the hypoxic state might be due to decreased tissue perfusion. Our assumption is consistent with previous histological analyses showing that muscle capillarity is reduced in normal mice receiving a 4-month sActRIIB-Fc treatment [START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF] and in myostatin-deficient muscle [START_REF] Amthor | Lack of myostatin results in excessive muscle growth but impaired force generation[END_REF][START_REF] Rehfeldt | Effects of the compact mutant myostatin allele Mstn (Cmpt-dl1Abc) introgressed into a high growth mouse line on skeletal muscle cellularity[END_REF]. Consequently, it is possible that sActRIIB-Fc limits oxygen delivery to the muscle, which in return would reduce mitochondrial respiration. Noteworthy, we found that sActRIIB-Fc treatment did not alter basal ATP level but reduced that of ADP. Considering the CK reaction that reversely transfers high-energy phosphate from PCr to ADP to form ATP via the reaction: PCr + ADP + H + ↔ ATP + creatine [START_REF] Lawson | Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions[END_REF], the reduced ADP level could be interpreted as a compensatory mechanism allowing to maintain ATP homeostasis in the face of both an intracellular acidosis and a reduced PCr content.

The mouse gastrocnemius muscle is predominantly glycolytic. However, considering that energy metabolism plays a critical role in muscle contraction, one can now ask whether the mitochondrial impairment induced by ActRIIB blockade causes functional alteration in exercising muscle, i.e., when energy demand is substantially larger. This question is legitimate given the contrasting effect of sActRIIB-Fc on mechanical performance. Firstly, we shown that force increase (Fmax-Fstart) and both maximal (Fmax) and total absolute force produced during the 6-min in vivo fatiguing exercise were larger in animals treated with sActRIIB-Fc, which is in line with previous studies showing an increased forelimb grip strength in normal mice receiving sActRIIB-Fc [START_REF] Akpan | The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity[END_REF][START_REF] Lawlor | Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice[END_REF]. Importantly, our findings confirm that sActRIIB-Fc treatment might be beneficial for patients suffering from muscle wasting. Secondly, our results related to the specific force clearly indicated that the mechanical performance was reduced as compared to what could have been expected on the basis of the muscle hypertrophy. Actually, we showed that muscle hypertrophy was not accompanied by a proportional increase in absolute force. On the contrary, specific force was reduced in animals treated with sActRIIB-Fc in agreement with what has been already reported in hypertrophied muscle of myostatin-deficient mice [START_REF] Amthor | Lack of myostatin results in excessive muscle growth but impaired force generation[END_REF][START_REF] Giannesini | Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo[END_REF][START_REF] Mendias | Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation[END_REF]. Thirdly, we observed that sActRIIB-Fc treatment did not improve maximal tetanic force in vivo. Although counterintuitive, this finding has been already reported in isolated muscles from myostatin-deficient mice (2) or from wild-type mice treated with sActRIIB-Fc [START_REF] Akpan | The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity[END_REF][START_REF] Chiu | Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents[END_REF] or myostatin neutralizing antibodies [START_REF] Lebrasseur | Myostatin inhibition enhances the effects of exercise on performance and metabolic outcomes in aged mice[END_REF]. While the underlying cause for this phenomenon has not been documented, one must be aware that myostatin deficiency reduces the relative collagen content, which alters muscle mechanistic by increasing the stiffness of muscle aponevrosis and tendon [START_REF] Mendias | Contractile properties of EDL and soleus muscles of myostatin-deficient mice[END_REF][START_REF] Welle | Skeletal muscle gene expression after myostatin knockout in mature mice[END_REF]. It is then conceivable that the long-term ActRIIB blockade produces similar alterations, hence ultimately lowering the ability to generate tetanic tension. Lastly, we found that ActRIIB signaling inhibition reduced fatigue resistance during the in vivo fatiguing exercise, which corroborates other studies showing that sActRIIB-Fc delivery increases fatigability in hindlimb muscle electrically stimulated in situ [START_REF] Chiu | Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents[END_REF] and decreases the time to exhaustion during incremental speed running tests [START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF].

Despite the mitochondrial and mechanical abnormalities reported herein, several lines of evidence led us to believe that sActRIIB-Fc treatment is not deleterious for energy metabolism in exercising muscle. Intramyofibrilar PCr concentration is controlled by the CK reaction and the PCr-CK system is considered to have a crucial role in ATP homeostasis during exercise. The system actually functions as an energy buffer at the transition from rest to exercise and -with continuation of exercise -as an energy carrier directly involved in the transport of high-energy phosphate between the sites of production (mitochondrial matrix) and utilization (cytosol) of ATP [START_REF] Hochachka | Cellular metabolic homeostasis during large-scale change in ATP turnover rates in muscles[END_REF]. In the present study, sActRIIB-Fc treatment did alter neither the initial rate of PCr degradation nor the PCr cost of contraction at the start of the fatiguing exercise, thereby indicating that the energy buffering function of the PCr-CK system remained unaffected. At the end of the exercise, there were no differences between both groups for oxidative cost of contraction and extents of both PCr consumption and [ATP] reduction, which demonstrates that (i) sActRIIB-Fc treatment does not alter the energy transport function of the PCr-CK system and (ii) energy metabolism entirely fits ATP demand for contraction. Also, it is noteworthy that the time-dependent changes in pH during the fatiguing exercise were similar between both groups. Considering that intracellular acidosis in contracting muscle mainly reflects glycolytic flux, our data suggests that glucose metabolism was not disturbed by ActRIIB blockade, which is in line with previous experiments showing that sActRIIB-Fc treatment does affect neither muscle glucose uptake (1) nor transcription of gene involved in glucose metabolism [START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF]. Overall, one can affirm that the fact that muscle capacity for generating force does not take full advantage of muscle hypertrophy is not due to alteration in energy metabolism, and other mechanism(s) should then occur but this issue has to our knowledge never been documented.

Typological changes, calcium fluxes alteration and muscle architecture changes have been put forward as accounting factors of the increased fatigability and the decreased specific force reported in myostatin-deficient animals [START_REF] Amthor | Lack of myostatin results in excessive muscle growth but impaired force generation[END_REF][START_REF] Bodnar | Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling[END_REF][START_REF] Mendias | Contractile properties of EDL and soleus muscles of myostatin-deficient mice[END_REF][START_REF] Personius | Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice[END_REF][START_REF] Welle | Skeletal muscle gene expression after myostatin knockout in mature mice[END_REF]. In these animals, the relative fraction of glycolytic fibers -which produce more force but conversely are more susceptible to fatigue -is increased at the expense of both oxidative and oxidative-glycolytic fibers [START_REF] Gentry | Hindlimb skeletal muscle function in myostatin-deficient mice[END_REF][START_REF] Girgenrath | Loss of myostatin expression alters fiber-type distribution and expression of myosin heavy chain isoforms in slow-and fast-type skeletal muscle[END_REF][START_REF] Matsakas | Myostatin knockout mice increase oxidative muscle phenotype as an adaptive response to exercise[END_REF]. We have checked fiber composition in the gastrocnemius muscle and found that ActRIIB blockade did not cause fiber-type conversion, as previously shown in other sActRIIB-Fc treated mouse muscles such as quadriceps, EDL, soleus and plantaris [START_REF] Cadena | Administration of a soluble activin type IIB receptor promotes skeletal muscle growth independent of fiber type[END_REF][START_REF] Lawlor | Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice[END_REF][START_REF] Relizani | Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy[END_REF]. Our finding therefore rules out any typological change as an accounting factor of the altered mechanical performance in sActRIIB-Fc treated muscle. Accumulation of tubular aggregates at the calcium-release channel of the sarcoplasmic reticulum has also been proposed to alter calcium handling during excitation-contraction cycles in myostatin-deficient mice thereby altering mechanical performance [START_REF] Amthor | Lack of myostatin results in excessive muscle growth but impaired force generation[END_REF][START_REF] Bodnar | Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling[END_REF]. Nevertheless, this issue remains unlikely herein because we found that ActRIIB blockade did not affect f50 (the electrostimulation frequency for which 50% of the maximal tetanic force was exerted) an in vivo indicator of calcium sensitivity [START_REF] Gineste | Combined MRI and (3)(1)P-MRS investigations of the ACTA1(H40Y) mouse model of nemaline myopathy show impaired muscle function and altered energy metabolism[END_REF][START_REF] Russ | Central activation and force-frequency responses of the lumbar extensor muscles[END_REF]. On the other hand, it is possible that the reduced mechanical capacity in sActRIIB-Fc treated animals was linked to hypertrophy-induced alterations of fiber pennation angles caused by increased muscle cross sectional area. This muscle architecture alteration would thus impair the transmission of force by myofilaments [START_REF] Hughes | Effects of aging, exercise, and disease on force transfer in skeletal muscle[END_REF][START_REF] Maxwell | Estimation of number of fibers in guinea pig skeletal muscles[END_REF].

In conclusion, our study demonstrates in vivo that the pharmacological blockade of ActRIIB impairs mitochondrial function possibly via muscle oxygenation limitation, but this impairment does not compromise bioenergetics status and gastrocnemius muscle function during sustained muscle activity. However, it must be kept in mind that the gastrocnemius muscle is predominantly glycolytic and our results cannot be extrapolated to type I oxidative muscle fiber. On the other hand, it is noteworthy that despite reduced specific force and increased fatigability in the late phase of muscle activity, ActRIIB blockade also increases absolute force-generating capacity. Although fatigue resistance and specific force are important variables of muscle function, patients' performance is usually assessed according to whether they produce enough force to accomplish a short-duration task. In that context, it is obvious that the increased absolute force-generating capacity represents a functional advantage. Therefore, beyond the demonstration that ActRIIB signaling plays key roles in the balance between muscle mass, force generation, oxidative metabolism and resistance to fatigue, our data strengthen ActRIIB blockade as a promising clinical application for muscle wasting disorders.

  C57BL/6 3-month old mice (Janvier Labs, Le Genest Saint Isle, France) were used for these experiments conducted in strict accordance with the guidelines of the European Communities Council Directive 86/609/EEC for Care and Use of Laboratory Animals. All animal experiments were performed according to protocols reviewed and approved by the Institutional Animal Care Committee of Aix-Marseille University (permit number 93-21122012) and under the supervision of BG (license number 13.164 2008/11/25). Every attempt was made to minimize the number and the suffering of animals. Mice were socially housed as four per cage in an environmentally controlled facility (12-12 h light-dark cycle, 22°C) with free access to commercial standard food and water until the time of experiment. After experiments, animals were euthanized by cervical dislocation following isoflurane anesthesia.

.

  Explorations were performed in the 4.7-Tesla horizontal magnet of a 47/30 Biospec Avance MR system (Bruker, Karlsruhe, Germany) equipped with a Bruker 120-mm BGA12SL (200 mT/m) gradient insert. Ten consecutive noncontiguous 0.5-mm spaced axial slices (1-mm thickness) covering the region from the knee to the ankle were selected across the lower hindlimb. RARE images of these slices (8 echoes; effective echo time = 67.9 ms; actual echo time = 16.7 ms; 2000-ms repetition time; one accumulation; 20 x 15 mm field of view; 256 x 256 matrix size) were recorded at rest. 31 P-MR spectra (8 kHz sweep width; 2048 data points) from the gastrocnemius region were continuously acquired at rest (6 min), during the 6-min fatiguing exercise and during the following 15-min of recovery period. MR data acquisition was gated to muscle electrostimulation in order to reduce potential motion artifacts due to contraction. A fully relaxed spectrum (12 scans, 20-s repetition time) was acquired at rest, followed by a total of 768 saturated free induction decays (FID) (1.875 s repetition time). The first 64 FIDs were acquired at rest and summed together. The next 192 FIDs were acquired during the fatiguing exercise and were summed by packets of 32, allowing a 60-s temporal resolution. The remaining 512 FIDs were obtained during the postexercise recovery period and were summed as 7 packets of 32 FIDs followed by 3 packets of 64 FIDs (120-s temporal resolution) and one packet of 96 FIDs (160-s temporal resolution).

  equation: [PCr]t = [PCr]end+[PCr]cons*e -kt , where [PCr]end is the concentration of PCr measured at the end of the exercise. Similarly, VPCrrec (the initial rate of PCr resynthesis at the start of the recovery period following the fatiguing exercise, in mM/min) was calculated as the product of krec (the pseudo-first-order rate-constant of PCr resynthesis) and [PCr]cons. In order to determine krec, the PCr time-course during the recovery period was fitted with a least meansquared algorithm to a single exponential curve described by the equation: [PCr]t = [PCr]rest-[PCr]cons*e -kt , where [PCr]rest is the concentration of PCr measured at rest. The maximal rate of

Figure 1 .

 1 Figure 1. Effect of a 8-week treatment with PBS or sActRIIB-Fc on body weight (A),

Figure 2 .

 2 Figure 2. Absolute force produced during the in vivo force-frequency protocol in gastrocnemius muscle (A). Treatment with sActRIIB-Fc did not affect the maximal tetanic force

Figure 3 .

 3 Figure 3. Changes in absolute force production (A) and fatigue resistance (B) throughout the 6-min in vivo fatiguing exercise performed simultaneously to metabolic MR acquisition. Maximal force (Fmax; panel C), force increase (Fmax-Fstart; panel D) and the total amount of force

Figure 4 .

 4 Figure 4. Changes in specific force production throughout the 6-min in vivo fatiguing exercise

Figure 5 .

 5 Figure 5. Effect of a 8-week treatment with PBS or sActRIIB-Fc on gastrocnemius muscle mitochondrial DNA content expressed as the cytochrome c oxidase II (COXII) relative copy numbers normalized to NADH deshydrogenase uboquinone flavoprotein 1 (Ndufv1) genomic

Figure 6 .

 6 Figure 6. In vivo changes in gastrocnemius muscle [PCr] (A), [Pi] (B), [ATP] (C) and pH (D) during the 6-min fatiguing exercise and the 15-min recovery period. For each panel, the first

Figure 7 .

 7 Figure 7. Initial rate of PCr consumption (A) and PCr cost of contraction (B) at the start of the

  Figure 1

  Figure 3

  Figure 7

Table 1

 1 Effect of a 8-week treatment period with PBS or sActRIIB-Fc on mouse gastrocnemius bioenergetics assessed in vivo using 31 P-MR spectroscopy.

	PBS	sActRIIB-Fc

ACKNOWLEDGEMENTS

We are grateful to Acceleron Pharma Inc. for the gift of sActRIIB-Fc. The authors also thank Ms Laurence Louis (Plate-Forme Génomique et Transcriptomique, Aix-Marseille Université, INSERM, UMR_S910, Marseille, France) for its helpful assistance with infrared imaging.

GRANTS

This work was financially supported by the French Muscular Dystrophy Association (AFM-Téléthon).

DISCLOSURE

The authors have no conflicts of interest, financial or otherwise, to declare. Acceleron Pharma Inc. did not influence data collection, analysis, interpretation and decision to publish the present study.


Arnaud Bourdin

Non-optimal methodology questions indirect treatment comparison of dupilumab vs. other biologics in severe asthma


Romain Terrier
email: romain.terrier@b-com.com

Valérie Gouranton

Cédric Bach

Nico Pallamin

Bruno Arnaldi

Scenario-based VR application for collaborative design

Keywords: VR, Framework, Scenarios, Collaborative design

Virtual reality (VR) applications support design processes across multiple domains by providing shared environments in which the designers refine solutions. Given the different needs specific to these domains, the number of VR applications is increasing. Therefore, we propose to support their development by providing a new VR framework based on scenarios. Our VR framework uses scenarios to structure design activities dedicated to collaborative design in VR. The scenarios incorporate a new generic and theoretical collaborative design model that describes the designers' activities based on external representations. The concept of a common object of design is introduced to enable collaborations in VR and the synchronization of the scenarios between the designers. Consequently, the VR Framework enables the configuration of scenarios to create customized and versatile VR collaborative applications that meet the requirements of each stakeholder and domain.

INTRODUCTION

Virtual reality (VR) is largely used to support design in industry (e.g., product design, interior design). However, depending on the designers and the sector needs, each VR application has to be personalized and has to offer various design functionalities. The development time is becoming longer due to the requirement to script new behaviors from the beginning and devise new rules for each need. As an alternative to building each application from scratch, in this paper, we propose a unique VR Framework based on scenarios to create various VR multi-user applications for collaborative design.

VR is an appropriate tool that the designers can use to cooperate. The shared and interactive environments enable the designers to work on complex systems [START_REF] Wang | A comprehensive survey of ar/mr-based co-design in manufacturing[END_REF]. There is a great variety of VR applications that can be utilized to work on these complex systems. This variety is explained by the necessity to take into account the knowledge and the work tools of multiple stakeholders in VR, and by the integration of the different types of collaborations that occur during the design process [START_REF] Falzon | Collective design process[END_REF]. The reasons for this diversity highlight the complexity of building VR applications for effective collaborative design and underline the necessity of supporting the development of such applications.

To support the development of VR applications for collaborative design, we propose a new VR multiuser Framework that offer several services to the developers (Fig. 1). The Framework is based on scenarios to sequence and to define the services in compliance with our theoretical design model. First, we describe the generic design activities and the levels of collaboration in a theoretical model. Then, the theoretical model is incorporated into various scenarios "to depict all the possible sequencing" of design activities in VR [START_REF] Claude | Versatile scenario guidance for collaborative virtual en-vironments[END_REF]. The construction of the scenarios is based on Petri-Net, which enables the developers to adjust this process in the event of changes to the theoretical model. Finally, the new concept of a common object of design is implemented in the scenarios as a service to the developers to enable different collaborations during the de-sign process, including functionalities to display design state information in VR.

In this way, the developers are provided with a tool to create VR multi-user and personalized applications for collaborative design.

Related work

This section presents the state of the art of the various design activities, that are part of a general process, and the collaboration between designers. These activities, collaborations and processes are structured into theoretical design models that are subsequently outlined. Finally, multiple Frameworks are presented to facilitate the development of VR applications based on activities.

Design: activities and process

The design process could be defined either as a problem-solving process [START_REF] Simon | Problem forming, problem finding and problem solving in design[END_REF] or as a situated and reflective practice [START_REF] Schön | The reflective practitioner: How professionals think in action[END_REF]. Regardless of the definition adopted, the design process is composed of a succession of design activities [START_REF] Gero | An approach to the analysis of design protocols[END_REF] during which the designer's knowledge is reflected in practice [START_REF] Schön | Designing as reflective conversation with the materials of a design situation[END_REF]. These activities act on and occur in the designer's mind or in the physical world, that is, the activities are internal or external [START_REF] Zhang | The interaction of internal and external representations in a problem solving task[END_REF]. The designers use the external activities to materialize their internal representations [START_REF] Eastman | Chapter 8 -new directions in design cognition: Studies of representation and recall[END_REF], and the external activities are easier to implement in a VR application than internal activities.

In the literature, scholars mostly divide the design process into two main spaces, namely the problem space and the solution space [START_REF] Lonchampt | Supporting Problem Expression within a Co-evolutionary Design Framework[END_REF]. A third space, called the evaluation space, has been introduced to characterize the activities involved in the evaluation of a design [START_REF] Terrier | Scenario-based VR Framework for Product Design[END_REF]. The designers perform multiple sequences of activities to refine the design (cos, 2003). The three spaces are linked [START_REF] Brissaud | Design process rationale capture and support by abstraction of criteria[END_REF], and they co-evolve. Thus, all the spaces are refined together, and the design process ends when the final state of compromise is reached [START_REF] Simon | Problem forming, problem finding and problem solving in design[END_REF]. Regardless of the design process and the design domains involved, the problem, the solution, and the evaluation are shared concepts among the design domains.

The users' design actions are described by activities that produce external representations that are categorized into generic design spaces.

Collaboration and cognitive synchronization

In the collaborative design process, the design activities involve several designers cooperating simultaneously. In VR, this cooperation is classified into three levels [START_REF] Margery | A general framework for cooperative manipulation in virtual environments[END_REF]. In level 1, the users are able to see each other and to communicate. In level 2, the users are able to act on the scene by changing the scene individually. In level 3, the users are able to act on the same entities in the scene. But these levels are never used to describe the collaborative design process. The users' success depends on their ability to establish a cognitive synchronization [START_REF] Détienne | Collaborative design: Managing task interdependencies and multiple perspectives[END_REF]. Thus, in VR collaboration, the design systems have to support communications between the designers and provide a mutual workspace that enables a "shared understanding of the design artifact among a design team" [START_REF] Saad | Shared understanding in computer-supported collaborative design[END_REF]. A deficiency in shared understanding may lead to misinterpretation, or annoyance, consequently slowing down the design process [START_REF] Valkenburg | Shared understanding as a condition for team design[END_REF]. Representation and interaction metaphors can be utilized to avoid this drawback. The relevant ideas and knowledge are externalized through the metaphors, thus facilitating and supporting communication between the designers [START_REF] Perry | Coordinating joint design work: the role of communication and artefacts[END_REF]. For these reasons, common descriptions and explanations of a system are key factors for effective collaboration, which emphasizes the necessity of synchronicity and communication between the designers [START_REF] Smart | Shared understanding within military coalitions: A definition and review of research challenges[END_REF]. External representations are important for sharing information to establish a shared understanding between the designers in VR and for easing communication between them, which could also be supported by the use of a verbal channel in virtual environments (VE) [START_REF] Gabriel | Coding and modelling communication in architectural collaborative design[END_REF].

Theoretical design models

Different models have been proposed to structure and understand the design process. During the design process multiple iterations occur, and these aspects of design can be described by models. Some models envisage design as consisting of multiple activities [START_REF] Girod | Decision making in conceptual engineering design: An empirical investigation[END_REF] without defining it as involving a series of events. Others group activities by family and link the activities with events to describe a generic design process [START_REF] Gero | The situated function behaviour structure framework[END_REF]. All of these models enable iterations and describe activities, but none depict collaboration.

To overcome this gap in the existing models, the individual function-behaviour-structure (FBS) model has been extended for collaborative design [START_REF] Gero | The situated function-behavior-structure co-design model[END_REF]. The junction between the individual activities occurs in the external world, thereby allowing the development of shared understanding. The FBS model is a powerful tool for understanding and encoding the collaborative design mechanism, but it describes internal and external activities. The internal activities occur in the mind of the people, thus the development of these activities are not possible. A generic model has been proposed for individual design describing the co-evolution of the problem, solution, and evaluation space [START_REF] Terrier | Scenario-based VR Framework for Product Design[END_REF]. This model describes generic activities categorized according to the three spaces delineated in Section 2.1. Moreover, the described activities acts on external representations and have been used to structure VR activities. Nevertheless, this model does not describe collaborative activities, the common understanding process, and the three levels of collaboration. A solution to this limitation is to extend this individual model following the multi-user FBS extension.

Framework for VR

The development of VR applications requires support provided by generic and reusable systems [START_REF] Mollet | Storytelling in virtual reality for training[END_REF]. Several frameworks exist. MAS-SIVE [START_REF] Greenhalgh | Massive: A collaborative virtual environment for teleconferencing[END_REF] enables the developers to immerse the users into a shared virtual environment with rules to define different collaborative states. Another framework [START_REF] Gonzalez-Franco | Framework for remote collaborative interaction in virtual environments based on proximity[END_REF] enables the developers to build multiuser application that engages the users in three levels of collaboration according to their proximity of each other. But the developers cannot use these frameworks to drive and to sequence the activities of the users, and to implement personal tools into the application. VHD++ [START_REF] Ponder | Vhd++ development framework: towards extendible, component based vr/ar simulation engine featuring advanced virtual character technologies[END_REF] enables the developers to create and use personal tools.

Another solution is to use scenarios. The scenarios are able to fully constrain, partially constrain, or completely free the users' actions by listening to and interacting with the VE. A graphical representation can be used to model scenarios, for example, the Petri-Nets in #SEVEN [START_REF] Claude | Short paper: #seven, a sensor effector based scenarios model for driving collaborative virtual environment[END_REF]. In this way, communication is facilitated between the developers and the users. An advantage of the scenariosbased approach lies in the fact that the actions described are similar to the external activities depicted in the theoretical design models. Here, the actions described are the interactions between the users and the objects or the virtual world. In addition, the scenario of each user can run independently from one user to another, allowing the users to temporarily work individually [START_REF] Jota | Immiview: a multi-user solution for design review in real-time[END_REF]. Consequently, the scenar-ios are able to reflect the collaborative design activities of multiple users and enable iterations. Solutions exist to create a Framework for collaboration in VR, but none of these solutions have been used for collaborative design based on generic activities occurring in VR.

The related works surveyed in this section reveal the need to propose a new theoretical collaborative design model that depicts external activities categorized in the three spaces. To the best of our knowledge, the scenarios have not previously been used to describe a collaborative theoretical model with the goal of structuring collaborative design activities in a synchronized VR application.

Solution overview

In this paper, the VR Framework supports the developers' work towards building personalized, multitool, and multi-user VR applications intended to meet the needs of multiple design domains. The solution needs to drive the generic users' actions in VR independently of the domain to structure the collaborations of and to support the communication between designers in VR.

Consequently, the solution we propose here uses a theoretical design model to define the generic design activities of several users. The collaboration is modelled through the common object of design (Fig. 2, part 1). Next, the theoretical model is naturally described by the three-level scenarios in a VR Framework, with each level managing functionalities making possible specific activities in the collaborative design process (Fig. 2, part 2). For example, a user is able to select an object for the team and each user is able to perform evaluation or to generate new solutions. Finally, the implementation of the scenarios in a VE leads to the production of VR applications for industrial design (Fig. 2, part 3).

For example, the developer configures the scenario to provide access to specific tool metaphors to enable the creation or modification of a 3D object in VR (Fig. 1). Scenarios are the cornerstone of our VR Framework. All the design activities are correlated to the use of a set of design tools. Each user has their own independent instance of the scenarios. The scenarios can be adapted to depict other models and alternative spatial or conceptual organizations of the VE.

Theoretical collaborative design model

Our multi-user VR Framework is driven by a generic and theoretical collaborative design model to cover the needs of various domains. It is composed of a multiple single-user theoretical model linked by design objects and logical space for collaboration.

Single user dimension

The model depicts the three logical design spaces: problem, solution, and evaluation [START_REF] Terrier | Scenario-based VR Framework for Product Design[END_REF]. In each logical space, two activities are distinguishable: the creation and the iteration of contents. In addition, the evaluation logical space includes the act of evaluating and the act of defining or modifying the criteria of evaluation. The next step is to depict the multi-user dimension in the theoretical model.

Multi-user model

As the collaborative design implies multiple designers, we propose to use one instance of the theoretical single user design model for each designer. Since the activities are based on the creation of external representations, the internal mental process of building of a shared understanding is not represented in our multi-user model. However, the collaboration and the shared understanding can be facilitated. To this end, the model introduces the concept of a common object of design. This concept describes the object of design on which the designers are working in unison.

Once a common object is defined, all the activities of each designer focus on this specific object. In this way, the collaboration level 3 occurs between the designers (Fig. 3, top). For example, two designers are able to modify the same mockup of a room to propose new solutions. The model also describes the collaboration level 2, which occurs when no common object is selected, thereby allowing the designers to interact with any object of the mockup. In this situation, the designers still collaborate to refine the design but are not constrained to work on the same object (Fig. 3, down). For example, one designer is able to modify a workstation while the second moves screens around the room to adjust together the overall layout of the room. Thus, our theoretical collaborative design model is able to depict various collaborative design situations and their associated activities. The next step is to depict this model with a scenario model that will be used by developers and interpreted by a scenario engine to play the events in the VE.

Scenario model for collaborative design 5.1 Scenario details

Our VR Framework uses a scenario creation tool integrated into Unity [START_REF] Claude | Short paper: #seven, a sensor effector based scenarios model for driving collaborative virtual environment[END_REF]. Based on a Petri-Nets [START_REF] Petri | Kommunikation mit Automaten[END_REF] language, the scenarios are edited into Unity with graphical representations to model user events, environment states, and object behavior. The scenario model is a graph depicting a series of events made up of places, transitions, and subscenarios. The scenario model is able to listen to and modify the state of the VE through sensors and effectors. The associated scenario engine is able to play the events in the VE and, thus, to sequence the design actions (e.g., collaboration level and common object selections) depending on conditions (e.g., roles).

Three-levels scenario model

The theoretical collaborative design model is split into three levels of scenarios (see Fig. 4). An instance of the three-levels scenarios is attached to each user. The first level manages the functionalities that the developers can use to enable selection of the common object of design in the VE by the designers. The framework provides a service of synchronization that occurs only between each first level and that depends on the state of the selection of a common design object in the shared VE. As described in the theoretical model, the collaboration level 3 occurs when a common object is selected. All of the activities are performed on this object. The framework integrates a service based on roles for developers to enable the functionality of selection only for the "supervisor". The other role can only select a design object when the collaboration is level 2 (i.e., no common object is selected). For example, the service enable only the "supervisor" to select the collaboration mode level 3 or level 2. Once the selection is executed, the selection state is detected by all the instance of scenarios at the same time engaging all the designers to work jointly or independently. If the level 3 is activated by the scenarios for each designers, only the "supervisor" is able to select the common object of design. The services enable the designers to change their selections (i.e., collaboration mode and object).

The second level manages the functionalities that the developers can use to enable the users to access one of the three logical spaces of design (i.e., problem, solution, or evaluation) and to pick a tool. For example, independently of the choice of the other designers, each scenario is able to provide the capacity for a designer to select the solution space and a virtual pen to later propose a new solution of a workstation. This level is activated only when an object is selected. The scenario enables the developers to allow the designers to switch from one space to another and to switch from one tool to another.

The third level manages the functionalities that the developers can use to enable the users to access the design activities (i.e., creation and modification) of the space. The level is activated only when a space and a tool are selected. The developers are able to constrain the user to select one activity at a time. The scenario enables the designers to switch between activities for a single tool.

Technical details

Many items need to be synchronized between each user's applications: the scenario, the VE state, the avatars, and the common design object state. Besides making sure that things are running smoothly, the synchronization supports the building of a shared understanding between the designers. The framework implement an existing software component to provide this service to the developers. The networking is managed by the component TNET31 . Moreover, the framework meets the developers' need to share information regarding each user's own activity state to the others in the VE by adding effectors to provide these awareness information. This information takes the shape of a text displayed above the user's avatar with the following information: the space of the activity, the tool in use, and the current activity performed by the user (see Fig. 5). All this awareness information is updated according to the user's scenario. The networking setup also enables the developers to implement audio sharing among the designers. In this way, the VR Framework enables distant and co-localized collaborations.

Use case

The use case stems from the industrial partner's activities, which deal with the collaborative design of new working spaces. In this use case, the developers immerse two designers in a VE and to reflect on a layout (see Fig. 5), considering that the surface of the room remains unmodified and that a workforce increase of 50% is expected over the next 5 years. Based on these details, the designers propose and analyze new layout configurations. The following sections present: (1) the designers' series of events in the VE and (2) the functioning of the VR application, including the link between the VE and the scenarios.

Designers activities

In the application, the two designers propose multiple layouts according to the relevant specifications and norms. In this case, the problem is already defined and the designers focus on the creation or the modification of solutions. The designers focus also on the creation or the modification of evaluation according to the solutions they have proposed.

Considering the design activities, the designers need tool metaphors to interact in the VE. Thus, the developers are able to implement several tools in the application to move the objects, to navigate among space configurations, to change the material, to check the safety space between the furniture, to simulate the different users' heights, to add points of interest, and to have a top view.

Before building the application, the developers are able to attach the "supervisor" role to one instance of the application to only allow one designer to select a common object once the application is running. The designer is then able to show the different space configurations to the other designer. The collaboration level 3 is activated.

Meanwhile, the service in charge of the space management enables the developer to create an application that let the users to switch between evaluation and solution activities during the session, for example, to iterate on the materials of the common object and check the global harmony of the room.

By using the common object and collaboration services, the developers are able to build an application that let the "supervisor" designer switching from one common object to another or to change the collaboration mode for the level 2.

At that point, both designers are able to select distinct design objects. Each designer performs activities on a different design object with the same goal of proposing a new layout that takes into account the anticipated workforce increase and the relevant specifications.

The framework and the TNet3 component are able to synchronize all modifications of the VE between each instance of Unity.

Scenarios and VE relationships

The following describes the step during which one designer switches between configurations, while the second evaluates the evacuation criterion.

Space and tool detection. The developers enable each user to navigate between the three logical spaces thanks to the second level of scenarios. The second level of scenarios and the space service enable the developers to allow each user to navigate between the three logical spaces : Problem, Solution, and Evaluation. Each logical space contains a batch of dedicated tools implemented by the developers. A sensor detects that the user has selected the solution space, and an effector activates the third level of scenarios (see Fig. 6). In this way, the tool selection functionality is activated enabling the user to pick the configuration tool. Simultaneously, the second user selects and activates the tool to evaluate the safety distance between objects. Depicting the design activities. The configuration tool is activated for the first user, and the possible activities are depicted in a branch of the third level of scenarios, Solution Activities (see Fig. 7). The functionalities of this level enable the user to modify the configuration of the object (e.g., a workstation). A sensor detects the action, while another sensor displays the activity over the user's head to inform the second user about this action. The position and the active state of each workstation configuration are synchronized between the Unity instances. The scenario is thus able to discriminate between the designers' activities, their space, the tools involved, and the creation or modification of contents. Finally, the three-level scenarios enable design iterations on the same object for a level-3 collaboration.

In a customization context to meet the needs of an industry, new VR tools and/or activities can be implemented in the model. The developers are also able to use only a specific part of the scenario to constrain the users' activities during a design session (e.g., adding or deleting branches, transitions, etc.).

Conclusion

Our solution supports the development of VR collaborative design applications involving multiple designers. The Framework implements scenarios and services according to a theoretical collaborative design model to drive the users' activities in VR. In this paper, only the functionalities of the Framework are described and illustrated in a use case.

The collaboration is supported by two functionalities. The Framework works over the network by being implemented individually for each user. The VE, the communications, and the scenarios are synchronized between each instance of the Framework. Then, the concept of a common object of design enables the users to switch between types of collaboration. The users are consequently able to work on the same object or on different objects with the same goals.

Moreover, our solution meets the requirement to personalize the applications dedicated to design. The Framework enables the implementation of various design tools in VR without modifying the scenarios. The functionalities to access the activities remain operational independently of the design tools.

Presently, generic activities have been implemented and the developers are able to personalize the scenarios. However, a new functionality could be implemented to enable the selection of the activities needed by the designers, the selection of each user's role during the design session, and the selection of restricted access concerning specific activities according to the user's role. At this point, an evaluation of the use of the Framework is needed in comparison to other existing Frameworks. Moreover, the concurrent edition of the same object is not yet managed by our Framework and should be integrated in a future version. The persistence of the scene and the saving/loading are already implemented via the network asset TNET3 but they need to be implemented as functionalities in the Framework.

To conclude, our scenario-based Framework supports the development of various collaborative VR applications for design and can be upgrade to manage more functionalities.

Figure 1 :

 1 Figure 1: Virtual reality (VR) framework based on scenarios for collaborative design applications.

Figure 2 :

 2 Figure 2: In red, our contribution: (1) a new theoretical collaborative design model defining generic design activities and collaborations, (2) the three-level scenarios depicting the new theoretical model, providing services to the developers (blue dots) and enabling the implementation of tools (green dots), and (3) an implementation of the scenarios in a virtual environment (VE).

Figure 3 :

 3 Figure 3: The theoretical collaborative design model introducing the common design object. It depicts, top, the collaboration level 3, and down, the collaboration level 2.

Figure 4 :

 4 Figure 4: Three-level scenarios depicting the theoretical collaborative design model. An instance of the three-level scenarios is attached to each user. The synchronization between each instance is performed through the common 3D object in the VE.

Figure 5 :

 5 Figure 5: The collaborative design VE scene for the use case of the working space layout.

Figure 6 :

 6 Figure6: Left: the space selection functionality is displayed to the user. Right: the second level of scenarios that manages the selection of the space. Since the space is selected, the scenario activates the third level.

Figure 7 :

 7 Figure 7: Up: the green user's third level in the Evaluation Activities branch is active. Down: the red user's third level in the Solution Activities branch is active. It has detected the use of a solution tool to modify contents. Thus, the token in the Modif. content place indicates that the user is modifying the current mock-up. The transitions are colored blue as they are activated but not triggered.

TNET3: www.tasharen.com