V Mazzocchi
email: vincent.mazzocchi@cea.fr

P G Sennikov

A D Bulanov

M F Churbanov

B Bertrand

L Hutin

J P Barnes

M N Drozdov

J M Hartmann

M Sanquer
email: marc.sanquer@cea.fr

28Si CVD-grown epilayer on 300 mm substrates for large scale integration of silicon spin qubits

Keywords: A1. isotopes separation and enrichment 28.60.+s, A3. Chemical vapor deposition processes, B2. Semiconducting silicon, B3. Quantum devices, silicon spin quantum bits, B3. Microlectronics 85.40.-e

99.992% 28Si CVD-grown epilayer on 300 mm substrates

for large scale integration of silicon spin qubits

Introduction

Spin quantum bits in isotopically purified 28 Si present remarquably longer (by two to four orders of magnitude) inhomogeneous dephasing time as compared to their natural Si counterparts (T * 2 = 120µs for quantum bits based on MOS quantum dots [START_REF] Veldhorst | An addressable quantum dot qubit with fault-tolerant control fidelity[END_REF], 140µs/270µs for quantum bits based on donors [START_REF] Muhonen | Storing quantum information for 30 seconds in a nanoelectronic device[END_REF], 20µs for quantum bits based on 28 Si-SiGe heterostructures [START_REF] Yoneda | A quantum-dot spin qubit with coherence limited by charge noise and fidelity higher than 99.9[END_REF], for 99.92% 28 Si crystals). Two-quantum bit-gates fabricated with isotopically purified 99.92% 28 Si exhibit also high fidelities [START_REF] Yoneda | A quantum-dot spin qubit with coherence limited by charge noise and fidelity higher than 99.9[END_REF][START_REF] Veldhorst | A twoqubit logic gate in silicon[END_REF], much better than those based on natural Si [START_REF] Kawakami | Electrical control of a long-lived spin qubit in a Si/SiGe quantum dot[END_REF][START_REF] Takeda | A fault-tolerant addressable spin qubit in a natural silicon quantum dot[END_REF]. Recently qubits based on isotopically purified MOS structures based on 300 mm wafers covered with a 100 nm thick layer 28 Si purified at 99.92% have been reported [7]. Long spin relaxation time T1 (≈ 145ms) has been measured.

It is still unclear what are the mechanisms for limiting the dephasing time T 2 for silicon spin qubit (residual 29 Si nuclear spins, electrical noise mediated by spin-orbit or spinvalley coupling, etc.) and if a higher level of isotopic purification is needed. In bulk 28 Si ( 29 Si = 0.005%), very long T 2 for electron spins on phosphorus donors at a concentration of 1.2x10 14 cm -3 has been measured by electron spin resonance techniques: T 2 = 600 ms, extrapolated to 10s by using a magnetic field gradient impeding flipflop relaxation with residual nuclear 29 Si spins [START_REF] Tyryshkin | Electron spin coherence exceeding seconds in high-purity silicon[END_REF]. The fundamental limit is T 2 ≈ T 1 = 2000s at low temperature, indicating that progress can still be made if one reduces the 29 Si content beyond 0.08% in the silicon crystal used to date for spin quantum bits [START_REF] Tyryshkin | Electron spin coherence exceeding seconds in high-purity silicon[END_REF]. The general objective of the presented study is to obtain 28 Si crystalline layers with enrichment ≥ 99,992 % and a very low level of other contaminants to fulfill the strict requirements of preindustrial Complementary Metal Oxide Semiconductor (CMOS) foundries. A first CMOS qubit fabricated in a pre-industrial foundry has been recently published by some of us [START_REF] Maurand | A CMOS silicon spin qubit[END_REF]. For instance the content of P and B without indication of the reducing chemical agent. The enriched silane was purified by distillation and thermally decomposed to polycrystalline 28 Si. The latter was used for growth of 28 Si single crystal by float-zone or Czokhralski method. The enrichment of silicon depends on the enrichment of the starting 28 SiF 4 . Nowadays the best value is 99.99930 % [START_REF] Abrosimov | A new generation of 99.999% enriched 28Si single crystals for the determination of Avogadros constant[END_REF] for a ntype 28 Si single crystal with a mass larger than 5 kg (concentration of 29 Si: 0.000658%). For the purest part of this crystal, the concentrations of carbon and oxygen were less 10 15 cm -3 and that of boron and phosphorus less than 10 13 cm -3 . In [START_REF] Sennikov | Towards 0.99999 28Si[END_REF], the deposition of a thick layer of microcrystalline 28 Si with a 99.9986 % enrichment on a 28 Si (99.995 %) substrate was achieved thanks to an enriched 28 SiF 4 in electron cyclotron resonance discharge. An extremely high-enriched thin film (100-200 nm) of 28 Si 99.99983 % with a 29 Si concentration less 0.0001% was grown using hyperthermal energy ion beam deposition system from SiH 4 of natural isotope abundance [START_REF] Dwyer | Enriching 28Si beyond 99.9998 % for semiconductor quantum computing[END_REF].

In studies [START_REF] Takyu | Growth and Characterization of the Isotopically Enriched 28Si Bulk Single Crystal[END_REF][START_REF] Ruf | Thermal conductivity of isotopically enriched silicon[END_REF][START_REF] Ladd | All-Silicon Quantum Computer[END_REF][START_REF] Itoh | High Purity Isotopically Enriched 29Si and 30Si Single Crystals: Isotope Separation, Purification, and Growth[END_REF][START_REF] Shimizu | Experimental Evidence of the Vacancy-Mediated Silicon Self-Diffusion in Single-Crystalline Silicon[END_REF][START_REF] Lo | Device fabrication and transport measurements of FinFETs built with 28Si SOI wafers toward donor qubits in silicon[END_REF], layers of 28 Si were epitaxially grown, notably by Pr. K. Itoh's Group, using 28 SiH 4 produced in 1998-2005 by the Isonics Corporation, USA, in cooperation with the Voltaix company. The enrichment of this silane gas was as follows: 28 Si (99.924%), 29 Si (0.073%) and 30 Si (0.003%) [START_REF] Itoh | Isotope engineering of silicon and diamond for quantum computing and sensing applications[END_REF]. In our experiments we have used silane produced from 28 SiF 4 according to [START_REF] Bulanov | The Highly Isotopic Enriched (99.9%), High-Pure 28Si Single Crystal[END_REF]. The details of its synthesis and characterization are succintly described below.

Experimental methods

Fabrication of the 28 SiH 4 precursor

Gas centrifugation is the only effective method of isotope separation with a high enrichment level. Other methods, such as magnetic mass separation, ion exchange and laser technology, are more expensive. These methods do not allow a high isotopic enrichment to be reached. There are some limitations for gases to be used in gas centrifuges. One of the most important limitations is the molecular mass of the gas. Silane is too light for this purpose. This is why silicon tetrafluoride SiF 4 is used as a process gas for the centrifugal separation. Two other reasons are as follows: only one isotope exists for fluorine and this substance has sufficiently high vapour pressure at room temperature. Synthesis and enrichment of SiF 4 took place in Russia at SC "PA ElectroChemical Plant" (ECP), Zelenogorsk, Krasnoyarsk region.

The natural abundance silicon ( 28 Si = 92.23%) of electronic grade quality was manufactured and supplied by Wacker Polysilicon Europe, Wacker Chemie AG, Germany. SC PA ECP produced high-purity fluorine (F 2 ) by itself. A special installation with a reactor was constructed and used for the synthesis of the initial SiF 4 and for silicon isotope separation in centrifugal cascades. Other details of enrichment procedure and purification of SiF 4 are given in [START_REF] Abrosimov | A new generation of 99.999% enriched 28Si single crystals for the determination of Avogadros constant[END_REF]. Then 28 SiF 4 was converted into silane, 28 SiH 4 . First, the synthesis of silane was carried out by the reaction of high-purity silicon tetrafluoride with calcium hydride: 28 SiF 4 + 2CaH 2 → 28 SiH 4 + 2CaF 2 A mixture of isotopically enriched silicon tetrafluoride with hydrogen of special grade B purity was passed through a layer of mechanically dispersed calcium hydride. The reactor was made of high purity Si-free stainless steel to prevent the diffusion of boron compounds and natural Si into the highly enriched gas. The content of hydrocarbon impurities in silane after synthesis was at the level of 10 -5 mol/mol. The concentration, around 10 -3 mol/mol, of polysilanes and disiloxanesis was the largest component according to gas-chromatographicmass-spectrometric (GCMS). Calcium hydride seems to be the main source of impurities, in particular carbon. The produced 28 SiH 4 was freed from impurities by cryofiltration and periodic low-temperature rectification.

Growth of the 28 Si epilayer

A 300mm Epsilon 3200 single wafer tool from ASM America was used in CEA-LETI for the epitaxial growth of natural and isotopically pure Si layers. Software and hardware modifications were implemented on the tool to be able to (i) switch from natural to isotopically pure SiH 4 and (ii) deliver precise amounts of silane (whatever its nature) in the epitaxy chamber thanks to a Mass-Flow Controller (MFC). Given the cost of 28 SiH 4 , a dedicated gas box was installed in the basement of our cleanroom as close as possible to the tool to minimize the length of the gas line (≈ 10 m against ≈ 70 m). As we will be targeting in the coming years the deposition of layers several nm to several tens of nm thick on bulk Si or Silicon-On-Insulator substrates, we have decided to investigate the properties of layers grown at 650 • C, 20 Torr with a F( 28 SiH 4 )/F(H 2 ) mass-flow ratio of 0.012. The Si growth rate is then typically around 10 nm min. -1 [START_REF] Hartmann | A benchmarking of silane, disilane and dichlorosilane for the low temperature growth of group IV layers[END_REF], which should be handy for thin layers. The temperature is otehrwise low enough to enable epitaxy on thinned-down SOI substrates, with a starting Si layer thickness as low as 3 nm [START_REF] Jahan | Agglomeration control during the Selective Epitaxial Growth of Si raised sources and drains on ultra-thin Silicon-OnInsulator substrates[END_REF].

Results

Characterization of the 28 SiH 4 precursor.

The isotope concentrations in silane in the transport vessel were measured by laser source mass spectroscopy (LIMS) and are as follows: 28 SiH 4 = 99.99748 (± 0.00026) at.%, 29 SiH 4 = 0.00250 (± 0.00026) at.% and 30 SiH 4 = 0.00002 (± 0.00001) at.%. Chemical analysis of unintentional species was performed by gas chromatography and chromatomassspectroscopy from the transport vessel. High purity 28 SiH 4 was prepared with an overall hydrocarbon content less than 2 × 10 -7 mol/mol, less than 4 × 10 -8 mol/mol of disiloxane and around 10 -7 mol/mol of higher order silanes (see Table 1). The hydrocarbon contents were otherwise very low. This is a prerequisite in order to obtain superior quality epitaxial layers.

Stoichiometric SiC nano-precipitates, which are detrimental to layer quality, might indeed be formed if the C content in the bottle is too high (notably for growth temperatures above 600°C). We have first profiled with secondary ion mass spectrometry (SIMS) the various Si isotopes in a 60 nm thick Si layer grown at 650•C, 20 Torr with 28 SiH 4 on a natural abundance Si(001) wafer (after a 1100 • C, 2 min. H 2 bake in order to get rid of native oxide). SIMS measurements of silicon isotope concentrations in Grenoble and Nizhny Novgorod (IPM RAS) were close to each other (see Figure 1). The 28 Si isotopic concentration in the epilayer from SIMS, 99.99337 (± 0.00026) at.% was slightly lower than the LIMS in the 28 SiH 4 bottle, i.e. 99.99748 (± 0.00026) at.%. This was associated with a two fold increase of the harmful 29 Si isotopic concentration from 0.00250 (± 0.00026) at.% up to 0.00524 (± 0.0009) at.% (from LIMS to SIMS). Metrology techniques were not the same, however, which might partly explain those differences. The epitaxy chamber quartz wall, the SiC-covered susceptor plate (on which wafers lay during growth) and other inner components are cleaned between depositions thanks to high temperature, high pressure etches with gaseous HCl. A memory effect stemming from the epitaxy beforehand of a large number of natural abundance Si layers cannot be fully excluded, however. Finally, although the fraction of the gas panel common to natural and purified silane was purged between switches, some unwanted molecules might have been still present in the Mass-Flow Controller and in the injection line during the deposition of that 28 Si layer. The increase of the 29 Si and 30 Si isotopic concentrations in the 10 nm close to the substrate is likely due to the diffusion of natural silicon from the latter towards the epilayer.

In order to gain access to the Si growth rate with those process conditions and assess the structural quality of Si layers grown with 28 SiH 4 , we have proceeded as follows. We have started from natural abundance Si(001) substrates and grown at 650 • C, 20 Torr (SiGe 30% 19 nm / Si cap 30 nm) bilayers. As before, a 1100 • C, 2 min. H 2 bake was used beforehand to get rid of native oxide. A SiH 2 Cl 2 + GeH 4 chemistry was used for the growth of the SiGe 30% marker layers, while natural abundance or purified silane was used for the growth of the Si caps on top. We have first used X-Ray Reflectivity (XRR) to (i) gain access to the individual layer thickness of those stacks (and thus determine the Si growth rate) and (ii) qualitatively see if the Si caps were smooth. XRR profiles are provided in Figure 2. The presence of welldefined thickness fringes up to high incidence angles is a clear sign that the surface of the Si cap was smooth and the interfaces (between SiGe 30% and Si) abrupt irrespectively of the silane source used. The presence of two sets of thickness fringes is due to constructive and destructive interferences of the X-rays reflected at the Si cap / SiGe 30% and the SiGe 30% / Si substrate interfaces as omega, the angle of incidence, changes.

Their angular period is inversely proportional to (i) the Si cap thickness and (ii) the SiGe 30% / Si cap thickness. We extracted from the Si cap thickness the following Si growth rate: 10.1 nm min. -1 . The perfect superposition of the XRR profiles is a clear sign that growth proceeds exactly the same way with both sources of silane. We have completed the characterization of those stacks with tapping-mode Atomic Force Microscopy (AFM) and surface haze measurements (in Bruckers FastScan and KLA Tencor SP2 metrology tools, respectively). or purified silane (right column). Smooth, featureless surfaces were obtained whatever the scan size and the source of silane, with a Root Mean Square (RMS) roughness and a Z range (Z max. -Z min. ) equal on average to 0.153 nm and 1.47 nm for natural silane and 0.151 nm and 1.34 nm for purified silane. wafers in a SP2 tool [START_REF] Hartmann | Critical thickness for plastic relaxation of SiGe on Si(001) revisited[END_REF]. The DW and DN haze values are very low and once again strikingly similar (DW haze = 0.325 parts per million (ppm) and DN haze = 0.0810 ppm for natural silane, to be compared with 0.329 ppm and 0.0817 ppm for purified silane).

We thus confirm at the wafer scale the conclusions reached with XRR and AFM, i.e. that the Si cap surfaces are smooth at the atomic scale and featureless whatever the source of silicon. Finally, we have also had a look, in our SP2 tool, at the particules (which might be large size epitaxy defects coming from imperfect gas sources) detected after the epitaxy of those stacks. Maps at the wafer scale are provided in Figure 3, bottom right. The number of counts and defects are quite similar whatever the source of silane, which is very reassuring.

We have otherwise assessed whether or not we were faced with metallic impurities after the growth of numerous 28 Si layers on various types of substrates. It might indeed have happened that, because of the numerous very specific process steps used to obtain 28 SiH 4 , the gas was contaminated. To that end, we have loaded, after the growth of numerous 28 SiH 4 layers, bulk Si wafers inside the epitaxy chamber, left them in it for several minutes at 900 • C, 760 Torr and unloaded them. We have then used Total X-Ray Fluorescence (TXRF) spectroscopy to detect a metallic contamination on the front side and the back side of those wafers. We were in every case below the TXRF detection threshold (typically around 10 10 atoms/cm 2 ) for the various metals tracked, i.e. Al, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni and Ti. The number of added particules on the front side (120 nm detection threshold in the SP2 tool) after this loading/unloading was barely 3. From all those experiments, we can safely state that the purified silane source we used yields the same epitaxial quality layers than microelectronics grade silane, without any deleterious metallic and/or particular contamination afterwards. The isotopic purity in epilayers is very high, which should definitely be an advantage in quantum electronics. In the next section, we will quantify, in specially tailored stacks, the impact of thermal budget on the self-diffusion of Si.

Si self-diffusion in natural Si / 28Si heterostructures

In order to confirm that Si self-diffusion was the same in our CVD-grown layers and in the literature, we have deposited at 650 • C, 20 Torr on 300mm Si(001) wafers the following stacks (from bottom to top): 10 nm of 28 Si / 10 nm of natural Si / 10 nm of 28 Si. It is not practical to switch from natural silane to purified silane in the same growth run. Using natural dichlorosilane (SiH 2 Cl 2 ) is otherwise not an option as the Si growth rate at 650 • C is prohibitively low. We have thus taken advantage of the fact that we also have natural disilane (i.e. Si 2 H 6 ) connected to our tool to grow the natural Si layer, which is sandwiched between the two 28 Si layers. The natural disilane mass-flow used, the lowest one that could reproducibly be delivered in our tool, was 11 times less than that of purified silane. The 650 • C, 20 Torr growth rate from disilane was nevertheless twice higher (i.e. 20 instead of 10 nm min. -1 ). This is due to the ability disilane molecules have of creating their own absorption sites on H-terminated Si surfaces (through the following chemical reaction: Si 2 H 6 (g) + H → SiH 3 + SiH 4 (g)). Si growth rates are then, for a given silicon flow, several times higher with disilane than with silane for T < 675°C [START_REF] Hartmann | Impact of Si precursor mixing on the low temperature growth kinetics of Si and SiGe[END_REF]. The SIMS depth profile of the 28 Si, 29 Si and 30 Si isotopes in the as-grown stack can be found in Figure 4. Several things are obvious:

(i) The 29 Si and 30 Si concentrations are, as expected, the same in the 10 nm thick natural Si layer than in the Si substrate;

(ii) Most likely because of intermixing in the SIMS crater during profiling, the 29 Si and 30 Si concentrations are nearly one decade lower in the 28 Si cap than in the bottom 28 Si layer. The latter layer was indeed profiled after the natural Si spacer.

(iii) The chemical width of the interface is several nm only, i.e. the same kind of width found during the Atom Probe Tomography of Si/SiGeC superlattices [START_REF] Estivill | Quantitative investigation of SiGeC layers using atom probe tomography[END_REF][START_REF] Mukherjee | Atomistic and Optical Properties of Group IV Ultrathin Superlattices[END_REF]. We indeed have steady 29 Si and 30 Si concentrations in the majority of the top 28 Si layer and the natural Si layer just below. This is important for device processing, as it means that a natural SOI substrate could behave almost as a 28 SOI substrate provided that a 28 Si epilayer 10 nm thick or more is deposited on top.

Having seen that, we have submitted the same stacks to 2 min. H 2 anneals at various temperatures (925 • C, 1050 • C and 1175 • C) in the epitaxy chamber to emulate the thermal budget stacks made of natural Si and 28 Si would be submitted to during device fabrication.

The ramping-up and ramping-down rate (from 550 • C up to the annealing temperature), 2.5 • C/s, was low enough to minimize temperature overshoot. SIMS depth profiles of the 28 Si, 29 Si and 30 Si isotopes can be found in Figure 5. The 29 Si and 30 Si depth profiles are almost the same for the as-grown sample and for the sample annealed for 2 min. at 925 • C. Moving over to 1050 • C has a huge impact on isotopic profiles, which are flattened with definitely broader interfaces from the chemical point of view. Annealing such a stack at 1175 • C for 2 min. completely suppress the fluctuations because of intermixing. The only differences with bulk Si are then, as expected, the isotopic concentrations which are different from the natural ones in the first 50 nm or so of Si (the 28 Si isotope is more abundant and the 29 Si and 30 Si isotopes less abundant, then, which is logical given that 20 nm of 28 Si was deposited). We have extracted what could be called "steady state" 28 Si isotopic concentrations in the middle of each 10 nm thick layer (i.e. at 5 nm, 15 nm and 25 nm SIMS depths) for the various samples investigated. Data are plotted in Figure 6. While 28 Si concentrations are barely lower in the two 28 Si layers after a 2 min. H 2 anneal at 925 • C than in the as-grown layers, it is obvious that concentrations go back to the natural abundance for higher thermal budgets.

A 1D finite difference time domain (FDTD) model for the calculation of Si selfdiffusion was used to simulate the SIMS profiles shown in figure 5. We assume infinitely sharp interfaces between the 28 Si and nat Si layers of the stacks. Temperature ramp-up and down of the annealing are included in the simulations. For the self-diffusion coefficient, a single Arrhenius law: D SD Si = 423 × exp(-4.73eV / k B T) cm 2 s -1 is assumed, fully in line with ref. [START_REF] Südkamp | Self-diffusion in crystalline silicon: A single diffusion activation enthalpy down to 755•C[END_REF] findings. A standard MRI (ion Mixing surface Roughness Information depth, first developed in ref. [START_REF] Hofmann | Atomic Mixing, Surface Roughness and Information Depth in Highresolution AES Depth Profiling of a GaAs/AlAs Superlattice Structure[END_REF]) depth resolution function is then convoluted to the simulation output to reproduce the finite depth resolution of SIMS data. The MRI parameters are set with a single fitting procedure to the whole data set (see figure caption figure 7). As shown in Figure 7, the measured 29 Si SIMS profiles are in good/excellent agreement with our simulations based on previously reported Si self-diffusion coefficients.

Conclusions

We reported the growth of high structural quality 28 should be highlighted that the y-scale, linear for 28 Si, is logarithmic for 29 Si and 30 Si isotopes.

Figure 6: (color online). 28 Si isotopic concentration in the middle of the two 28 Si layers and in the natural Si spacer in-between for the as-grown sample or the stacks annealed for 2 min.

3. 2 .

 2 Isotopical, Chemical, morphological and physical characterization of the28 Si epilayer.

Figure 3 ,

 3 Figure 3, left panel shows various scan size (1 µm x 1 µm, 5 µm x 5 µm and 20 µm x

Figure 3 ,

 3 top right shows Dark Wide (DW) and Dark Narrow (DN) haze maps associated with diffuse light scattering (by surface roughness) by our 300mm

Figure 2 :

 2 Figure 2: (color online). X-Ray Reflectivity profiles associated with (SiGe 30% 19 nm / Si 30 nm) bilayers grown at 650 • C, 20 Torr on Si(001) substrates. The 30 nm thick Si caps were

Figure 3 :

 3 Figure 3: (color online). Left panel: Tapping-mode Atomic Force Microscopy images of the surface of the 30 nm thick Si caps grown at 650 • C, 20 Torr with natural or purified silane.

Figure 4 :

 4 Figure 4: (color online). Si isotopes atomic percentages from SIMS in a 28 Si 10 nm / natural or standard Si 10 nm / 28 Si 10 nm stack grown at 650 • C, 20 Torr with 28 SiH 4 and natural Si 2 H 6

Figure 5 :

 5 Figure 5: (color online). Si isotopes atomic percentages from SIMS in a 28 Si 10 nm / natural or standard Si 10 nm / 28 Si 10 nm stack (i) grown at 650 • C, 20 Torr on a bulk Si(001) substrate (no anneal) or (ii) annealed for 2 min. under H 2 at 925 • C, 1050 • C or 1175 • C. It

under H 2

 2 at 925 • C, 1050 •C or 1175 • C.

Figure 7 :

 7 Figure 7: (color online). SIMS data (symbols) and simulation results (lines) of Si self-

Figure 1 Figure 2 25 Figure 3

 12253 Figure 1 Mazzocchi et al.

Table 1 :

 1 Si crystalline layers with enrichment ≥ 99,992 % and with a very low level of other contaminants. The isotopically purified epilayers are grown on 300mm substrates which are the standard wafers for CMOS foundries. The epilayers have been characterized in depth by several complementary techniques in different places. The level of28 Si enrichment is larger than in previous reports in the context of silicon qubits and constitutes a record for silicon films of large area. A study hydrocarbon contents and others species (gas chromatography) in the28 silane (from the transport vessel). Right panel: (Top) SIMS depth profiling in Grenoble of the Si isotopes in the resulting layer. The concentration of the29 Si isotope which contains a nuclear spin 1/2 is less than 0.006 % in the top layer and the concentration of the nuclear spin-free30 Si isotope lower than 0.002 %. A SIMS depth profiling in Nizhny-Novgorod of the Si isotopes (not shown) gives: 28 Si = 99.99337 (± 0.001) at.%, 29 Si = 0.00524 (± 0.0009) at.% and 30 Si = 0.00139 (± 0.0005) at.% for the epilayer, in very good agreement with Grenoble SIMS experiments. Both were performed on ION TOF TOF-SIMS 5 instruments. There is otherwise a small isotopic dilution when going from the natural abundance Si(001) wafer towards the28 Si epilayer.

	Species	Concentration (µmol/mol)
	CH 4	≤ 0.05
	C 2 H 6	≤ 0.02
	C 2 H 4	≤ 0.02
	C 3 H 6	≤ 0.02
	C 4 H 8	≤ 0.01
	i-C 4 H 10	≤ 0.02
	n-C 4 H 10	≤ 0.02
	More than 52 other species	≤ 80.35 ± 20.135

Si isotope > 99,992%

Acknowledgements

This work was supported by the EU H2020 program (under the MOSQUITO project No 688539), by the Russian Federal Agency of Scientific Organizations (Project No AAAA-A17-117030910056-1) and by the Russian Foundation for Basic Research (Project No 18-03-00235).


Roberto Silva-Rojas

Vasugi Nattarayan

Francisco Jaque-Fernandez

Raquel Gomez-Oca

Alexia Menuet

David Reiss

Marie Goret

Nadia Messaddeq

Valentina M Lionello

Christine Kretz

Belinda S Cowling

Vincent Jacquemond

Jocelyn Laporte
email: jocelyn@igbmc.fr

Mice with muscle-specific deletion of Bin1 recapitulate centronuclear myopathy and acute downregulation of dynamin 2 improves their phenotypes

Keywords: myopathy, antisense oligonucleotides, membrane curvature, dynamin, amphiphysin, therapy

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

Introduction

A plethora of muscle diseases impair different steps of muscle formation or maintenance.

Centronuclear myopathies (CNM) are rare genetic diseases associated with a severe generalized muscle weakness associated with myofiber hypotrophy and premature death. [START_REF] Jungbluth | Centronuclear (myotubular) myopathy[END_REF][START_REF] Gonorazky | The genetics of congenital myopathies[END_REF] The name reflects the typical intracellular disorganization of myofibers with organelle mis-positioning including the centralization of nuclei that are normally present at the periphery of muscle fibers. [START_REF] Romero | Centronuclear myopathies: a widening concept[END_REF][START_REF] Lawlor | Skeletal Muscle Pathology in X-Linked Myotubular Myopathy: Review With Cross-Species Comparisons[END_REF] In addition, myofibers are smaller, rounder or heterogeneous in size. Despite the significant impact on morbidity and mortality, to date there is no specific therapy available for CNM patients.

CNM can be due to mutations in either MTM1, DNM2, BIN1 or SPEG genes, while mutations in additional genes as RYR1, TTN, CACNA1S, ZAK and PYROXD1 combine CNM features with other histological defects. [START_REF] Schartner | Abnormal Excitation-Contraction Coupling and Calcium Homeostasis in Myopathies and Cardiomyopathies[END_REF][START_REF] Lornage | Clinical, histological, and genetic characterization of PYROXD1-related myopathy[END_REF][START_REF] Pelin | Update on the Genetics of Congenital Myopathies[END_REF] Of note, several of these gene products directly regulate excitationcontraction coupling at the skeletal muscle triad (RYR1, CACNA1S/Cav1.1) or membrane remodeling (BIN1, DNM2), leading to the hypothesis that defects in triad structure and function form a common disease cause. [START_REF] Toussaint | Defects in amphiphysin 2 (BIN1) and triads in several forms of centronuclear myopathies[END_REF] BIN1 encodes amphiphysin 2, a protein sensing and controlling membrane curvature through its N-BAR (N-amphipathic Bin/Amphiphysin/Rvs) domain and recruiting through its SH3 (Src Homology) domain effectors like dynamins (DNM1 and DNM2) which tubulate and potentially fission membranes. [START_REF] Hohendahl | Structural insights into the centronuclear myopathy-associated functions of BIN1 and dynamin 2[END_REF][START_REF] Lee | Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle[END_REF] While BIN1 is not the most frequent gene mutated in CNM, it is mutated in both dominant and recessive forms. [START_REF] Bohm | Adultonset autosomal dominant centronuclear myopathy due to BIN1 mutations[END_REF][START_REF] Nicot | Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy[END_REF] Moreover, a founder mutation leads to a potentially high prevalence in the Roma population (also known as Gypsies) that is the most numerous ethnic minority in Europe with an estimated population of 11 million. [START_REF] Cabrera-Serrano | A Roma founder BIN1 mutation causes a novel phenotype of centronuclear myopathy with rigid spine[END_REF] BIN1 mutations either modify the N-BAR domain leading to decreased membrane tubulation, or truncate or extend the SH3 domain altering the binding to DNM2. [START_REF] Nicot | Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy[END_REF][START_REF] Gowrisankaran | Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge[END_REF] Regarding the understanding and treatment of BIN1-CNM, the two main bottlenecks are the absence of a faithful and easy-to-handle mammalian model and the lack of therapeutic proof-ofconcept necessary to trigger clinical development.

A drosophila mutant for Amph, the BIN1 ortholog, is flightless with severe structural defects of the triads, the structural basis of excitation-contraction coupling formed by a T-tubule contacting 2 terminal cisternae of sarcoplasmic reticulum (SR). [START_REF] Razzaq | Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila[END_REF] Downregulation of bin1 with antisense morpholino in zebrafish led to myofiber disorganization with centralized nuclei and structural and functional defects of the triads. [START_REF] Smith | Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy[END_REF] A spontaneous dog model was found with a splice mutation of the muscle-specific exon 11 of BIN1 and muscle atrophy, myofibers with centralized nuclei and radial organization of the sarcoplasmic reticulum. [START_REF] Bohm | Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Dogs with Highly Progressive Centronuclear Myopathy[END_REF] In mice, constitutive Bin1 knockout presented embryonic or perinatal lethality probably due to feeding defects. [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF][START_REF] Muller | Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation[END_REF] Similarly, such lethal phenotype was reproduced in a skeletal muscle specific knockout using the Cre recombinase under the control of the human skeletal actin (HSA) promoter. [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF][START_REF] Prokic | Differential physiological role of BIN1 isoforms in skeletal muscle development, function and regeneration[END_REF] Conversely, knockout of the musclespecific exon 11 led to viable mice that did not display CNM phenotypes. [START_REF] Prokic | Differential physiological role of BIN1 isoforms in skeletal muscle development, function and regeneration[END_REF] Acute reduction of Bin1 with shRNA in adult mice however showed disruption of the triad structure linked to alterations of intracellular Ca 2+ release. [START_REF] Tjondrokoesoemo | Disrupted membrane structure and intracellular Ca(2)(+) signaling in adult skeletal muscle with acute knockdown of Bin1[END_REF] Some potential therapies for centronuclear myopathies have been proposed. [START_REF] Tasfaout | Centronuclear myopathies under attack: A plethora of therapeutic targets[END_REF] Moreover, downregulation of Dnm2 with antisense oligonucleotides was shown to efficiently ameliorate the skeletal muscle phenotype in MTM1-and DNM2-CNM mouse models, [START_REF] Buono | Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice[END_REF]24 an approach that is currently in clinical trials (NCT04033159). However, it was never tested for BIN1-CNM due to lack of a faithful viable mouse model, albeit genetic crosses recently suggested that decreasing Dnm2 prevent the neonatal death of Bin1 knockout mice. [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF] Here, we generated and characterized a viable Bin1 knockout mouse faithfully reproducing most motor, histological and structural hallmarks of CNM, and validated Dnm2 antisense oligonucleotides as a first translatable approach to efficiently cure this model.

Results

Generation of a viable muscle-specific Bin1 knockout mouse with decreased muscle force.

To generate a viable mouse model without Bin1 expression in muscle, we crossed mice floxed for Bin1 exon 20 (Bin1 fl/fl ) with Bin1 heterozygous mice (Bin1 mck+/-) expressing the Cre recombinase under the control of the muscle creatine kinase promoter (MCK-Cre) that expresses from 17 days post-coitum (dpc) (Fig. 1A). In comparison, the HSA promoter used to obtain perinatal lethal mice expresses from 9 dpc. [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF][START_REF] Prokic | Differential physiological role of BIN1 isoforms in skeletal muscle development, function and regeneration[END_REF] Bin1 -/-MCK-Cre (Bin1 mck-/-) mice were generated with normal Mendelian ratio and were viable to at least 12 months of age (Fig. 1B-C), the maximum age analyzed in this study. RT-qPCR confirmed the absence of Bin1 mRNA levels in Bin1 mck-/-tibialis anterior (TA) muscle and western blot revealed the absence of BIN1 from Bin1 mck-/-animals at 8 weeks of age (Fig. 1D-E).

Bin1 mck-/-mice did not present differences in body or muscle weight at 8 weeks while body weight was reduced at 4 months and 12 months, and TA weight ratio was reduced at 4 months only (Fig. 1F andS1). Hanging capability until 8 weeks of age, endurance and fatigue in treadmill at 4 months, and performances in the string test and rotarod test at 6-7 weeks of age was comparable between Bin1 fl/f and Bin1 mck-/-animals, suggesting normal locomotor coordination (Fig. S2A-E).

For phenotypical and histological studies, we used TA as this muscle is commonly affected in other CNM mouse models. [START_REF] Buono | Reducing dynamin 2 (DNM2) rescues DNM2-related dominant centronuclear myopathy[END_REF][START_REF] Cowling | Reducing dynamin 2 expression rescues X-linked centronuclear myopathy[END_REF][START_REF] Munoz | Physiological impact and disease reversion for the severe form of centronuclear myopathy linked to dynamin[END_REF] We quantified the TA muscle force in situ following sciatic nerve stimulation. At 8 weeks of age and at 4 months, Bin1 mck-/-developed reduced maximal and specific force compared to controls. While Bin1 fl/fl mice at 8 weeks of age reached an average specific force of 18 mN/mg, Bin1 mck-/-mice only produced a specific force of 10.6 mN/mg, a 41 % reduction (Fig. 1G andS2F). Analysis at different stimulation frequencies revealed a clear reduction at several frequencies tested in 8 weeks and 4 months old animals (Fig. 1H and S2G-J). Therefore the absence of BIN1 didn't have a significant impact on the overall locomotor abilities while it clearly affected the muscle force at 8 weeks and 4 months of age.

Bin1 mck-/-mice develop most of the histological hallmarks of centronuclear myopathy.

We further assessed the presence of CNM features at the histopathological level. Staining of Bin1 mck-/-TA sections revealed the presence of darker hematoxylin eosin (HE) staining in the center of the fibers that were enriched in mitochondrial oxidative activity as stated in SDH and NADH staining while Gomori staining remained normal at 8 weeks (Fig. 2A andS3A). Similar defects in HE and SDH staining were observed in 4 months and 12 months TA from Bin1 mck-/-mice (Fig. S3B-G). The gastrocnemius presented with similar histological defects as observed in the TA, while heart histology didn't reveal any obvious defect at 8 weeks of age (Fig. S4A). Heterozygous knockout animals (Bin1 mck+/-) didn't present any histological defect in TA, showing total reduction of Bin1 is required to generate the histological phenotype and confirming this is a model for recessive BIN1-CNM (Fig. S4B). Bin1 mck-/-TA presented smaller fibers and a strong reduction in the number of large fibers when compared to Bin1 fl/f controls at 8 weeks, 4 months and 12 months of age (Fig. 2B-C andS3D-E). Bin1 mck-/-mice also presented a slight but significant increase in the percentage of fibers with high circularity at 8 weeks of age (Fig. 2D). The decrease in large fibers at 8 weeks of age was in line with the reduction of fast-twitch IIb fibers and the slight but not significant increase in type I fibers (Fig. S4C). Bin1 mck-/-did not present increased nuclear centralization at 8 weeks of age but the rate of TA fibers with internalized nuclei increased in 4 months and particularly in 12 months old Bin1 mck-/-mice (Fig. 2E, S3B and S3F). Of note, the Dnm2 RW/+ mouse mimicking the most common R465W mutation found in DNM2-CNM also displays CNM hallmarks but has normal nuclei positioning at 8 weeks. [START_REF] Durieux | A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice[END_REF] Altogether, Bin1 mck-/- mice display muscle weakness and most of the histological hallmarks of CNM, highlighting the relevance of Bin1 mck-/-mice as a model for BIN1-CNM.

Bin1 mck-/-myofibers display mitochondria disorganization.

To better characterize the pathomechanism linked to the defects in muscle force and histology in Bin1 mck-/-mice, we performed electron microscopy on TA muscles and found a general intracellular disorganization with altered sarcomere width and spacing (Fig. 3A). Nuclei position and shape were normal. Local accumulations of mitochondria were observed, together with increased prohibitin protein levels, an inner mitochondrial membrane protein that also presented a central and sub-sarcolemma accumulation pattern in Bin1 mck-/-mice (Fig. 3B, C and S5A). To determine the cause of mitochondrial accumulation, mitochondrial biogenesis and removal were assessed. mRNA levels from the mitochondrial biogenesis master regulator Ppargc1a were comparable between Bin1 mck-/-and Bin1 fl/fl mice (Fig. 3D). In Bin1 mck-/-muscles, electron microscopy revealed autophagosomes in regions where mitochondria accumulated and LC3 immunofluorescence showed increased accumulation of LC3-positive dots (Fig. 3E). In line with this, the protein expression level of P62 and lipidated LC3 were slightly, albeit not significantly, increased in Bin1 mck-/-mice while their gene expression in Bin1 mck-/-TA was mostly comparable to Bin1 fl/fl littermates (Fig. 3F, G andS5B-C), suggesting mild defects in mitochondrial positioning consistent with mitochondria accumulation. Therefore, the muscle weakness observed in Bin1 mck-/-muscles may arise in part from sarcomere and mitochondria disorganization.

Bin1 mck-/-myofibers present abnormal T-tubules and deficient excitation-contraction coupling.

BIN1 is a key player in the biogenesis of T-tubule in skeletal muscle. [START_REF] Lee | Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle[END_REF][START_REF] Razzaq | Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila[END_REF] To better understand the decreased muscle force observed in Bin1 mck-/-mice, T-tubules were imaged and excitationcontraction coupling assessed in isolated muscle fibers from flexor digitorum brevis (FDB), a muscle from which intact isolated fibers can be easily obtained. While Bin1 mck-/-FDB histology is similar to Bin1 fl/fl littermates at 8 weeks of age (Fig. S6A-B), di-8-anneps staining of plasma membrane invaginations revealed an altered network and a decreased density of T-tubules in isolated Bin1 mck-/-fibers (Fig. 4A-B). The Ca 2+ current through the T-tubule CaV1.1 Ca 2+ channel/voltage sensor and the release of SR Ca 2+ through RYR1 channels were simultaneously detected in isolated fibers under voltage-clamp conditions (Fig. 4C-H). The CaV1.1 current density was smaller in the Bin1 mck-/-fiber, accompanied by an increased residual leak component visible in response to the lowest amplitude pulses (Fig. 4C). Mean values for the voltage dependence of the peak current in the two groups are reported in Figure 4D. In each fiber the voltage dependence was fitted by the standard equation plus a linear outward component. Mean values from the fits revealed that the maximum conductance of the CaV1.1 channel population in the Bin1 mck-/-fibers was reduced by ~30 % and as compared to the mean value in Bin1 fl/fl fibers (Fig. 4E), while corresponding mean values for the apparent reversal potential (Fig. 4E), and for the half-activation voltage and steepness factor did not statistically differ between the two groups (Fig. S6C). The effect on the conductance was paralleled by a slight while not significant increase in the residual outward leak current (slope) in the Bin1 mck-/-fibers (Fig. 4E) indicating that besides affecting CaV1.1 channels, another conductance component of the plasma membrane may be affected.

Rhod-2 Ca 2+ transients from Bin1 mck-/-fibers exhibited a reduced peak amplitude as compared to Bin1 fl/fl fibers (Fig. 4F). The rate of SR Ca 2+ release calculated from the rhod-2 transients indicated that the mean value for maximum rate was reduced by almost 60 % (Fig. 4G-H) in the Bin1 mck-/- fibers while values for the mid-activation voltage and steepness factor were unaffected (Fig. S6D).

Overall, myofibers lacking BIN1 have an abnormal T-tubule network and a strong defect in excitation-contraction coupling, correlating with decreased muscle force.

Dnm2 downregulation improves the force production of Bin1 mck-/-mice.

Our previous studies suggested BIN1 is a negative regulator of DNM2 in muscle, as decreasing Dnm2 through genetic cross prevented the neonatal death of Bin1 -/-mice. [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF] We took advantage of the Bin1 mck-/-mice as a viable and faithful BIN1-CNM model to test a therapeutic approach by injecting antisense oligonucleotides (ASO) targeting Dnm2. We selected a dose of 25mg/kg that was previously validated as the safest and most efficient dose in other CNM mouse models. 24,[START_REF] Buono | Reducing dynamin 2 (DNM2) rescues DNM2-related dominant centronuclear myopathy[END_REF][START_REF] Munoz | Physiological impact and disease reversion for the severe form of centronuclear myopathy linked to dynamin[END_REF] Bin1 mck-/-muscles presented a slight increase in DNM2 protein and RNA levels (Fig. 5A-B andS7A). Dnm2 ASO treatment clearly reduced Dnm2 mRNA and protein levels in both ASO-treated Bin1 fl/fl and Bin1 mck-/-mice (Fig. 5B-C andS7B). While the muscle force of PBS-treated Bin1 mck-/- mice was significantly different from the one of PBS-treated Bin1 fl/fl mice, Dnm2 downregulation slightly increased the muscle force of ASO-treated Bin1 mck-/-mice to a level not significantly different from the ASO-treated Bin1 fl/fl controls (Fig. 5D andS8A). At low stimulation frequencies (1, 25 and 50 Hz), the force produced by ASO-treated Bin1 mck-/-mice was comparable to the ASOtreated Bin1 fl/fl controls and significantly increased compared to PBS-treated Bin1 mck-/-mice (Fig. 5E). Overall, Dnm2 downregulation increases the force production of ASO-treated Bin1 mck-/-mice.

Dnm2 downregulation improves the CNM histological hallmarks of Bin1 mck-/-mice.

As Dnm2 downregulation increased muscle force production of Bin1 mck-/-muscle, we then assessed rescue at the histopathological level. The Dnm2 ASO treatment fully normalized the central accumulations of mitochondrial oxidative activity observed in HE, SDH and NADH stainings of Bin1 mck-/-muscle sections, further confimed by mitochondrial TOMM20 immunofluorescence (Fig. 6A-B and S8B-D) Moreover, desmin, an essential regulator of myofibril integrity, with aberrant localization in MTM1-CNM patients and mice, [START_REF] Hnia | Myotubularin controls desmin intermediate filament architecture and mitochondrial dynamics in human and mouse skeletal muscle[END_REF][START_REF] Tasfaout | Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice[END_REF] is mislocalized in the center of PBStreated Bin1 mck-/-fibers and its localization is fully restored in ASO-treated Bin1 mck-/-mice (Fig. S8C-D). While myofibers from the PBS-treated Bin1 mck-/-mice were smaller compared to Bin1 fl/fl controls , Dnm2 ASO treatment normalized fiber size distribution and the percentage of large fibers (Fig. 6C-E). Correlating with the effect on large fibers, a slight but not significant decrease in Myh7 mRNA levels (specific for type I fiber) and increase of Myh4 mRNA levels (type IIb fiber) was noted in ASO treated Bin1 mck-/-muscles compared to PBS treated Bin1 mck-/-muscles (Fig. S8E).

Altogether, Dnm2 downregulation improved both motor function and the CNM histological hallmarks of Bin1 mck-/-mice, highlighting Dnm2 ASO treatment as an efficient injectable therapeutic approach to treat recessive BIN1-CNM.

Discussion

In this study, we generated and characterized the Bin1 mck-/-mouse as a first viable mammalian model reproducing most signs of BIN1-CNM. It provided a unique opportunity to test a potential treatment. Dynamin 2 is a binding partner of BIN1 and also mutated in CNM. Injection of antisense oligonucleotides targeting Dnm2 led to about 50% reduction in dynamin 2 compared to PBStreated mice of the same genotype, and to the improvement of motor and histopathological phenotypes of the Bin1 mck-/-mice. In these experiments, dynamin 2 levels in both ASO-treated genotypes are below the baseline of PBS-treated Bin1 fl/fl mice. These results provide novel insights in the role of BIN1 in skeletal muscle and validate a therapeutic proof-of-concept.

Bin1 mck-/-mice represent a faithful model for autosomal recessive BIN1-CNM with a clear decrease in muscle force, and most histological hallmarks as smaller and rounder myofibers with typical central accumulation of oxidative activity and a general intracellular disorganization. Alteration of the T-tubule network and the strong defect in excitation-contraction coupling most probably explain the decreased muscle force. However, such defects appear compatible with a conserved locomotor activity and survival into adulthood. In contrast to the neonatal lethality observed when Bin1 was deleted using Cre recombinase under the control of CMV or HSA promoters, [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF][START_REF] Muller | Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation[END_REF][START_REF] Prokic | Differential physiological roles for BIN1 isoforms in skeletal muscle development, function and regeneration[END_REF] Bin1 mck-/-mice are viable and fertile and display a disease onset similar to patients, making this model suitable for the establishment of treatment protocols. BIN1-CNM patients present high rates of fibers with centralized nuclei while Bin1 mck-/-mice only display increased nuclear internalization at 12 months of age and not at 8 weeks, [START_REF] Nicot | Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy[END_REF] suggesting progressive deterioration of muscle morphology in Bin1 mck-/-mice. Similarly, in the mouse models for DNM2-CNM, [START_REF] Munoz | Physiological impact and disease reversion for the severe form of centronuclear myopathy linked to dynamin[END_REF][START_REF] Durieux | A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice[END_REF] the abnormal nuclei centralization seen in patients is not reproduced. Indeed, centronucleation might be an easier-to-see manifestation of the same problem that causes mislocalization of other organelles in these disorders Furthermore, the percentage of centralized nuclei in CNM patients is highly variable and does not correlate with disease severity. [START_REF] Pierson | Myofiber size correlates with MTM1 mutation type and outcome in X-linked myotubular myopathy[END_REF] A few studies linked centronucleation to muscle pathology. [START_REF] Roman | Myofibril contraction and crosslinking drive nuclear movement to the periphery of skeletal muscle[END_REF] However, centronucleation during muscle regeneration does not lead to CNM, and muscle weakness is not often associated to central nuclei (e.g. nemaline myopathy). Therefore, the strong EC coupling defects observed in Bin1 mck-/-muscles is sufficient to cause the myopathy. Yet, several lines of evidence support the direct role of BIN1 in nuclei positioning. In particular, BIN1 controls nuclei and triad position with N-WASP in myofibers differentiated in vitro. [START_REF] Falcone | N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy[END_REF] Moreover, BIN1 links the nuclear envelope protein nesprin to both actin and microtubules cytoskeleton to locate nuclei in C. elegans seam cells. [START_REF] D'alessandro | Amphiphysin 2 Orchestrates Nucleus Positioning and Shape by Linking the Nuclear Envelope to the Actin and Microtubule Cytoskeleton[END_REF] Altogether it suggests that BIN1 and DNM2 may be less important for nuclei position in mice than in human.

Conversely, BIN1 loss strongly alters mitochondria positioning in muscle, leading to accumulation of mitochondria in the center of myofibers as typically seen in patients. [START_REF] Romero | Centronuclear myopathies: a widening concept[END_REF] T-tubules start forming at around 14 dpc in mouse embryo and reach their final orientation soon after birth. [START_REF] Takekura | Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle[END_REF] Deleting Bin1 with the HSA promoter expressing from 9 dpc lead to perinatal death, [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF] while using the MCK promoter expressing from 17 dpc is compatible with survival into adulthood.

In addition, both the intramuscular injection of shRNA or U7 small nuclear RNA targeting Bin1 expression or splicing, respectively, in adult mice lead to structural T-tubule defects and alteration of intracellular Ca 2+ release. [START_REF] Tjondrokoesoemo | Disrupted membrane structure and intracellular Ca(2)(+) signaling in adult skeletal muscle with acute knockdown of Bin1[END_REF][START_REF] Fugier | Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy[END_REF] Altogether, it suggests BIN1 is a major protein for T-tubule biogenesis, as proposed in differentiating muscle cells and in cardiac muscle. [START_REF] Lee | Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle[END_REF][START_REF] Hong | Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia[END_REF][START_REF] Caldwell | Dependence of cardiac transverse tubules on the BAR domain protein amphiphysin II (BIN-1)[END_REF] BIN1 has also an important role in T-tubule maintenance in adult. Noteworthy, decreasing DNM2 by about 50% in Bin1 -/-mouse through a genetic cross led to normal T-tubule and intracellular myofiber structure. [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF] Similarly, as shown here, decreasing DNM2 by about 50% in Bin1 mck-/-mouse with antisense oligonucleotides led to normal oxidative staining, fiber size and morphology, and increased muscle force. Hence, it is still unknown how T-tubules can form and/or maintain without BIN1. Either the BIN1-DNM2 balance is the major regulator, or DNM2 alone or with another BAR protein could control T-tubule biogenesis and maintenance.

Here, amelioration all of the phenotypes tested correlate with the decrease of DNM2 protein levels.

Decreasing DNM2 in a time window from 3 to 8 weeks partially improved muscle force defects while fully rescuing organelle mispositioning of Bin1 mck-/-muscle fibers. This treatment period corresponds to the childhood to early adulthood transition, when most BIN1-CNM patients are affected. It supports that DNM2 may be overactive in muscle lacking BIN1, and more generally that BAR domain proteins could be negative regulators of dynamins, as shown before for dynamin 1 and endophilin. [START_REF] Hohendahl | Structural inhibition of dynamin-mediated membrane fission by endophilin[END_REF] BIN1 and DNM2 form a complex and the absence of BIN1 in Bin1 mck-/-mice could result in an increase in DNM2 protein levels. [START_REF] Nicot | Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy[END_REF] In this line, DNM2 reduction via antisense oligonucleotides may represent a first translational approach to treat CNM related to BIN1 mutations. The existence of at least one founder BIN1 mutation in the Roma/Gypsy ethnic minority should foster further pre-clinical development. Noteworthy, as we previously showed that DNM2 downregulation rescues mouse models for myotubular myopathy (MTM1-CNM) and dominant DNM2-CNM, it becomes clear that DNM2 antisense oligonucleotides could represent a common therapy to treat these different CNM forms. [START_REF] Tasfaout | Centronuclear myopathies under attack: A plethora of therapeutic targets[END_REF] Noteworthy a 50% decrease in DNM2 in mice, either from embryogenesis in the Dnm2 +/-knockout or upon postnatal ASO treatment, [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF]24,[START_REF] Buono | Reducing dynamin 2 (DNM2) rescues DNM2-related dominant centronuclear myopathy[END_REF][START_REF] Munoz | Physiological impact and disease reversion for the severe form of centronuclear myopathy linked to dynamin[END_REF] does not lead to detectable toxic effects. DNM2 downregulation is on clinical trial for MTM1 and DNM2-CNM patients of more than 16 years old (NCT04033159) by Dynacure, and our present data on this novel BIN1-CNM model supports the inclusion of BIN1-CNM patients in such trial.

BIN1 is also strongly associated with other diseases: 41 BIN1 mis-splicing was proposed to be a main cause of the skeletal muscle phenotypes of myotonic dystrophy, [START_REF] Fugier | Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy[END_REF] decreased BIN1 plasma levels correlate with arrhythmia and with heart failure, 42 BIN1 was found downregulated in several cancers including breast and colon cancers, [START_REF] Prendergast | Bar the door : cancer suppression by amphiphysin-like genes[END_REF] and BIN1 is the second most important risk factor after APOE for late-onset Alzheimer disease. [START_REF] Seshadri | Genome-wide analysis of genetic loci associated with Alzheimer disease[END_REF] The therapeutic proof-of-concept validated here for centronuclear myopathy may trigger additional avenues of research for these and other diseases.

Materials and Methods

Animals.

Animal care and experimentation was in accordance with French and European legislation and approved by institutional ethics committee (project numbers 2016031110589922 and 2016052510176016). Mice were placed in ventilated cages with free access to food and water in temperature-controlled rooms with 12h day light/dark cycles. Bin1 mck-/-mouse line was established by crossing mice floxed for exon 20 (Bin1 fl/fl ) [START_REF] Cowling | Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation[END_REF] with Bin1 fl/+ mice expressing the Cre recombinase under the control of the muscle creatine kinase promoter (MCK-Cre; The Jackson laboratory, USA). [START_REF] Bruning | A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance[END_REF] Both lines were on a pure C57BL/6J background. Tail biopsies were used for genotyping testing for the floxed allele with 5'AGTGACCTAGGACTGTACCCAGAG3' and 5'ACAGGTAGGTGAAAGAGACTTGG3' and the Cre recombinase gene with 5'GAACCTGATGGACATGTTCAGG3' and 5'AGTGCGTTCGAACGCTAGAGCCTGT3'.

Male littermates were analyzed in this study.

Antisense oligonucleotides treatment.

Antisense oligonucleotides (ASO) used in this study (Ionis Pharmaceuticals, Carlsbad, USA) have phosphorothioate backbone and constrained ethyl (cET) modifications on the wings with a 3-10-3 gapmer design. They were designed to target all the murine isoforms of Dnm2 and were previously validated. [START_REF] Buono | Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice[END_REF]24 Bin1 fl/fl and Bin1 mck-/-male mice were treated weekly from 3 weeks to 8 weeks of age with intraperitoneal injections of 25 mg/kg body weight of ASO diluted in sterile PBS or sterile PBS only. Mice were randomly allocated in the ASO and PBS groups at weaning (3 weeks old).

Mice were killed 3 days after the last injection for analysis.

RNA extraction and RT-qPCR.

RNA was extracted from TA of 8 weeks old mice using TRI Reagent (Molecular Research Center, Cincinnati, USA) and cDNA synthesis performed with SuperscriptTM IV Transcriptase (ThermoFischer Scientific, Whaltham, USA). For quantitative PCR, cDNAs were amplified with SYBR Green Master Mix I (Roche Diagnostics, Basel, Switzerland) using 0.1 µM of forward and reverse primers in a Lightcycler® 480 (Roche Diagnostics, Basel, Switzerland). Primers specificity was validated by melting curve and sequencing of amplicons. Several primers were used in previous studies and are summarized in table S1. [START_REF] Durieux | A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice[END_REF][START_REF] Honda | Vestigiallike 2 contributes to normal muscle fiber type distribution in mice[END_REF][START_REF] Kuno | Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice[END_REF][START_REF] Thomas | Evidence based selection of commonly used RT-qPCR reference genes for the analysis of mouse skeletal muscle[END_REF] RT-qPCR was performed using technical triplicates and relative mRNA level was calculated with Rpl27 as a housekeeping gene.

The data displayed in the figures are the average of the triplicates and the result of a single experiment.

Protein expression studies.

TA from 8 weeks old mice were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1 mM PMSF and complete mini-EDTA-free protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein concentrations were determined using DC TM Protein Assay Kit (BioRad, Hercules, USA). Samples were denaturated 5 minutes at 95 °C with 5X Lane Reducing Buffer (ThermoFischer Scientific, Whaltham, USA) and loaded on 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose or PVDF membrane using Transblot® TurboTM RTA Transfer Kit (BioRad, Hercules, USA). Loading was controlled by Ponceau S staining before to membrane blocking. Membranes were blocked 1h with 5% non-fat dry milk in 0.1% Tween 20 prior to incubations with primary and secondary antibodies. The primary and secondary antibodies used were: pan-BIN1 (Rabbit, 1:1000, R3623; homemade anti-BIN1 SH3 domain), pan-DNM2 (Rabbit, 1:700, R2865; homemade anti-DNM2 PRD), [START_REF] Cowling | Increased expression of wild-type or a centronuclear myopathy mutant of dynamin 2 in skeletal muscle of adult mice leads to structural defects and muscle weakness[END_REF] Prohibitin (Rabbit, 1:500, ab28172), P62 (Mouse, 1:1000, H00008878-M01), LC3 (Rabbit, 1:1000, NB100-2220), peroxidase-coupled goat anti-rabbit (Goat, 1/10000, 112-036-04) and peroxidase-coupled goat anti-mouse (Goat, 1/10000, 115-036-068). The data displayed in the figures are the result of a single experiment.

Mice phenotyping.

Hanging test was performed weekly from 4 weeks to 8 weeks of age. Bin1 fl/fl and Bin1 mck-/-male mice were suspended on a cage lid for up to 60 seconds and the time to fall was recorded. The test was replicated 3 times with a time interval of 5 minutes to rest and the average hanging time was calculated. At 6 weeks of age, the mice performed a rotarod test with increasing speed (4-40 rpm) for a maximum of 5 minutes on a rotarod apparatus (Bioseb, Vitrolles, France). The mice were trained on day 1 and tested on the following 4 days when the latency time to fail was recorded three times with 5 minutes resting intervals and the average latency was calculated for each experimental day. At 7 weeks of age, the mice performed the string test. Mice were suspended on a wire by their forelimbs and the time to reach the wire with their hindlimbs was recorded with a maximum time of 20 seconds and any fall was recorded as 20 seconds. The experiment was replicated 3 times with a resting time of 5 minutes. At 4 months of age, exhaustion test was performed using treadmill apparatus (Bioseb). The experiment was initialized with 5 degrees of inclination and 25 cm/s of speed that increased up to 15 degrees of inclination and 41 cm/s over 150 minutes. Stimulation to keep the mouse on track was performed with an individual grip per lane delivering mild electric shocks (less than 0.2mA). Test was stopped when mice receive more than 100 shocks/5 minutes or they stayed 20 consecutive seconds next to the electrodes, and maximal distance covered and latency time were recorded. Habituation for this test was done the day before the experiment for 10 minutes with an inclination of 5 degrees and speed of 25 cm/s.

In situ muscle force.

The force production of the TA was assessed with the Complete1300A Mouse Test System (Aurora Scientific, Aurora, Canada). 8 weeks, 4 months and 12 months old Bin1 fl/fl and Bin1 mck-/- male mice were anesthetized through a triple shot cocktail by intraperitoneal injection of 1) domitor/fentanyl mix (2/0.28 mg/kg), 2) diazepam (8 mg/kg) and 3) domitor (0.28 mg/kg). The distal tendon was excised and attached to the muscle transducer while knees and feet were fixed.

The sciatic nerve was stimulated by electrical pulses with increasing frequency from 1 to 125 Hz in order to measure maximal force. The specific force was determined by dividing the maximal force with the muscle weight (mg).

Histology, immunofluorescence and electron microscopy.

TA, gastrocnemius, FDB andheart were dissected and frozen in liquid nitrogen-cooled isopentane and 8 µm cryosections were stained with hematoxylin-eosin (HE), succinate dehydrogenase (SDH), nicotinamide adenine dinucleotide dehydrogenase (NADH) or modified Gomori. Fiber size distribution, circularity and nuclei counting were determined on 8 µm cryosections from 8 weeks TA cryosections stained with Hoechst (Sigma-Aldrich, St Louis, USA) and Wheat Germ Agglutinin Alexa Fluor TM 555 conjugate (ThermoFischer Scientific, Whaltham, USA) to stain nuclei and plasma membrane, respectively. Images were recorded using the Nanozoomer 2HT slide scanner (Hamamatsu, Japan) and the fiber size and nuclei counting analysis was performed using a homemade ImageJ plugin as described. [START_REF] Silva-Rojas | STIM1 over-activation generates a multi-systemic phenotype affecting the skeletal muscle, spleen, eye, skin, bones and immune system in mice[END_REF] For FDB from 8 weeks old mice and TA from 4 months and 12 months old mice, fiber segmentation was performed using HE images and Cellpose software for segmentation algorithm. [START_REF] Stringer | Cellpose: a generalist algorithm for cellular segmentation[END_REF] Fiber MinFere diameter and nuclei counting were calculated or counted using ImageJ. For fiber circularity distribution, the fibers were classified with a value from 0 to 1 where 0 corresponds to a line and 1 to a circle, and one transversal section of the whole muscle was quantified. For immunofluorescence, 8 µm TA cryosections were fixed with paraformaldehyde 4%, blocked with fetal calf serum and sequentially incubated with antiprohibitin (Rabbit, 1:500, ab28172),anti-LC3 (Rabbit, 1:1000, NB100-2220), anti-desmin (Rabbit, 1:200, ab15200) or anti-TOMM20 (Rabbit, 1:100, ab78547), and Alexa-Fluor 594-coupled goat anti-Rabbit (Goat, 1/250, A11012) and or Alexa-Fluor 488-coupled goat anti-Rabbit (Goat, 1/250, A11008) and Hoechst (Sigma-Aldrich, St Louis, USA). Confocal microscopy images were recorded on a SP8 confocal microscope (Leica, Wetzlar, Germany) and image analysis and counting performed with ImageJ.

For electron microscopy, small pieces of TA were fixed in 2.5% glutaraldehyde, 2.5% paraformaldehyde, 50 mM Ca 2+ in cacodylate buffer (0.1M, pH 7.4). For inclusion, samples were washed in cacodylate buffer for 30 minutes, postfixed in 1% osmium tetroxide in 0.1M cacodylate for 1 h at 4°C. Samples were dehydrated through graded alcohol (50%, 70%, 90% and 100%) and propylene oxide for 30 minutes each and embedded in Epon 812. Seventy nm ultrathin sections were cut on a Leica Ultracut microtome (Leica, Wetzlar, Germany), contrasted with uranyl acetate and lead citrate, and examined at 70kv with a Morgagni 268D electron microscope (FEI, Electron Optics, Eindhoven, Netherlands). Images were captured digitally by a Mega View III Camera (Soft Imaging System, Münster, Germany).

Electrophysiology, intracellular Ca 2+ measurements.

Single fibers were isolated from the flexor digitorum brevis (FDB) and interosseus muscles of 3 Bin1 fl/fl and 3 Bin1 mck-/-mice using previously described procedures. [START_REF] Lefebvre | Whole-cell voltage clamp on skeletal muscle fibers with the silicone-clamp technique[END_REF] In brief, 8 weeks old mice were anaesthetized by isoflurane inhalation and killed by cervical dislocation. Muscles were removed and incubated for 45 minutes at 37 °C in Tyrode solution supplemented with type 1 collagenase (Sigma-Aldrich, St Louis, USA). Single fibers were then separated by gentle mechanical trituration within a culture µ-dish (Ibidi, Planegg / Martinsried, Germany).

For electrophysiology measurements fibers from the interosseus muscles were used (5-6 fibers per mouse). The bottom of a µ-dish was first covered with a thin layer of silicone grease, and muscle trituration was performed in culture medium containing 10% fetal bovine serum (MI199; Eurobio, France). Isolated fibers were painted with silicone so that only a short portion of one fiber end remained out of the silicone, in contact with the bathing solution. The culture medium was then replaced by the experimental extracellular solution containing 140 mM TEA-methane-sulfonate, 2.5 mM CaCl2, 2 mM MgCl2, 1 mM 4-aminopyridine, 10 mM HEPES and 0.002 mM tetrodotoxin (pH 7.2). Voltage-clamp was achieved with a glass micropipette connected to a RK-400 patchclamp amplifier (Bio-Logic, Claix, France) in whole-cell configuration, in combination with an analog-digital converter (Digidata 1440A, Axon Instruments, Foster City, CA) controlled by pClamp 9 software (Axon Instruments). The micropipette was filled with a solution containing 120 mM K-glutamate, 5 mM Na2-ATP, 5 mM Na2-phosphocreatine, 5.5 mM MgCl2, 5 mM glucose, 15 mM EGTA, 6 mM CaCl2, 0.1 mM rhod-2 and 5 mM HEPES (pH 7.2). The pipette tip was inserted into the silicone-insulated part of the fiber. It was gently crushed against the bottom of the µ-dish to ease dialysis and reduce series resistance. The fiber cytosol was dialyzed for 30 min to allow for intracellular equilibration of the solution before starting measurements.

CaV1.1 Ca 2+ current and rhod-2 (Thermo Fisher Scientific, Illkirch, France) intracellular Ca 2+ transients were simultaneously measured in response to 0.5 s-long depolarizing steps from a holding voltage of -80 mV. For CaV1.1 current analysis, the linear leak component of the current during the test pulses was removed by subtracting the adequately scaled current value measured during a 20 mV hyperpolarizing step. A residual leak component (referred to as slope) was found to be larger in fibers from Bin1 fl/fl mice than in fibers from Bin1 mck-/-mice. Thus, for each fiber, the voltage-dependence of peak Ca 2+ current values (normalized to fiber capacitance) was fitted with the following equation: I(V) = Gmax(V-Vrev)/(1+[exp(V0.5-V)/k]) + slope(V+80), with I(V) the peak current density at the command voltage V, Gmax the maximum conductance, Vrev the apparent reversal potential, V0.5 the half-activation potential, k the steepness factor, and slope the conductance of the residual linear component.

Fluorescence imaging was conducted with a Zeiss LSM-800 system equipped with a 63× oil immersion objective (numerical aperture 1.4). For detection of rhod-2 fluorescence, excitation was from a 561 nm diode laser and fluorescence was collected above 570 nm. Fluorescence changes were imaged using the line-scan mode (x,t) of the system with a scanning frequency of 1.02 ms per line and expressed as F/F0 where F0 is the baseline fluorescence. Quantification of the Ca 2+ release flux underlying the rhod-2 Ca2+ transients was performed as previously described. [START_REF] Lefebvre | Whole-cell voltage clamp on skeletal muscle fibers with the silicone-clamp technique[END_REF][START_REF] Kutchukian | Phosphatidylinositol 3-kinase inhibition restores Ca2+ release defects and prolongs survival in myotubularin-deficient mice[END_REF] In each fiber, the voltage-dependence of the peak rate of Ca 2+ release was fitted with a Boltzmann function.

For imaging the T-tubule network, flexor digitorum brevis muscle fibers were incubated for 30 minutes in the presence of 10 µM di-8-anepps (Thermo Fisher Scientific, Illkirch, France). Di-8anepps was excited with a 488 nm diode laser and fluorescence was collected above 510 nm. The T-tubule density index (9-13 fibers per mouse) was estimated as described previously. [START_REF] Kutchukian | Impaired excitation-contraction coupling in muscle fibres from the dynamin2(R465W) mouse model of centronuclear myopathy[END_REF]

Statistical analysis.

All experiments were performed and analyzed in a blinded manner and the investigators were unaware of the genotype of the mice. The normal distribution of the data was assessed using the Shapiro-Wilk test and presented as mean ± standard error of the mean (SEM) with exception of data compared with Kruskal-Wallis test where the data is presented as median ± interquartile range. For normally distributed data the significance of changes was examined by two-tailed Student's t-test (with or without Welch's correction if variance was different or not) for comparison of 2 groups or by one-way ANOVA followed by Tukey's post hoc test for comparison of more than 2 groups. In case of not-normally distributed data, Mann-Whitney test was used to compare 2 groups and Kruskal-Wallis followed by Dunn's multiple comparison test to compare more than 2 groups. The significant difference of birth ratio was assessed by chi-square test.

Significant differences are illustrated as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Figure 1 .

 1 Figure 1. Muscle-specific Bin1 mck-/-mice are viable with impaired TA muscle force. (A) Homozygous Bin1 mck-/-mice were generated by crossing Bin1 fl/fl with heterozygous floxed Bin1 mice expressing the Cre recombinase under the control of the muscle creatine kinase promoter (MCK-Cre). The represented situation refers to skeletal muscle. (B) Genotype proportion obtained 10 days after birth; expected ratio is 50/50. (C) Bin1 fl/fl and Bin1 mck-/-mice at 8 weeks of age. (D) Bin1 mRNA pan-isoform relative levels in TA from Bin1 fl/fl and Bin1 mck-/-mice at 8 weeks of age (n=4; Student t-test with Welch's correction). (E) Western blot with TA muscle protein extracts probed with anti-BIN1 pan-isoform antibody. BIN1 is not detected in Bin1 mck-/-muscle. (F) Body weight evolution of Bin1 fl/fl and Bin1 mck-/-mice (n=8-9; two-way ANOVA and Bonferroni post hoc test). (G) Specific muscle force produced by TA of Bin1 fl/fl and Bin1 mck-/-at 8 weeks of age (n=8-9; Student's t test). Bin1 mck-/-mice produce 41% less specific force than Bin1 fl/fl controls. (H) Specific force produced by Bin1 fl/fl and Bin1 mck-/-TA when stimulated at different frequencies (n=8-9; two-way ANOVA with Bonferroni's post hoc test). ****P<0.0001; ***P<0.001; **P<0.01.

Figure 2 .

 2 Figure 2. Bin1 mck-/-mice develop most of the histopathological hallmarks of CNM in TA. (A) Representative HE and SDH images of TA from Bin1 fl/fl and Bin1 mck-/-mice at 8 weeks of age. Bin1 mck-/-muscle sections present SDH accumulations in the center of fibers (arrows; n=3). Scale bar =50 µm. (B) MinFeret perimeter distribution of TA fibers (n=4-5). (C) The percentage of large

Figure 3 .

 3 Figure 3. Bin1 mck-/-mice present myofibrils disorganization and mitochondria accumulation in TA. (A) Electron microscopy images from Bin1 fl/fl and Bin1 mck-/-at 8 weeks of age reveal overall myofibrils disorganization with higher inter-myofibril spaces (left) while nuclear shape and position remain normal in Bin1 mck-/-mice (right) (n=2). Scale bar = 1 µm. (B) Mitochondria accumulations in Bin1 mck-/-muscle observed by electron microscopy (left) and revealed with prohibitin staining (right) (n=2). Scale bar = 1 µm for electron microscopy and 10 µm for confocal microscopy. (C) Quantification of prohibitin protein expression (n=3; Student's t test). (D) Ppargc1a mRNA relative levels (n=6; Student's t test). (E) Regions with mitochondria accumulation (arrow) in Bin1 mck-/-muscle present autophagosomes (arrowhead; left), and immunofluorescence labelling reveals increased number of autophagic LC3 positive clusters (right) (n=2). (F-G) P62 and lipidated LC3 (II) protein levels (n=4; Student's t test for P62 and Student t-test with Welch's correction for lipidated LC3). **P<0.01.

Figure 4 .

 4 Figure 4. Deficient voltage-dependent Ca 2+ signaling in muscle fibers isolated from Bin1 mck- /-mice FDB. (A) Representative confocal images of the T-tubule network in muscle fibers from Bin1 fl/fl and Bin1 mck-/-mice at 8 weeks of age. Scale bar =10 µm. (B) Mean values for the T-tubule network density index in the two groups (n= 3 mice per genotype, 9-13 fibers analyzed per mice; Student's t test). (C) Examples of CaV1.1 Ca 2+ current records from muscle fibers from a Bin1 fl/fl mouse (left) and from a Bin1 mck-/-mouse (right) in response to 0.5 s-long depolarizing pulses to the range of indicated values (voltage increment between -40 and +50 is 10 mV). (D) Mean values for the peak Ca 2+ current vs voltage in the two groups. The voltage dependence of the peak Ca 2+ current in each fiber was fitted as described in Methods (n= 3 mice per genotype, 5-6 fibers analyzed per mice; Student's t test). (E) Mean values, from fitting the voltage-dependence of the peak Ca 2+ current, for the maximum conductance (Gmax), reversal voltage (Vrev), and for the additional linear conductance (slope). Mean values for Vrev and for the two other fitted parameters (half-activation voltage and steepness factor) did not statistically differ between the two groups

Figure 5 .

 5 Figure 5. Dnm2 downregulation ameliorates the TA muscle force of Bin1 mck-/-mice. (A) DNM2 protein expression in TA at 8 weeks of age (n=4; Student t-test with Welch's correction). (B) Dnm2 mRNA levels in TA at 8 weeks of age by RT-qPCR for pan-isoforms (n= 4; two-way ANOVA with Bonferroni's post hoc test). (C) DNM2 protein expression in TA at 8 weeks (n=4-7; two-way ANOVA with Bonferroni's post hoc test). (D) Maximal specific force produced by Bin1 fl/fl and Bin1 mck-/-TA. Only muscle force produced by PBS-treated Bin1 mck-/-muscles show a significant decrease compared to Bin1 fl/fl controls (n=4-6; Kruskal-Wallis with Dunn's post hoc test). (E) Specific force produced when stimulated at increasing frequencies (n=4-6; one-way ANOVA with Tukey's post hoc test for 1, 25 and 50 Hz frequencies and Kruskal-Wallis with Dunn's post hoc test for 100 and 125 Hz frequencies). * between Bin1 fl/fl PBS and Bin1 mck-/-PBS, and β between Bin1 mck-/-PBS and Bin1 mck-/-ASO. **P<0.01; *P<0.05; ββP<0.01. We found no statistical difference between Bin1 mck-/-ASO and Bin fl/fl ASO.

Figure 6 .

 6 Figure 6. Dnm2 downregulation rescues the TA CNM histopathological hallmarks of Bin1 mck- /-mice. (A-B) Representative HE and SDH images of TA from PBS and ASO-treated Bin1 fl/fl and Bin1 mck-/-mice at 8 weeks of age (n=4-6). Scale bar =50 µm. ASO-treated Bin1 mck-/-mice do not present the central SDH accumulations seen in PBS-treated Bin1 mck-/-mice (arrows). (C) MinFeret perimeter distribution of TA fibers from PBS and ASO-treated Bin1 fl/fl-and Bin1 mck-/-mice (n=4-5). (D) Percentage of fibers with MinFeret diameter higher than 40 µm are comparable in PBS and ASO-treated Bin1 fl/fl and Bin1 mck-/-mice (n=4-7; one-way ANOVA with Tukey's post hoc test).

Acknowledgements

We thank IGBMC and ICS platforms for animal care, histology, photonic and electron imaging, Shuling Guo and Brett Monia for providing the Dnm2 antisense oligonucleotide, Quentin Giraud for his help with mice. and Doulaye Dembélé for the statistical analysis assistance. The creation of the Bin1 fl/fl mouse was done with Phenomin-ICS, Illkirch, France. This study was supported by institute funding from Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de Strasbourg and by grants from the Agence Nationale de la Recherche ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d'Avenir ANR-10-IDEX-0002-02, Réseau National des Génopoles (RNG), the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (675392), Muscular Dystrophy Association (576154), Fondation pour la Recherche Médicale (201903007992), and AFM-Téléthon (21267, 22734, MyoNeurALP # 5.3.4.4). R.G.O. had a CIFRE fellowship obtained with Dynacure, and R.S.R. a fellowship from Fondation pour la Recherche Médicale (PLP20170939073).

Data availability.

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary material.

Authors' contributions

All experiments were done in IGBMC. J.L. designed the study; B.S.C., V.J. and J.L. supervised research; V.N., R.S.R., F.J-F., A.M., R.G.O., N.M., V.M.L., D.R., C.K. performed research; V.N., R.S.R., F.J-F., R.G.O., M.G., N.M., D.R, V.J. analyzed data; R.S.R. and J.L. wrote the manuscript. S1. List of primers used for RT-qPCR.

Supplemental Table