Laurent Brochard
email: laurent.brochard@enpc.fr

György Hantal

Hadrien Laubie

J.-M Pellenq

Franz J Ulm

FRACTURE PROPERTIES OF KEROGEN AND IMPORTANCE FOR ORGANIC-RICH SHALES

Introduction

Oil and gas produced from organic-rich shales have become in the last ten years one of the most promising sources of unconventional fossil fuels. The oil and gas are trapped in rocks of very small permeability, but hydraulic fracturing enables to operate those reservoirs with competitive costs. The global reserves of shale oil and gas that are potentially recoverable are equivalent to tens of years of world consumption. However, hydraulic fracturing is facing many challenges regarding the productivity but also the security and the environment. One of those challenges is to understand how the fractures propagate underground. The propagation depends on the mechanical stress prevailing in the reservoir and on the fracture properties of the rocks. Regarding the fracture properties, the oil and gas industry developed brittleness indicators to distinguish between brittle rocks (containing mostly calcite and silica) and ductile rocks (containing a significant proportion of clay and kerogen). During fracturing, a brittle rock shatters easily leading to a well-distributed network of fractures, whereas a ductile rock deforms instead of shattering leading to few fractures and in some situations acting as a barrier to the fracture propagation. In this work, we study the role of kerogen in the ductility of shale. The ultimate objective is to develop a fine understanding of the fracture properties of shales.

Methodology

Organic-rich shales are heterogeneous materials containing a small fraction of kerogen. The typical size of the kerogen heterogeneities is a micrometer or less, and, therefore, mechanical tests on these materials are usually performed at the scale of the heterogeneous medium. Measuring the fracture properties of kerogen and of its interface with minerals is very challenging. As an alternative to laboratory experiments, we use molecular simulations to estimate fracture properties at the nanometer scale. As a preliminary work, we develop a methodology to estimate fracture properties and validate it against experimental data in the case of silica, a common mineral in shale reservoirs and a good benchmark in our work.

By simulating fracture propagation by molecular simulations one can estimate the fracture properties of a material. At the atomic scale, crack propagation results of complex atomic rearrangements at the crack tip whereas, at the macroscopic scale, one refers to the simplified notions of toughness and ductility. In this work, we propose an approach by thermodynamic integration to capture the toughness and ductility from the results of a molecular simulation. This approach relies on the energetic theory of fracture mechanics [START_REF] Leblond | Mécanique de la rupture fragile et ductile[END_REF].

We consider a molecular system in which we initiate a crack and we load the system to propagate this initial crack. Assuming isothermal condition, this loading leads to an increase of free energy F of the system which can be converted either into mechanical energy P by deforming the solid or into fracture energy G c by propagating the crack. The energy balance of the system is

dF = dP + G c dA (1)
The quantity G c , also known as critical energy release rate, characterizes the fracture properties of the material: G c is related to the fracture toughness K Ic through Irwin's formula [START_REF] Leblond | Mécanique de la rupture fragile et ductile[END_REF] and its extension to anisotropic materials [START_REF] Sih | [END_REF]. For a perfectly brittle material, G c = 2γ where γ is the surface energy of the material; but for ductile materials G c is much larger than 2γ because of significant irreversibility at the crack tip during the crack propagation, e.g., plasticity. Therefore, G c is also an indicator of the level of ductility of the material. We consider systems whose general geometry is displayed in Figure 1: the system is periodic with an elliptic hole at the center of the system which corresponds to a through-crack. We also display in Figure 1 a schematic description of the procedure we follow for the thermodynamic integration. The derivative of the free energy Σ = 1 LxLz ∂F ∂Ly T,Lx,Lz can be interpreted as the average stress along the top and bottom boundaries of system. The main steps of the procedure are the following: 1 -At the beginning of the simulation, the system is relaxed mechanically and, accordingly, P = 0. 2 -We load the system by increasing the length L y in the direction orthogonal to the crack. 3 -At the beginning, the loading is too small to propagate the crack and all the free energy is stored in the form of mechanical energy. 4 -When the loading is large enough, some irreversible processes may occur at the crack tip and the crack starts to propagate. At that point, Σ reaches its maximum and mechanical energy starts to be converted into fracture energy. 5 -At the end of the procedure, the crack fully propagated over the whole system. As at the beginning of the procedure, P = 0.

The thermodynamic integration consists in integrating Σ over the whole loading procedure. Since P = 0 both at the beginning and at the end of the procedure, all the variation of free energy (Eq. 1) corresponds to fracture energy only:

G c = ∆F ∆A = L x L z ∆A Lymax Ly0 ΣdL y (2)
Therefore, with this methodology, we can estimate the fracture properties, i.e., toughness and ductility, starting from the elementary inter-atomic interactions. The detailed description of the methodology and its main limitations are presented in [START_REF] Brochard | Fracture toughness from molecular simulation: Application to bulk silica, microporous carbons and their interface[END_REF].

The relevance of this estimation relies on the reliability of the inter-atomic potential we consider. In particular, to properly simulate fracture processes, this energy potential must be able to predict complex atomic rearrangements such as bond breaking / formation or transfer of charges. In this work, we consider the reactive potential reaxFF [START_REF] Van Duin | [END_REF] parameterized for system made of Si-O-C-H [5]. We benchmarked the applicability of this potential to silicakerogen system by comparing the energy computed with reaxFF and that computed with ab-initio methods for a series of elementary chemical bonds that are present in those systems [START_REF] Hantal | Chemistry and molecular reconstruction of kerogen-silica interfaces[END_REF].

Validation

Before applying this methodology to kerogen, we validate it against experimental data and against the usual theory of Linear Elastic Fracture Mechanics (LEFM). Silica, which is a common mineral in shale reservoirs, is a good benchmark for the methodology we proposed: experimental data are available, and LEFM applies well to this material even at small scale since it is a homogeneous brittle material.

We consider α-cristobalite, a silica crystal metastable in ambient conditions. This particular silica is convenient regarding the forthcoming study of the kerogen-silica interface, because its crystalline structure is orthorhombic and its size adapts very well to the lattice of the kerogen molecular reconstruction we consider later on. We investigate a total of 33 different systems varying the size of the initial crack, the size of the whole system, the orientation of the crack. The detail of the systems studied and the numerical aspects of the simulations are available in [START_REF] Brochard | Fracture toughness from molecular simulation: Application to bulk silica, microporous carbons and their interface[END_REF]. In this work, we present the results of 18 of those systems (long procedure in [START_REF] Brochard | Fracture toughness from molecular simulation: Application to bulk silica, microporous carbons and their interface[END_REF]). We display in Figure 2 the evolution of Σ during the loading procedure for two of the systems studied, one with the crack in the (001) orientation, and the other with the crack in the (100) orientation. In addition we illustrate the strain-stress curve with a few snapshots of molecular configurations for the first system (001) orientation. The crack propagation in α-cristobalite is very sudden, characteristic of a brittle behavior, although we observe some arrest in the propagation as is the case for the second system in Figure 2. Since the different systems studied have different geometries, the strain and the stress at the onset of propagation always differ from one system to another. However, the thermodynamic integration should provide a similar energy release rate G c for all the systems. We display in Figure 3 the fracture toughness estimated in function of the area of crack created ∆A. The fracture toughness K Ic is derived from the critical energy release rate G c with the extension of Irwin's formula to orthotropic materials [START_REF] Sih | [END_REF]. The values of all those systems are very consistent and independent of the geometry. Based on thermodynamic integration, the average toughness is 1.03 MPa.m 1/2 . This result compares well with the value generally measured experimentally for silica [START_REF] Lucas | [END_REF]: 0.82 ± 0.07 MPa.m 1/2 .

In addition, we can validate the methodology against LEFM. Silica is a linear elastic brittle material and therefore LEFM should be valid in the case of silica. According to LEFM, the critical stress Σ cr at the onset of crack propagation is related to the toughness K Ic according to:

K Ic Σ cr √ πa = C ( 3
)
where C is a coefficient that depends on the geometry of the system. In the case of the periodic system we consider here (see Figure 1), C depends only on the ratios a/W and H/W and can be computed numerically [8]. We represent in Figure 4 the critical stress in function of the effective crack length πa ef f = πaC 2 for the various systems of α-cristobalite simulated. The relationship between the critical stress and the effective length is consistent with Eq. 3. From Eq. 3, we estimate toughnesses of 0.87 and 0.81 MPa.m 1/2 for the crack orientations (001) and (100), respectively. These results compare well with that obtain from thermodynamic integration and with the experimental results. Accordingly, the approach we propose applies well in the case of α-cristobalite.

Effective half length of the crack πaeff , Å Critical stress Σcr, GPa

Fracture properties of kerogen

Kerogen is a generic word that encompasses all the solid organic matter that is present in shale reservoir. There is not a unique molecular structure of kerogen but a large variety of possible structures. In this work, we study a molecular structure of microporous carbon, CS1000 [9], that is similar to dense mature kerogen, i.e., dense kerogen with low hydrogen and oxygen contents. Accordingly, the results of this section cannot be considered as representative of all types of kerogen. Nevertheless, the most interesting reservoirs in terms of shale gas recovery are those filled with mature kerogen, for which CS1000 is a reasonable molecular representation. We consider a series of 25 systems for CS1000 varying the size of the crack and of the whole system and the positions of the crack in the heterogeneous structure. An example of strain-stress curve is displayed in Figure 5. As is readily visible, the crack propagation is not sudden like in the case of α-cristobalite: the material exhibits a significant ductility because of large rearrangements in the atomic structure at the crack tips. These rearrangements are irreversible processes that dissipate energy. Accordingly, we expect the fracture energy of CS1000 to be much larger than the surface energy. We computed the fracture energy (Eq. 2) and we display the results in function of the area of crack created in Figure 6. The results show a correlation with the area, that we interpret as a size effect: the overlap between the process zones at the periodic boundaries cannot be neglected. We propose a correction of the results to account for this overlap by reducing the actual area of crack created with a simplistic model (see [START_REF] Brochard | Fracture toughness from molecular simulation: Application to bulk silica, microporous carbons and their interface[END_REF]). The corrected results are displayed in Figure 6 and exhibit no more trend. By thermodynamic integration, we estimate a toughness of 1.61 MPa.m 1/2 for CS1000 which is about two times the value for α-cristobalite. As for α-cristobalite, we also use LEFM to provide an alternative estimate of the toughness. We display in Figure 7 the critical stress in function of the effective crack length. It appears that the trend is not as consistent with Eq. 3 as in the case of α-cristobalite. The best fit with Eq. 3 leads to a toughness of 0.80 MPa.m 1/2 two times smaller than the estimate from thermodynamic integration. This discrepancy stems to the fact that LEFM is not valid in the case of CS1000 at this scale because the size of the process zone cannot be neglected as was the case for silica. In contrast, the thermodynamic integration does not assume any material behavior and is valid even if the process zone is significant. The value of 1.61 MPa.m 1/2 can be considered as a good estimation of the actual toughness of CS1000. This toughness corresponds to an energy release rate G c about 12 times larger than the surface energy of CS1000, that is a very significant ductility. The ductility is readily visible in Figure 5 (strain-stress curve): the ratio between the maximum strain (0.4) and the maximum stress at failure (14 GPa) is two times larger than in the case of α-cristobalite for similar geometries. These results are consistent with the ductility observed at the scale of shale rocks (increase of ductility with kerogen content). However, the absolute value of toughness obtained for CS1000 should not be considered as representative of all mature kerogen since it is very dependent on the density; and the density of CS1000 (1.6 kg.m -3 ) corresponds to the density of the microporous part of kerogen, i.e., kerogen excluding meso-and macro-pores. Accordingly the toughness of CS1000 is in the upper range of values for mature kerogen.

Fracture at the kerogen-silica interface At the scale of the heterogeneous material, the effective fracture properties may strongly depend on the properof the interface and not only on the properties of the elementary phases. If cracking at the scale of the heterogeneous shale never occurs in the kerogen phase, but only at the kerogen / mineral interface, the ductility of kerogen itself is not enough to explain the ductility of the shale. For this reason, this section is dedicated to the CS1000 / α-cristobalite interface. In a separate work, we reconstructed a series of 11 typical interfaces by studying the bonding at the interface between kerogen and silica by quantum calculations [START_REF] Hantal | Chemistry and molecular reconstruction of kerogen-silica interfaces[END_REF]. We then applied the same methodology to these interfaces as we did for CS1000 and α-cristobalite, with an initial crack initiated along the interface. In Figure 8, we display the strain-stress curve for one of the system simulated. The interface exhibits some ductility as CS1000, but mostly on the side of CS1000 and not in α-cristobalite. The crack propagation is not sudden and the shape of the strain-stress curve looks similar to that of CS1000 (Figure 5). However, the value of the critical stress in much smaller than for the bulk materials. As a consequence, the thermodynamic integration leads to an average toughness of 0.57 MPa.m 1/2 , smaller than for the bulk materials. Now, this toughness value is not small when considering the number of covalent bonds at the interface: only 37% of the Si-O terminations of the α-cristobalite surface are bonded to the kerogen.

Even though the absolute toughness is small, the interface is still ductile. Indeed, applying the LEFM Eq. 3, we obtain a value of toughness for the interface of 0.26 MPa.m 1/2 , more than two times smaller than the value obtain by thermodynamic integration. Therefore, the ductility of the interface looks very similar to the ductility of bulk CS1000, which supports the experimental observations at the scale of shale rocks.

From kerogen to heterogeneous shale rocks

In the previous section, we focused on two elementary constituents of shales rocks and their interface, and we found that kerogen and kerogen-silica interface are quite ductile. However, what matters for field applications is the ductility of the shale rock which depends not only on the fracture properties of the elementary constituents and their interface, but also on the microstructure. As an illustra-tion of the potential increase of ductility due to the presence of kerogen, we consider here the case of a particular geometry in which the kerogen is located in spherical pockets distributed periodically in a homogeneous mineral matrix (Figure 9). In such geometry, the ductile pockets may form bridges in the wake of a propagating crack [10]. Since the fracture energy of the interface is 14% of the fracture energy of bulk kerogen, the bridges eventually debond at their interface with the brittle matrix [11]. This phenomenon, called particle pull out, is known to significantly increase the resistance to fracture propagation. In this work, we use the numerical approach of Bower et al. [10] to compute numerically the increase of the effective toughness K ef f Ic due to the particle pull out phenomenon. Each pocket of kerogen exerts a bridging force P on the crack faces that depend on the crack opening δ. Following the numerical approach of Bower et al., the relative increase in toughness K ef f Ic /K matrix Ic depends on the pull out law P = P (δ), and on the geometry of the system (R/L, H/L, x 0 /L). In our work, we considered the pull out law displayed in Figure 9 which is inspired from the shape of the strain-stress curve obtained by molecular simulation of crack propagation at the interface (Figure 8).The values of the parameters K p.o. and δ p.o. are related to the energy release rate at the interface. Assuming spherical particles debonded on half of their interface with the matrix, we have:

2πR 2 G interf ace c = int 2δp.o. 0 P (δ)dδ = K p.o. (δ p.o. ) 2 .
In what follows, we varied K p.o. and δ p.o.

while ensuring this condition on energy release. As a starting value, we considered a high value of the elasticity of the particle pull-out equal to the elasticity of the matrix:

K ref p.o. = (2πRE)/ 2 1 -ν 2
, where E and ν are the Young's modulus and Poisson's ratio of the matrix.

In Figure 10, we display the relative increase in toughness in function of the surface fraction of ductile particle for various pull out elasticity K p.o. . The increase in toughness is important, up to a factor of 3 for as little as 20% of ductile particles. In the specific case of kerogen and silica, K interf ace Ic /K matrix Ic = 0.7 from the molecular simulations, and the elasticity of kerogen is usually estimated around 10 GPa, i.e. K p.o. /K ref p.o. ≈ 0.125. In those conditions, the relative increase of toughness is 2 for a fraction of kerogen of 10% and 3 for a fraction of kerogen of 20%.

Of course not all kerogen in shale does form bridges and the geometry considered is very simplistic. But this calculation shows that even a small fraction of ductile particle can significantly affect the fracture properties of the shale rock. Kerogen is not the only ductile material in shale: clays also increase the ductility of shale. Therefore a general picture of the ductility of shale requires accounting for the presence of clay and not only of kerogen.

Conclusion

We performed molecular simulations to study the fracture properties of kerogen, silica and their interface. First, we developed a specific methodology based on a thermodynamic integration to estimate the fracture properties by molecular simulation and we validated this approach against experimental data and LEFM in the case of silica, a brittle mineral common in shales. We have shown that the kerogen and kerogen-silica interface exhibit a significant ductility. The ductility at the scale of the constituents of shale can significantly affect the ductility of the shale rock itself even with small fraction of ductile inclusions. We illustrate this potential effect by considering the example of the particle pull out phenomenon.

Fig. 1

 1 Fig. 1 Schematic description of the geometry of the molecular systems and of the procedure of thermodynamic integration.

Fig. 2

 2 Fig.2Strain-stress curves for two of the α-cristobalite systems studied.

Fig. 3

 3 Fig.3Fracture toughness of estimated by thermodynamic integration.

Fig. 4

 4 Fig.4Critical stress in function of the crack length and fit with Eq. 3 (LEFM) for α-cristobalite.

Fig. 5

 5 Fig.5Strain-stress curve for one of the CS1000 systems studied.

Fig. 6

 6 Fig.6Fracture toughness of CS1000 estimated by thermodynamic integration.

Fig. 7

 7 Fig. 7 Critical stress in function of the crack length and fit with Eq. 3 (LEFM) for CS1000.This toughness corresponds to an energy release rate G c about 12 times larger than the surface energy of CS1000, that is a very significant ductility. The ductility is readily visible in Figure5(strain-stress curve): the ratio between the maximum strain (0.4) and the maximum stress at failure (14 GPa) is two times larger than in the case of α-cristobalite for similar geometries. These results are consistent with the ductility observed at the scale of shale rocks (increase of ductility with kerogen content). However, the absolute value of toughness obtained for CS1000 should not be considered as representative of all mature kerogen since it is very dependent on the density; and the density of CS1000 (1.6 kg.m -3 ) corresponds to the density of the microporous part of kerogen, i.e., kerogen excluding meso-and macro-pores. Accordingly the toughness of CS1000 is in the upper range of values for mature kerogen.

- 1 Fig. 8

 18 Fig.8Strain-stress curve for one of the α-cristobalite / CS1000 interfaces studied.

Fig. 9

 9 Fig.9Model considered for the particle pull out phenomenon.In this work, we use the numerical approach of Bower et al.[10] to compute numerically the increase of the effective toughness K ef f

Fig. 10

 10 Fig.10Relative increase in toughness.


Jean-Baptiste Reisqs

Adrien Moreau

Yvonne Sleiman

Azzouz Charrabi

Antoine Delinière

Francis Bessière

Kevin Gardey

Sylvain Richard

Philippe Chevalier
email: philippe.chevalier@chu-lyon.fr

Spironolactone as a Potential New Treatment to Prevent Arrhythmias in Arrhythmogenic Cardiomyopathy Cell Model

Keywords: arrhythmogenic cardiomyopathy, spironolactone, arrhythmias, hiPSC-CM

Arrhythmogenic cardiomyopathy (ACM) is a rare genetic disease associated with ventricular arrhythmias in patients. The occurrence of these arrhythmias is due to direct electrophysiological remodeling of the cardiomyocytes, namely a reduction in the action potential duration (APD) and a disturbance of Ca2+ homeostasis. Interestingly, spironolactone (SP), a mineralocorticoid receptor antagonist, is known to block K+ channels and may reduce arrhythmias. Here, we assess the direct effect of SP and its metabolite canrenoic acid (CA) in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) of a patient bearing a missense mutation (c.394C>T) in the DSC2 gene coding for desmocollin 2 and for the amino acid replacement of arginine by cysteine at position 132 (R132C). SP and CA corrected the APD in the muted cells (vs. the control) in linking to a normalization of the hERG and KCNQ1 K+ channel currents. In addition, SP and CA had a direct cellular effect on Ca2+ homeostasis. They reduced the amplitude and aberrant Ca2+ events. In conclusion, we show the direct beneficial effects of SP on the AP and Ca2+ homeostasis of DSC2-specific hiPSC-CMs. These results provide a rationale for a new therapeutical approach to tackle mechanical and electrical burdens in patients suffering from ACM.

Introduction

Arrhythmogenic cardiomyopathy (ACM) is a rare inherited heart condition, often linked to mutations in genes encoding desmosomal proteins [START_REF] Awad | Mechanisms of disease: Molecular genetics of arrhythmogenic right ventricular dysplasia/ cardiomyopathy[END_REF][START_REF] Brodehl | A homozygous DSC2 deletion associated with arrhythmogenic cardiomyopathy is caused by uniparental isodisomy[END_REF] (OMIM 610476). ACM is characterized by progressive mechanical and electrical myocardial dysfunction [START_REF] Caspi | Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells[END_REF][START_REF] El-Battrawy | Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy[END_REF]. In the concealed stage of this disease, there is no mechanical dysfunction but a risk of sudden cardiac death due cardiac arrhythmias, such as ventricular fibrillation [START_REF] Akdis | Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls[END_REF][START_REF] Corrado | Spectrum of Clinicopathologic Manifestations of Arrhythmogenic Right Ventricular Cardiomyopathy/ Dysplasia: A Multicenter Study[END_REF]. How this disease predisposes patients to ventricular fibrillation is not fully understood [START_REF] Basso | Arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Marcus | Multidisciplinary Study of Right Ventricular Dysplasia Investigators. Efficacy of antiarrhythmic drugs in arrhythmogenic right ventricular cardiomyopathy: A report from the North American ARVC Registry[END_REF]. In a transgenic mice model with a cardiac-specific overexpression of DSC2, severe myocardial necrosis, fibrosis, and inflammation mimicking ACM are found [START_REF] Brodehl | Transgenic mice overexpressing desmocollin-2 (DSC2) develop cardiomyopathy associated with myocardial inflammation and fibrotic remodeling[END_REF]. Recent studies on ACM patient specific induced pluripotent stem cells (ACM-hiPSC-CMs) have shown that profound electrophysiological remodeling of cardiomyocytes contributes to arrhythmias [START_REF] El-Battrawy | Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy[END_REF]. Recently, a thorough analysis of hiPSC-CMs derived from a patient with a heterozygous missense mutation (c.394C>T) in exon 3 of the DSC2 gene (NM_024422.6:c.394C>T[p.Arg132Cys], which encodes desmocollin 2, revealed abnormal cellular early repolarization dynamics in line with a short QT interval in some ACM patients [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. This observation is one of the major causes of triggering and maintaining ventricular arrhythmia in ACM patients and increasing dispersion repolarization, which is a pro-arrhythmogenic mechanism [START_REF] Giudicessi | Potassium-channel mutations and cardiac arrhythmias-Diagnosis and therapy[END_REF]. Normalizing the QT interval can be an interesting target to prevent arrhythmia in ACM patients.

For a long time, many studies have shown that angiotensin-converting enzyme inhibitors are associated with a lower incidence of ventricular arrhythmias in patients with heart failure or LV dysfunction [START_REF] Henein | Angiotensin-converting enzyme (ACE) inhibitors revert abnormal right ventricular filling in patients with restrictive left ventricular disease[END_REF][START_REF] Rouleau | Cardioprotective Effects of Ramipril and Losartan in Right Ventricular Pressure Overload in the Rabbit: Importance of Kinins and Influence on Angiotensin II Type 1 Receptor Signaling Pathway[END_REF]. In the Heart Failure Efficacy and Survival Study (EPHESUS) and Randomized Aldactone Evaluation Study (RALES), mineralocorticoid receptor antagonists (MRAs) reduced the risk of ventricular arrhythmias [START_REF] Fletcher | Enalapril decreases prevalence of ventricular tachycardia in patients with chronic congestive heart failure. The V-HeFT II VA Cooperative Studies Group[END_REF]. A direct anti-arrhythmic effect of MRAs has been suggested by several studies [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF][START_REF] Pitt | Spironolactone for Heart Failure with Preserved Ejection Fraction[END_REF][START_REF] Shafiq | Blocking aldosterone in heart failure[END_REF]. A phase II clinical study has been initiated in patients with ACM to investigate the impact of spironolactone (SP) on arrhythmias [START_REF] Morel | Blockade of the renin-angiotensinaldosterone system in patients with arrhythmogenic right ventricular dysplasia: A doubleblind, multicenter, prospective, randomized, genotype-driven study (BRAVE study)[END_REF]. Interestingly, SP is known to block K+ channels [START_REF] Caballero | Spironolactone and its main metabolite, canrenoic acid, block human ether-a-go-go-related gene channels[END_REF]. Our work aims to assess the direct effects of SP and its metabolite, canrenoic acid (CA), on patient-specific human DSC2 mutated hiPSC-CMs (ACM-DSC2-hiPSC-CMs) to assess the potential benefits to prevent or reduce ACM-induced electrical instability.

Methods

Clinical Patient Case

The patient, in the clinical case, harbored two mutations of the DSP gene. One was on exon 23, inducing a heterozygous DSP mutation (c.4565C>T) and coding for an amino acid replacement of threonine by methionine at position 1522 (T1522M). The second was a heterozygous deletion of exon 24. The deletion of this exon is a new variation whose implication cannot be affirmed with certainty but is highly probable. The search for the deletion was carried out by the MLPA method (kit P168, MRC Holland).

Cell Culture

The methods are described elsewhere [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. Briefly, we used two control hiPSC lines, one from the platform IPS from INMG (hiPSC AG08C5; Neuromyogen institute, Lyon, France) and the other from Dr. Xavier Nissan (hiPSC FSC03; I-Stem institute, Evry, France). We cultured the hiPSC with StemFlexTM in a DMEM/F12 medium for 5 days (ThermoFisher Scientific, Waltham, MA, USA) and differentiated them using the sandwich monolayer technique. We induced cardiac differentiation by adding 6 _M of CHIR99021 (LC Laboratories, MA, USA) and then 2 _M of Wnt-C59 (LC Laboratories) for each molecule for 2 days. Previously, we showed that the DSC2 phenotype is best expressed at 60 days (D60) [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. Finally, for "single-cell" experiments, we dissociated the cardiomyocytes at D53 using TrypLETM Express (ThermoFisher Scientific) and replated them on Matrigel-coated 35 mm Petri dishes for patch-clamp experiments. We incubated cells with SP at 20 M or canrenoic acid at 10 nM for one hour before performing the experiments.

Whole-Cell Current-Clamp Recordings

For the current-clamp studies, we recorded the action potential (AP) and global K+ currents in single cells at room temperature (22 +/-2 °C) using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). The resistance of patch pipettes ranged between 1 and 2 mW. We generated voltage-clamp command pulses using pCLAMP v10 (Axon Instruments). We filtered currents at 5 kHz, digitized them at 10 kHz, and stored them on a microcomputer equipped with an AD converter (Digidata 1440A, Axon Instruments). The intracellular solution in the patch pipette for AP and K+ currents recordings contained (in mmol/L) the following: 10 NaCl; 122 KCl; 1 MgCl2; 1 EGTA; and 10 HEPES. Furthermore, the pH was adjusted at 7.3 with KOH. The bath solution contained (in mmol/L) the following: 154 NaCl; 5.6 KCl; 2 CaCl2; 1 MgCl2; 8 Glucose; and 10 HEPES. The pH was adjusted at 7.4 with 1N NaOH.

Calcium Handling in hiPSC-CMs

To measure spontaneous calcium (Ca2+) transients, we incubated hiPSC-CMs with 3 M of Fluo-4 AM (Molecular Probes, Em/Ex: 494/506 nm) in a classic Tyrode solution (154 mM NaCl; 5.6 mM KCl; 2 mM CaCl2; 1 mM MgCl2; 8 mM Glucose; 10 mM HEPES; pH 7.4). We recorded fluorescence images using confocal microscopy (Zeiss LSM 810) in line-scan mode (i.e., x-t mode, 1.24 ms per line; 512 pixels x 5000 lines). To enable comparisons between cells, changes in the Fluo-4 fluorescence signal (F) were normalized by basal fluorescence (F0). We extracted all data using AIM 4.2 (Zeiss, Jena, Germany). We extracted maximal amplitudes and event frequencies and analyzed them from raw data through an inhouse-developed algorithm implemented in Python.

Quantitative Reverse Transcription PCR (RT-qPCR)

Total RNA was isolated from hiPSC-CMs at 60 days using Direct-Zol RNA Miniprep, including DNAse treatment following the protocol (Zymo Research, Irvine, USA, ref: R2051). We performed reverse transcription of 500 ng total RNA into cDNA using the Transcriptor Universal cDNA Master kit (Roche, Basel, Switzerland, ref: 05893151001), following the manufacturer's protocols. We carried out qPCR using LightCycler 480 SYBR Green I Master (Roche, ref: 04887392001), executed in quintuplicate. We used Ribosomal Protein Lateral Stalk Subunit P0 (RPLPO) as the internal control, and we normalized the mRNA levels for each sample. The primers for the interest genes are listed in Table 1.

Contraction of hiPSC-CMs'

We investigated the contraction of hiPSC-CMs in monolayer six-well dishes placed on the stage of an inverted microscope with a 10_ objective and an incubation chamber (37 °C, 5% CO2). We acquired videos at 30 frames per second and performed analyses using custommade Matlab scripts. We considered several contractile parameters as follows: the beat rate; the contraction and relaxation time; the amplitude of the contraction and area under the curve (AUC); and the summation of the contraction and the relaxation time. We also studied the asynchronous time, which is the time-fraction of each video area spent in asynchrony, and described the aberrant contractile events.

Data Analysis and Statistics

We analyzed the electrophysiological results using Clampfit (pCLAMP v10.0; Molecular Devices). We tested normality (Agostino test) to determine whether data followed a normal distribution. We expressed data with the median and a 95% confidence interval. We used the Kruskal-Wallis test to evaluate statistical differences and the stars * correspond to the difference from the control. We considered the differences significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****).

Results

Aldactone Decreases Arrhythmic Events in an ACM Patient

We prescribed SP (50 mg/day) to an ACM patient harboring a desmoplakin (DSP) variant. Figure 1 shows ECGs and the number of premature ventricular beats (PVBs) before and after this prescription. We measured the QTc durations initially at 406 milliseconds (ms), and the patient experienced a significant number of PVBs (1.7%; Figure 1,panel A). After seven days of SP therapy, the QTc interval increased to 436 ms and the number of PVBs decreased to 0.8% (Figure 1, panel B). Based on the available literature, this finding stimulated our investigation into SP impact at the cardiomyocyte level. To test the hypothesis of a direct effect of aldosterone antagonists in ACM patients experiencing arrhythmias, we used hiPSC-CMs obtained from an ACM patient affected by a missense variant (c.394C>T) in the DSC2 gene encoding desmocollin 2 (DSC2-hiPSC-CMs) [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF][START_REF] Reisqs | The PPAR pathway determines electrophysiological remodelling and arrhythmia risks in DSC2 arrhythmogenic cardiomyopathy[END_REF].

SP and CA Normalize the AP and Reduce Electrical Instability in ACM-DSC2-hiPSC-CMs

We examined the cellular electrical activity using the patch-clamp technique. Spontaneous APs in the control hiPSC-CMs, ACM-DSC2-hiPSC-CMs, and after one-hour incubation of ACM-DSC2-hiPSC-CMs in the presence of SP (20 M) and CA (10 nM) were recorded. We observed a difference in the firing of spontaneous APs with an increase in the frequency of APs per time unit between the control hiPSC-CMs (0.33+/-0.06 Hz) and the ACM-DSC2-hiPSC-CMs (0.51 +/-0.07 Hz) (Figure 2A,B). AP duration (APD) at 90% of repolarization (APD90) was shorter in the ACM-DSC2-hiPSC-CMs (224 +/-32 ms) than in the control hiPSC-CMs (432 +/-43 ms) (Figure 2C) in line with previous results [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF][START_REF] Reisqs | The PPAR pathway determines electrophysiological remodelling and arrhythmia risks in DSC2 arrhythmogenic cardiomyopathy[END_REF]. These alterations were similarly normalized following the acute application of SP or CA. Strikingly, we observed no difference in spontaneous AP frequency between the control hiPSC-CMs (0.33 +/-0.06 Hz) and the ACM-DSC2-hiPSC-CMs in the presence of SP (0.30 +/-0.03 Hz) or CA (0.40 +/-0.05 Hz) (Figure 2A,B). The APD normalized after the incubation of SP and CA in ACM-DSC2-hiPSC-CMs (471 +/-31 ms and 360 +/-29 ms for SP and CA, respectively, vs. 432 +/-43 ms for the control cells). We also measured the APD90 after pacing at 1 Hz and observed similar results (Figure S1).

The percentage of cells with aberrant electrical events, similar to early or delayed-after depolarization (EAD, DAD), was higher in the ACM-DSC2-hiPSC-CMs than in the control hiPSC-CMs (47% vs. 23%, respectively) (Figure 2D). Again, SP and CA attenuated the electrical instability of the ACM-DSC2-hiPSC-CMs. The number of ACM-DSC2-hiPSC CMs presenting ectopic electrical events decreased to 30% and 27% with exposure to, respectively, SP and CA (Figure 2D).

We assessed the direct effect of CA and SP on the global outward K+ current using the patchclamp technique. ACM-DSC2-hiPSC-CMs exhibited a larger K+ current than the control hiPSC-CMs (Figure 3). Acute exposure of ACM-DSC2-hiPSC-CMs to SP or CA decreased the K+ currents. The K+ currents were normalized for voltages ranging between -40 mV and 0 mV on one hand and further reduced between 0 and +40 mV with an amplitude lower than that of the control hiPSC-CMs (Figure 3A-E). For example, at +40 mV, the K+ currents were lower in ACM-DSC2-hiPSC-CMs in the presence of SP and CA (0.9 +/-0.1 pA/pF and 1.2+/-0.1 pA/pF, respectively) than in the control hiPSCCMs (2.3+/-0.3 pA/pF) (Figure 3E). RT-qPCR analysis of the K+ channels potentially involved suggested the presence of hERG and KCNQ1 proteins in hiPSC-CMs and unraveled an increase in their expression in ACM-DSC2-hiPSC-CMs compared to the control hiPSC-CMs (Figure 3F).

SP, CA, and Eplerenone Normalize Ca2+ Cycling in ACM-DSC2-hiPSC-CMs

We investigated Ca2+ cycling with the fluorescent Ca2+ dye fluo-4 AM. We observed distinct Ca2+ transient waveforms in the control hiPSC-CMs and the ACM-DSC2-hiPSCCMs. The amplitude was reduced in the ACM-DSC2-hiPSC-CMs compared to the control hiPSC-CMs (1.7 +/-0.1 F/Fo vs. 2.8 +/-0.1 F/Fo, respectively). The area under the curve (AUC) followed a similar trend (2.9 +/-0.2 x 10 6 A.U. vs. 4.0 +/-0.6 x 10 6 A.U.) (Figure 4A-F). Differences between the ACM-DSC2-hiPSC-CMs and the control hiPSCCMs were abolished following acute exposure of ACM-DSC2-hiPSC-CMs to SP (amplitude: 2.7+/-0.1 F/Fo; AUC: 3.5 +/-0.2 x10 6 A.U.) and CA (amplitude: 2.5 +/-0.1 F/Fo; AUC 3.3 +/-0.3 x 10 6 A.U.) (Figure 4E,F), which both normalized these parameters. The number o cells presenting aberrant Ca 2+ events was also higher in the ACM-DSC2-hiPSC-CMs than in control hiPSC-CMs (41% vs. 19%, respectively) (Figure 4G). Again, SP and CA normalized the occurrence of these Ca 2+ events (22% and 20%, respectively) (Figure 4G). Interestingly, these two compounds prevent microscopic aberrant calcium events, i.e., Ca2+ sparks (Figure 5). Of note, we tested eplerenone (500 nM), another aldosterone inhibitor specific to mineralocorticoid receptors, and we found similar results (Figure S2).

Effect of CA on Contractile Properties in ACM-DSC2-hiPSC-CMs

We assessed the contractile function of hiPSC-CMs from the cell monolayer after 60 days of differentiation using video-edge capture [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. The contractile activity was disturbed with an increase in the asynchrony rate of ACM-DSC2-hiPSC-CMs compared to the control hiPSC-CMs (Figure 6). After one hour of incubation with CA, the beating rates and the amplitude of contraction were normalized compared to the control hiPSC-CMs (Figure 6A,B). Various parameters, such as contraction, relaxation duration, and the AUC (representing all contractile parameters), were not significantly affected by CA, despite a trend in normalization, for example, for relaxation duration and AUC (Figure 6C-E). CA did not change the asynchronous rate (Figure 6F). These results show that CA was less successful in rescuing contractile defects than the pro-arrhythmogenic electrical and Ca2+ handling abnormalities in ACM-DSC2-hiPSC-CMs.

Discussion

This study documents, for the first time, the acute electrophysiological effects of the prototypic MR antagonist SP and its metabolite CA on cardiomyocytes derived from an ACM patient. Firstly, we produced a model of ACM using human-induced pluripotent stem cells, which describe the hallmark of this pathology. In fact, previous studies have suggested that the origin of arrhythmia in ACM is not exclusively due to morphological remodeling [START_REF] El-Battrawy | Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Cerrone | Sodium current deficit and arrhythmogenesis in a murine model of plakophilin-2 haploinsufficiency[END_REF].

The myogenic origin of electrical instability was recently suggested in ACM-DSC2-derived hiPSC-CMs with alterations of both Na+ and K+ currents, as well as Ca2+ dynamics [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF][START_REF] Reisqs | The PPAR pathway determines electrophysiological remodelling and arrhythmia risks in DSC2 arrhythmogenic cardiomyopathy[END_REF]. In this model, profound electrophysiological remodeling with a critical APD shortening reflects a large increase in repolarizing K+ currents. This APD shortening strengthens the concept that ACM involves a disorder of repolarization and fits with our previous observations of short QT intervals in ACM patients [START_REF] Chevalier | Short QT interval as a harbinger of an arrhythmogenic cardiomyopathy[END_REF]. hiPSCs allow us to discover novel potential treatments to prevent arrhythmias in cardiac pathology. The RALES (Randomized Aldactone Evaluation Study) showed that SP added to ACE inhibitor therapy reduces the risk of sudden cardiac death (SCD) in patients with severe heart failure and a reduced leftventricular ejection fraction [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF][START_REF] Weber | Aldosterone and spironolactone in heart failure[END_REF]. The effect of SP in the RALES is based on cortisoloccupied (and activated) MR in stressed cardiomyocytes [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF]. SP antagonizes the effect of aldosterone and can thereby reduce fibrosis and improve left-ventricular (LV) function. To our knowledge, SP has never been tested in ACM patients. ACM is highly arrhythmogenic and a significant cause of SCD in the young, particularly in athletes [START_REF] Asimaki | Arrhythmogenic Cardiomyopathy-New Insights into Disease Mechanisms and Drug Discovery[END_REF]. We have now established the acute favorable direct effects of SP, CA, and eplerenone on the cellular electrical activity and Ca2+ cycling in ACM-DSC2-hiPSC-CMs. SP and CA normalized AP duration and suppressed abnormal and irregular Ca2+ events, which are expected to decrease pro-arrhythmogenic risks of myogenic origin in ACM disease, independently of potentially favorable long-term effects on cardiac structural remodeling during disease progression.

A key result of our present study is that SP and CA acutely inhibited the global outward K+ current which, as an immediate consequence, normalized both the short APD and the firing of spontaneous APs in ACM-DSC2-derived hiPSC-CMs. In the human myocardium, the APD is controlled by several outward K+ channels, notably IKr (Kv11.1 protein encoded by the KCNH2 gene) and IKs (Kv7.1 protein encoded by the KCNQ1 gene), which we found to be overexpressed, in line with AP shortening, in our ACM-DSC2-derived hiPSC-CMs. These channels are important determinants of AP repolarization in the human ventricle and pacemaking activity [START_REF] Delpón | angiotensin II antagonists and spironolactone and their modulation of cardiac repolarization[END_REF][START_REF] Vandenberg | (+) channels: Structure, function, and clinical significance[END_REF]. Of major importance is that the effects reported here are not attributable to aldosterone receptor antagonism as suggested before for HERG currents [START_REF] Caballero | Spironolactone and its main metabolite, canrenoic acid, block human ether-a-go-go-related gene channels[END_REF][START_REF] Gómez | Spironolactone and its main metabolite canrenoic acid block hKv1.5, Kv4.3 and Kv7.1+minK channels: Spironolactone and canrenoic acid on K + channels[END_REF]. Indeed, SP and its metabolite CA can directly block the HERG channels expressed in stably transfected Chinese hamster ovary cells, i.e., in the absence of aldosterone, in addition to suppressing the native IKr in single guinea-pig ventricular myocytes [START_REF] Caballero | Spironolactone and its main metabolite, canrenoic acid, block human ether-a-go-go-related gene channels[END_REF]. Of note, SP and CA can target these channels in a voltage-and frequency-independent manner, preferentially in the closed and open states, according to the modulated receptor hypothesis [START_REF] Caballero | Spironolactone and its main metabolite, canrenoic acid, block human ether-a-go-go-related gene channels[END_REF][START_REF] Delpón | angiotensin II antagonists and spironolactone and their modulation of cardiac repolarization[END_REF][START_REF] Tamargo | Pharmacology of cardiac potassium channels[END_REF]. This aligns with our observed effects of K+ current normalization between 40 mV and 0 mV, coinciding with channel activation, and further inhibition above 0 mV (i.e., when channels are fully activated) in the ACMDSC2-derived hiPSC-CMs. We previously characterized the presence of the IKr current in our cellular model of ACM by a specific blocker of this current E4031 [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF][START_REF] Ma | High purity human-induced pluripotent stem cell-derived cardiomyocytes: Electrophysiological properties of action potentials and ionic currents[END_REF]. We found prolonging effects on the APD [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF] very similar to the prolonging effect of SP shown here. We did not investigate the effect of SP on the Ito current because hiPSC-CMs do not express the protein responsible for this current [START_REF] Honda | Electrophysiological characterization of cardiomyocytes derived from human induced pluripotent stem cells[END_REF]. However, a blocking effect has previously been reported in mice [START_REF] Gómez | Spironolactone and its main metabolite canrenoic acid block hKv1.5, Kv4.3 and Kv7.1+minK channels: Spironolactone and canrenoic acid on K + channels[END_REF], which could contribute to the effect of SP in patients These direct effects, modulated by voltage and pacing rate, may beneficially modify vulnerable parameters, such as the normalization of AP duration, to stop or prevent arrhythmias [START_REF] Carmeliet | Antiarrhythmic drugs and cardiac ion channels: Mechanisms of action[END_REF].

Abnormalities of Ca2+ handling and enhanced pro-arrhythmogenic microscopic and macroscopic irregular or ectopic Ca2+ events are known to increase cellular excitability in a variety of cardiac diseases [START_REF] Lehnart | Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias[END_REF]. These trigger DADs and spontaneous APs and increase the occurrence of EADs as well, increasing cellular excitability [START_REF] Laitinen | Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia[END_REF][START_REF] Roux-Buisson | Prevalence and significance of rare RYR2 variants in arrhythmogenic right ventricular cardiomyopathy/dysplasia: Results of a systematic screening[END_REF][START_REF] Ter Keurs | Calcium and arrhythmogenesis[END_REF]. Another important finding of our results is that SP, CA, and eplerenone normalized Ca2+ handling and limited the occurrence of EADs and DADs in ACM-DSC2-hiPSC-CMs. This result may be in line with a study showing that the main metabolite of SP, CA, decreased the beat rate by blocking the L-type calcium current in rabbit cardiomyocytes [START_REF] Alexandre | Electrophysiological and Antiarrhythmic Properties of Potassium Canrenoate During Myocardial Ischemia-Reperfusion[END_REF][START_REF] Costa | The negative inotropic action of canrenone is mediated by L-type calcium current blockade and reduced intracellular calcium transients: Negative inotropic action of canrenone[END_REF]. Our finding of an acute, direct effect of SP on Ca2+ events may also be reminiscent of a recent report showing the enhancing effect of short-term treatment with aldosterone on Ca2+ spark frequency in vascular myocytes from mesenteric arteries [START_REF] Salazar-Enciso | Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca2+ Pump Upregulation Counterbalances Cav1.2-Mediated Ca2+ Influx in Mesenteric Arteries[END_REF]. Because SP had effects on targets other than aldosterone receptors, proteins involved in Ca2+ homeostasis may be implicated. The ryanodine receptor (RYR) is a likely candidate because Ca2+ sparks reveal the activity of this receptor, and this hypothesis warrants further investigation. Interestingly, aldosterone has multiple cardiotoxic effects, including oxidation of CaMKII [START_REF] He | Oxidation of CaMKII determines the cardiotoxic effects of aldosterone[END_REF], which is expected to promote leakage through RyR2 with an increased occurrence of Ca2+ sparks and pro-arrhythmogenic Ca2+ waves [START_REF] Ai | Ca2+/calmodulindependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure[END_REF][START_REF] Uchinoumi | CaMKII-dependent phosphorylation of RyR2 promotes targetable pathological RyR2 conformational shift[END_REF].

Over-activation of the RAA system promotes cardiac structural remodeling (dilatation, fibrosis, and hypertrophy), which impairs impulse propagation and promotes reentrant arrhythmias [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF][START_REF] Delpón | angiotensin II antagonists and spironolactone and their modulation of cardiac repolarization[END_REF][START_REF] Matsumura | Role of aldosterone in left ventricular hypertrophy in hypertension[END_REF]. The use of an aldosterone antagonist, such as SP, combined with an ACE inhibitor thus allows a dual blockade of the RAA [START_REF] Sayer | The renin-angiotensin-aldosterone system and heart failure[END_REF]. SP slows the development of ventricular electrophysiological remodeling due to myocardial interstitial fibrosis and inflammation. It lowers arrhythmia inducibility in systolic heart failure. It is unclear whether SP or CA can potentially delay cardiac remodeling and slow the progression of the disease in ACM but their direct effects on K+ currents and Ca2+ cycling have excellent potential to prevent arrhythmias in ACM patients. Consistently, an ACM patient given SP for 2 weeks experienced fewer VPBs. This patient harbored a mutation of DSP, which may limit the conclusions of our study. Furthermore, this patient does not exhibit a short QT interval, as previously described by Chevalier et al. in 2021, as this is only visible at low heart rates [START_REF] Chevalier | Short QT interval as a harbinger of an arrhythmogenic cardiomyopathy[END_REF]. Unfortunately, we did not obtain an ECG recording from this patient at a low heart rate. However, in a group of ACM patients with different desmosomal mutations, we showed a similar ECG phenotype with a QTc shortening [START_REF] Moreau | Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy[END_REF]. Other studies in ACM-hiPSC-CMs with different desmosomal mutations also described similar cellular electrophysiological remodeling [START_REF] Caspi | Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells[END_REF][START_REF] El-Battrawy | Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy[END_REF][START_REF] Ma | Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy[END_REF]. Altogether, these results strengthen the use of SP as an antiarrhythmic in ACM patients, irrespective of the underlying mutation, which is shown in Figure 7. A new randomized clinical study comparing SP vs. placebo in ACM patients is ongoing (BRAVE) [START_REF] Morel | Blockade of the renin-angiotensinaldosterone system in patients with arrhythmogenic right ventricular dysplasia: A doubleblind, multicenter, prospective, randomized, genotype-driven study (BRAVE study)[END_REF]. SP could provide a new treatment to reduce electrical complications in patients suffering from ACM, a devastating disease for which there is no specific therapy. The results of the randomized BRAVE study are eagerly awaited.

Of note, in our investigations of contractile activity, CA failed to reduce the rate of asynchrony, which is the time-fraction of each video area spent in asynchrony. There is a gap between the beneficial effect observed in patients and at the cellular level and in the monolayer of several cells, despite a tendency to normalize certain parameters of the contraction. A possible explanation is that the immaturity of hiPSC-CMs would mask the beneficial effect of the drugs at the monolayer level. Moreover, we derived ACM-hiPSCCMs from one patient with a DSC2 mutation. It would have been interesting to extend our research with other hiPSCs carrying another mutation.

Figure 1 .

 1 Figure 1. ECG after prescription of Aldactone in one ACM patient.

  (A) Example of ECG and PVB recording before Aldactone and (B) after Aldactone in a mutated desmoplakin (DSP) patient.

Figure 2 .

 2 Figure 2. Effect of Canrenoic Acid and Spironolactone on the Action Potential parameters of DSC2 hiPSC-CM.

Figure 3 .

 3 Figure 3. Effect of Canrenoic Acid on Potassium Channels.

Figure 4 .

 4 Figure 4. Effect of Canrenoic Acid on Calcium handling. Examples of spontaneous Ca2+ transient recorded in control

Figure 5 .

 5 Figure 5. Effect of Canrenoic acid and Spironolactone on calcium sparks. Examples of spontaneous Ca2+ transient recorded showing the presence of calcium sparks in control

Figure 6 .

 6 Figure 6. Effect of Canrenoic Acid on the Contractile Properties of hiPSC-CM.The contractile activity was studied by video analyses from cell monolayer in control (black) (n=386), patient (red) (n = 275) and incubated with AC (green) (n = 166): beat rate (A), amplitude (B), contraction shown with the median and 95% confidence interval. The stars * correspond to the difference from the control. ns not statically significant, * p < 0.05, *** p < 0.001 **** p < 0.0001 (Kruskal-Wallis test).

Figure 7 .

 7 Figure 7. Schematic diagram of a benefits effects of Spironolactone on ACM-hiPSC-CM. HiPSC-CMs harboring a desmosomal mutation allow one to decipher the ACM disease. The electrophysiological and calcium-handling properties disturbed in this pathology. Application of Spironolactone prevents these effects, by a normalization of Action potential duration, electrical stability and a calcium handling restored. Spironolactone can reduce the risk of ventricular arrhythmias in ACM patients.

Conclusions

In summary, our cells models, based on hiPSC-CM, suggest that spironolactone prevents electrophysiological and calcium-handling disturbances in ACM. These effects are mediated by the normalization of action potential duration by inhibiting the K+ currents and contributing to the normalization of calcium handling. In this study, we show the link between a clinical case and the use of human-induced pluripotent stem cells-derived cardiomyocytes. This present study describes a good example of using hiPSCs to modelize a disease and to find a new treatment in a clinical context.

Acknowledgments:

We thank the "Montpellier Ressources Imagerie" (MRI) platform (https://www.mri.cnrs.fr/fr/, accessed on 1 January 2020). We thank Gilles Millat for the genotyping of the DSP patient.

Funding:

A CIFRE grant from SANOFI R&D supported this work for JBR. Fond Marion Elisabeth Brancher supported the post-doctoral fellowship of AM.

Institutional Review Board Statement:

The local ethics committee approved the study protocol (DC-2008-72/DC-2019-3464). This study was conducted according to the principle of the declaration of Helsinki.

Informed Consent Statement:

Informed consents were obtained from all subjects involved in the study.

Conflicts of Interest:

The authors declare no conflict of interest.


Joris Menard

MD Andreea M Seferian

MD Emmanuelle Fleurence

MD Audrey Barzic

MD Alexandra Binoche

MD Géraldine Labouret

MD Laurianne Coutier

PhD Carole Vuillerot

MD Blaise M Bieleu

MD Marta Gomez

Garcia De

Garcia De La Banda

MD Harriet Corvol

PhD Laurent Servais

PhD Jessica Taytard
email: jessica.taytard@aphp.fr

Respiratory management of spinal muscular atrophy type 1 patients treated with Nusinersen

Keywords: Spinal Muscular Atrophy type 1, Respiratory management, Nocturnal gas exchange, Non Invasive Ventilation, Nusinersen, Children

Introduction

Spinal muscular atrophy (SMA) is a rare genetic neuromuscular disease resulting from an autosomal recessive mutation in the Survival Motor Neuron 1 gene (SMN1) [START_REF] Kolb | Spinal muscular atrophy: a timely review[END_REF] . With an incidence of approximately 1 in 10 000 newborns and a carrier frequency of 1:40 in the Caucasian population, it is the most common genetic cause of death in infancy [START_REF] Sugarman | Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens[END_REF][START_REF] Finkel | Spinal Muscular Atrophy Type I: Is It Ethical to Standardize Supportive Care Intervention in Clinical Trials?[END_REF][START_REF] Kariyawasam | New and developing therapies in spinal muscular atrophy[END_REF] .

SMA is characterized by progressive muscle atrophy and proximal muscle weakness causing chronic respiratory insufficiency [START_REF] Farrar | The Genetics of Spinal Muscular Atrophy: Progress and Challenges[END_REF][START_REF] Kolb | Natural history of infantile-onset spinal muscular atrophy[END_REF] . Clinical presentation varies according to the functional status -illustrated by the highest motor milestone achieved -and age at onset [START_REF] Munsat | International SMA consortium meeting[END_REF][START_REF] Zerres | Natural history in proximal spinal muscular atrophy. Clinical analysis of 445 patients and suggestions for a modification of existing classifications[END_REF] . Thus, SMA is clinically classified into five subtypes (SMA types 0-4). SMA type 1 is the most frequent subtype, affecting approximately 58% of all SMA patients [START_REF] Talbot | The clinical landscape for SMA in a new therapeutic era[END_REF][START_REF] Lally | Indirect estimation of the prevalence of spinal muscular atrophy Type I, II, and III in the United States[END_REF] . These children present with severe hypotonia and muscle weakness, usually diagnosed during the first 6 months of life. Natural history studies have described a death rate of 68% in the first 2 years of life and 82% before the age of 4 years old without treatment [START_REF] Zerres | Natural history in proximal spinal muscular atrophy. Clinical analysis of 445 patients and suggestions for a modification of existing classifications[END_REF][START_REF] Munsat | Spinal muscular atrophy. 32nd ENMC International Workshop[END_REF] .

SMN1 gene is located on chromosome 5q [START_REF] Lefebvre | Identification and characterization of a spinal muscular atrophydetermining gene[END_REF] . Its mutation causes insufficient SMN protein production. In humans a second genenamed SMN2 -can also lead to the production of SMN protein but in insufficient quantities to compensate for the absence in SMN1 and maintain muscle function, due to alternative splicing of SMN2 mRNA [START_REF] Finkel | Spinal Muscular Atrophy Type I: Is It Ethical to Standardize Supportive Care Intervention in Clinical Trials?[END_REF][START_REF] Lorson | A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy[END_REF] . Copy number of SMN2 gene is correlated with disease severity [START_REF] Feldkötter | Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy[END_REF][START_REF] Butchbach | Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases[END_REF] . Children with SMA type 1 usually present with twosometimes three -copies of SMN2, those with three copies being usually less progressive or with later onset (type 1c) than those with two (type 1a or 1b) [START_REF] Feldkötter | Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy[END_REF][START_REF] Harada | Correlation between SMN2 copy number and clinical phenotype of spinal muscular atrophy: three SMN2 copies fail to rescue some patients from the disease severity[END_REF] .

The emergence of new disease-modifying treatments in the last decade has dramatically changed SMA natural history, in particular in SMA1 children treated at an early stage of the disease [START_REF] Baranello | Risdiplam in Type 1 Spinal Muscular Atrophy[END_REF][START_REF] Finkel | Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy[END_REF][START_REF] Mendell | Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy[END_REF][START_REF] Ramdas | New treatments in spinal muscular atrophy: an overview of currently available data[END_REF] . Nusinersen (Spinraza®), an antisense oligonucleotide administrated intrathecally and designed to increase the expression of SMN2 protein, was the first drug to be approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in 2017 and 2016. It is given in repeated doses, starting by four close doses in 2 months and maintenance doses thereafter every four months. The phase 3 efficacy and safety trial of Nusinersen showed a significant motor-milestone response and higher survival in treated patients [START_REF] Finkel | Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy[END_REF] . Respiratory evolution, however, remains less impressive, with 31% of treated patients on continuous respiratory support by 13 months of age, compared to 48% in the control group [START_REF] Finkel | Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy[END_REF] .

Along with these new therapies, the standards of care for SMA patients, and in particular in type 1 patients, have shifted from palliative to proactive. This is especially true for respiratory care, which now includes respiratory physiotherapy for airway clearance and, for most patients, nocturnal ventilatory support using Non-Invasive Ventilation (NIV) [START_REF] Finkel | Diagnosis and management of spinal muscular atrophy: Part 2: Pulmonary and acute care; medications, supplements and immunizations; other organ systems; and ethics[END_REF] . For most neuromuscular diseases, the decision to start NIV is based on nocturnal gas exchange recordings and the presence of nocturnal hypoventilation [START_REF] Hull | British Thoracic Society guideline for respiratory management of children with neuromuscular weakness[END_REF] , however, no study has evaluated the nocturnal gas exchange in SMA type 1 children treated with Nusinersen. There is currently a need for diseasecentered guidelines on when to start NIV, as recommendations used in other neuromuscular diseases do not seem to be appropriate for these patients.

In this study, we report the respiratory management and evolution of SMA type 1 children treated with Nusinersen, with the aim of assessing patients' nocturnal gas exchanges prior to NIV initiation and their subsequent clinical evolution in order to determine if capnia is a good criterion to decide when to introduce it.

Patients and Methods

Our retrospective multicenter study included SMA type 1 patients for whom treatment with Nusinersen was initiated between 2016 and 2018. We considered two centers where Nusinersen injections were first started (Paris, Lille) for patients who were afterwards followed in 8 French reference centers for neuromuscular diseases. All patients had a genetically confirmed diagnosis of SMA and onset before the age of 6 months, with no sitting position acquired before Nusinersen treatment.

The study was approved by the institutional Review Board of the French learned society for respiratory medicine -Société de Pneumologie de Langue Française-. Parental/legal representative consent was obtained prior to inclusion in the study.

Data collection

Data are part of the French Reference Center for Rare Lung Diseases (Respirare®) database. The database and data collection from the RespiRare® database have been approved by French national data protection authorities (CNIL n°908,324 and CCTIRS n°08.015bis).

At inclusion, the following data were collected and analyzed: sex, number of SMN2 copies, age at

Statistical analysis

Statistical analyses were performed using the GraphPadPrism® software. Categorical variables are expressed as numbers and percentages. Data are presented as median [IQR], minimum and maximum. The difference in the number of hospital admissions during treatment was tested using the Student t-test. Value of p < 0.05 was considered statistically significant.

Results

Between September 2016 and October 2018, 18 SMA type 1 patients were treated with Nusinersen in these two centers. One patient was excluded from the analysis because treatment was rapidly discontinued. General characteristics of the seventeen patients and their motor functional evaluations at baseline and at the end of the study are included in Table 1.

Median [IQR] length of follow-up was 38 [24;44] months, with a maximum of 64 and a minimum of 2 months. Only one patient died after a 2 months follow-up, at age 4.5 months.

NIV initiation

In total, thirteen (76%) patients were started on NIV. Median [IQR] age at NIV initiation was 12 [9;18] months. For two patients NIV was started before Nusinersen (five and six months respectively). Median [IQR] time between the first Nusinersen injection and NIV initiation was 4 [3;9] months, with NIV initiated more than one year after the first injection in only two patients (14 and 15 months after starting the treatment, respectively).

NIV indications

Eleven (65%) patients were started on NIV after requiring repeated hospitalizations and ICU admissions. In one patient NIV was started because of important airway secretions and, in another one, NIV was started with a proactive/preventive approach without any respiratory decompensation or sign of hypoventilation (at the age of six months, three months after Nusinersen was started). Nine (69%) patients had increased respiratory rate before NIV was initiated, with a mean (range) of 40 (25;80). Ten (77%) patients presented with paradoxical breathing and 6 (46%) with signs of increased work of breathing (i.e. subcostal or intercostal recession). No patient was discontinued from the NIV once initiated, four patients were not on ventilatory support at the end of the study.

Respiratory explorations before NIV

Blood gases were performed before NIV initiation in eight patients, and they did not show hypercapnia (maximum PaCO 2 of 42 mmHg). Nocturnal gas exchange recordings were performed prior to NIV initiation in eight patients, without showing markers of hypoventilation, nor spending time with PtcCO 2 > 50 mmHg during sleep. Respiratory explorations and their results before NIV are included in Table 2. All nocturnal gas exchange recording were performed in a hospital setting.

A chest X-ray was performed in 11/13 patients prior to ventilation initiation. It was either normal (n=4) or showed atelectasis (n=5) and/or signs of acute lower respiratory tract infection with images of lobar consolidation and alveolar pattern (n=4).

Thoracic circumference/Head circumference ratio

TC/HC at inclusion was available in five (29%) patients, with an initial median [IQR] ratio of 1.0 [0.91;1.02]. TC/HC at the end of follow-up was available in nine (53%) patients, with a median

[IQR] ratio of 1.03 [1.02;1.10]. For all these patients, TC/HC was stable or increased during follow-up.

Discussion

In this study we report results of nocturnal gas exchange prior to NIV initiation in SMA type 1 patients treated with Nusinersen. We observed that patients do not present with nocturnal hypoventilation before respiratory decompensations. More importantly, this study shows that a delay in ventilatory support initiation might result in pulmonary atelectasis and other respiratory complications that will negatively impact the patients' respiratory evolution.

Seventeen SMA type 1 patients treated with Nusinersen were included in this retrospective multicenter study, focusing on the respiratory management and outcome. Despite an increasing number of studies on respiratory outcomes and evolution since the development of diseasemodifying treatments, information on respiratory explorations and nocturnal gas exchange results before NIV initiation are very scarce. Here, we highlight that most children were started on NIV because of repeated respiratory exacerbations requiring hospitalizations, but not because of hypoventilation.

In our study, 13 (76%) children were started on NIV following repeated hospitalizations for respiratory failure (including in ICU) and one due to important airway secretions. This is in line with Chen et al. who reported that in three out of four newly-diagnosed SMA type 1 patients started on NIV, treatment initiation was decided after repeated hospitalizations. The median age at ventilatory support initiation was 12 months old in our study, similar to that reported by Chen et al. and Lavie et al. (13.8 and 13.5 months, respectively) [START_REF] Chen | Real-world respiratory and bulbar comorbidities of SMA type 1 children treated with nusinersen: 2-Year single centre Australian experience[END_REF][START_REF] Lavie | Nusinersen for spinal muscular atrophy type 1: Real-world respiratory experience[END_REF] . Unfortunately these studies do not provide information on nocturnal gas exchange prior to NIV [START_REF] Chen | Real-world respiratory and bulbar comorbidities of SMA type 1 children treated with nusinersen: 2-Year single centre Australian experience[END_REF] .

Nocturnal gas exchange recordings were performed before NIV initiation in eight out of 13 (61%) children, none of whom presented with hypoventilation prior to hospitalizations and NIV initiation, as shown by normal PtcCO 2 values. This is an important finding as none of the studies describing respiratory management and evolution in SMA type 1 patients treated with Nusinersen have provided results of nocturnal gas exchange recordings. Considering the recent shift in the standard of care for patients, namely the recommendation for a more proactive respiratory management, there is a need for new guidelines on when to consider ventilatory support in SMA type 1 patients [START_REF] Finkel | Diagnosis and management of spinal muscular atrophy: Part 2: Pulmonary and acute care; medications, supplements and immunizations; other organ systems; and ethics[END_REF] . Our results would encourage clinicians not to wait for signs of hypoventilation to initiate NIV, as opposed to recommendations for other neuromuscular diseases [START_REF] Hull | British Thoracic Society guideline for respiratory management of children with neuromuscular weakness[END_REF] . Indeed it is likely, as we show here, that patients will present with pulmonary atelectasis and repeated respiratory infections before showing signs of nocturnal hypoventilation. The two patients for whom the frequency of hospitalizations increased after one year of Nusinersen were those for whom NIV was started later (at 18 and 26 months of age). This underlines an increased risk of respiratory morbidity in case of postponing NIV, and favors a more proactive management.

Nocturnal recordings should be part of the decision-making but only as one of many tools including (but not limited to) clinical examination looking for paradoxical breathing, TC/HC ratio measurement, nutritional status assessment, and the necessity for hospitalizations secondary to respiratory distress. In five patients for whom we had TC/HC measures, NIV was started when the ratio had fallen under one. The ratio increased after treatment initiation for all patients. This suggests a favorable respiratory evolution under Nusinersen and/or NIV treatment. Ropars et al.

have previously suggested using TC/HC ratio as a specific respiratory outcome measure, as they found that SMA type 1 patients with a TC/HC ratio < 0.85 died within 3 months [START_REF] Ropars | Thoracic circumference: A new outcome measure in spinal muscular atrophy type 1?[END_REF] . We suggest using this measure to guide the clinician on when to start NIV and provide an indication treatment efficiency by means of chest wall growth. Respiratory scoring based on clinical observations could also be of great help to decide on the most appropriate respiratory management method. As an example, the Great Ormond Street Respiratory (GSR) score includes main clinical criteria (acute and recurrent chest infections, increased work of breathing, chronic or acute respiratory failure) and supportive clinical criteria (poor weight gain, chest wall deformity) to guide the decision-making [START_REF] Edel | A new respiratory scoring system for evaluation of respiratory outcomes in children with spinal muscular atrophy type1 (SMA1) on SMN enhancing drugs[END_REF] .

Our results are in agreement with the findings of other groups who evaluated the respiratory muscles function, showing that Nusinersen improves inspiratory muscle strength and increases accessory muscles performance [START_REF] De La Banda | Assessment of respiratory muscles and motor function in children with SMA treated by nusinersen[END_REF][START_REF] Lomauro | Effect of Nusinersen on Respiratory Muscle Function in Different Subtypes of Type 1 Spinal Muscular Atrophy[END_REF] . However, these findings were reported in less severe (type 1c and type 2) patients. Chacko et al. also showed a reduction in lung function decline in older SMA type 2 and 3 patients [START_REF] Chacko | Effect of nusinersen on respiratory function in paediatric spinal muscular atrophy types 1-3[END_REF] . Objective respiratory muscle function evaluation is difficult in this population as it involves invasive tests available only in a few centers. Nighttime recordings, such as polysomnography (PSG) and nocturnal gas exchange recordings, on the contrary, are more available and easier to perform in younger children. However, our results suggest that nocturnal gas exchanges might not be good indicators of when to start NIV in SMA type 1 patients. PSG recordings could be of interest as they provide information on hypopnea, the presence of paradoxical breathing and sleep structure.

Apart from two patients who presented with a more complicated respiratory evolution, all the remaining study patients had a significant decrease in the number of hospitalizations between the first and the second year of These results are in agreement with those reported by other authors [START_REF] Chen | Real-world respiratory and bulbar comorbidities of SMA type 1 children treated with nusinersen: 2-Year single centre Australian experience[END_REF][START_REF] Chacko | Effect of nusinersen on respiratory function in paediatric spinal muscular atrophy types 1-3[END_REF] . The reduction in the hospitalization rate is probably multifactorial, resulting from not only the stabilization of lung function due to Nusinersen treatment, but also from a combination of early NIV initiation, respiratory physiotherapy with secretion management techniques and oral suctioning. This result emphasizes the importance of a proactive approach in this population.

All patients except one had regular sessions of respiratory physiotherapy, including airway secretion management techniques (assisted cough or percussions). This is in line with what has been described in other studies and with standards of care recommendations [START_REF] Lavie | Nusinersen for spinal muscular atrophy type 1: Real-world respiratory experience[END_REF][START_REF] Finkel | Diagnosis and management of spinal muscular atrophy: Part 2: Pulmonary and acute care; medications, supplements and immunizations; other organ systems; and ethics[END_REF] . Conversely, only 1/3 of our patients received RSV immunization treatment. This could be explained by the fact that the standards of care recommending RSV immunization were published after the inclusion period of our study [START_REF] Finkel | Diagnosis and management of spinal muscular atrophy: Part 2: Pulmonary and acute care; medications, supplements and immunizations; other organ systems; and ethics[END_REF] .

This study has several limitations, mainly the low number of patients or incomplete or missing information, for example in TC/HC ratio measures, because of its retrospective nature. Along with the heterogeneity in the patients' management in the 8 national reference centers, which illustrates the lack of guidelines for the respiratory care for SMA type 1 patients. The small number of patients also makes it difficult to draw recommendations.

Respiratory management of SMA type 1 patients treated post-symptomatically remains nevertheless burdensome and resource-consuming. Inversely, pre-symptomatically treated patients present with much lower needs in terms of respiratory management [START_REF] Vivo | Nusinersen initiated in infants during the presymptomatic stage of spinal muscular atrophy: Interim efficacy and safety results from the Phase 2 NURTURE study[END_REF] . The high cost and burden for families and for patients should constitute additional reasons to rapidly move towards generalized newborn screening and presymptomatic treatment [START_REF] Dangouloff | Newborn screening programs for spinal muscular atrophy worldwide: Where we stand and where to go[END_REF] .

Conclusion

The latest recommendations standards of care for SMA emphasize the fact that NIV should be started in all symptomatic SMA type 1 patients [START_REF] Finkel | Diagnosis and management of spinal muscular atrophy: Part 2: Pulmonary and acute care; medications, supplements and immunizations; other organ systems; and ethics[END_REF] . In line with these recommendations, our results are in favor of not basing the decision to start NIV on nighttime recordings only. Clinical examination, TC/HC ratio and respiratory scoring systems are essential to decide when to start NIV in these patients.

When it comes to respiratory management, the burden of treatment should always be balanced with the risk of respiratory morbidity and socioeconomic impact in case of delayed treatment [START_REF] Dangouloff | Systematic literature review of the economic burden of spinal muscular atrophy and economic evaluations of treatments[END_REF] .

Currently, too much is left to the clinicians' judgement and we are in need for additional, clear recommendations specific to SMA type 1, especially as several of these patients might not benefit from the clinical expertise of reference centers. Further studies are needed to evaluate respiratory function evolution in this population, and to apprehend the long-term quality of life of patients, families and caregivers.

  symptoms onset, at diagnosis, at first Nusinersen injection and at last visit or at death and baseline motor function evaluation score using CHOP INTEND. Data collected during follow-up include: number of Nusinersen injections, CHOP INTEND scores, respiratory evolution (assessed by the number of hospitalizations, including the ones in intensive care unit (ICU) before Nusinersen and during the first two years of treatment), and thoracic circumference/head circumference (TC/HC) ratio. Data collected on respiratory assessment were: blood gas analyses and nocturnal gas exchange recordings (mean and maximum transcutaneous PCO 2 -PtcCO 2 -and percentage of total sleep time spent with a PtcCO 2 > 50 mmHg, mean and minimum pulse oxygen saturation -SpO 2 -and percentage of total sleep time spent with a SpO 2 < 90%) before NIV initiation, number of regular sessions of respiratory physiotherapy and airway secretion management techniques, use of NIV use and patient's age at onset of ventilation .

Figure 1 .

 1 Figure 1. Hospitalizations during the first and second years of Nusinersen

Table 1 . General characteristics and CHOP-intend score evolution of the 17 patientsTable 2 . Respiratory explorations before non-invasive ventilation initiationTable 3 . Respiratory physiotherapy management at the end of follow-upTable 4 . Age at initiation of respiratory and physiotherapy management -evolution between baseline and the end of study

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Acknowledgements

We thank all the patients and families. This research did not receive any funding from public, commercial or not-for-profit agencies.

No fundings were secured for this study. Coinvestigator for the ENDEAR study group and Avexis; LS has conducted consultancy/attented board for Roche, Novartis, Biogen, Scholar Rock. He is coordinating investigator of studies funded by Biogen, Roche and Avexis; CV has conducted consultancy/attented board for Roche, Novartis, biogen, is coordinating investigator of studies funded by Biogen and Roche. JM, EF, AB, AB, GL, LC, MBB, MGGB and HC have no conflict of interest to disclose.

data available on request from the

Hospitalizations secondary to respiratory decompensations

The number of hospitalizations before and during the first and second years of treatment is shown in Figure 1. We found a significant decrease (p = 0.04) in the total number of hospital admissions between the first and second year of treatment. The number of hospitalizations decreased for all patients but two in whom it increased, from 2 to 6 (including 3 hospitalizations in ICU) in one patient, and from 0 to 5 in the other one. For these two children, NIV was started following ICU hospitalization at 18 and 26 months of age and after 14 and 15 months of treatment with Nusinersen, respectively. They were the only patients started on NIV more than one year after Nusinersen was initiated. Interestingly, the child who was started on prophylactic NIV (patient 10) did not need any hospitalization for respiratory decompensation during followup.

Respiratory management (excluding NIV)

Respiratory physiotherapy and airway secretion management techniques are described in Table 3. By the end of follow-up, all patients but four were using a secretion management device (cough assist or intra-pulmonary percussive ventilation). The evolution of respiratory physiotherapy management between baseline and follow-up is shown in Table 4.

All patients received customary immunizations according to French recommendations, and 7 (41%) received additional RSV immunization. One patient received nebulized corticosteroids, and four were started on continuous antibiotics. Hypertonic saline nebulizations were not prescribed in our population. The use of a nasofeeding tube was necessary in 9 (53%) patients at a median [IQR] age of 10 [4;15] months. At the end of follow-up, 11 (65%) patients needed a gastric feeding tube at a median [IQR] age of 16 [14;23.5] months old.