Nicolas Blanchard
email: n.blanchard@unistra.fr

Vincent Bizet
email: vbizet@unistra.fr

Nicolas Brach
email: nicolas.brach@etu.unistra.fr

Lénaïc Rummler
email: lenaic.rummler@etu.unistra.fr

Krishna P Kaliappan

Oxetanes and Oxetenes: Fused-ring Derivatives

Keywords: Fused oxetane, Fused oxetene, Spiro-oxetane, Spiro-oxetene, b-Lactone, Spiro-b-Lactone, Heterocycles, Four-Membered rings

come

Introduction

This chapter reviews developments related to fused and spiro-oxetanes, oxetenes and b-lactones from 2008 to mid-2020. Their physical properties, the methods for their synthesis, their reactivity trends and their applications to the preparation of biologically relevant compounds are discussed.

Theoretical Methods

A physical organic chemistry study of the photochemical generation of ortho-naphthoquinone methides 5 from 1 and their reactivity in aqueous solution was reported by Arumugam and Popik. 1 Naphthoxete 3 was proposed as intermediate in the formation of ortho-naphthoquinone methide by irradiation of 3-hydroxy-2-naphthalenemethanol 1. The ring opening of naphthoxete 3 is a thermal reaction, slower than the decay of the excited state of 2a. All structures were investigated at the (B3LYP/6-311++G(d,p)//B3LYP/6-31++G(d,p) and MP2/6-31++G(d,p)//FC-MP2/6-31++G(d,p) levels of theory. <Scheme 1 near here> 1,8-Peribridged naphthalenes are highly strained structures that are often unstable although a few molecules have succumbed to X-ray diffraction. Novak reported ring strain energies (RSE) of series of single atom peribridged naphthalenes (SAPN) using high level quantum chemical methods. SAPN 6 has the highest RSE of 289.7 kJ/mol. A comparison was also proposed with compounds 3, 7 and 8 possessing a fused-oxetene at different positions of a naphthalene core. 2 <Scheme 2 near here> Ground state and excited state potential energy surfaces of the photoexcitation of orthohydroxybenzaldehyde have been studied in silico by Migani. 3 A potential product is an oxetene adduct arising from a decay at a twisted-pyramidalized intersection seam, although the barrier for its formation was higher than for other decay products. Oxetenes undergo conrotatory electrocyclic ring opening as demonstrated as early as 1971. 4 In 2010, Venuvanalingam and Chattaraj reported a quantum chemical mechanical study at the B3LYP level with 6-31G(d) and 6-311+G(d,p) basis sets on this kind of perturbed pericyclic isomerizations. 5 This study proposed hardness indices based on closely lying frontier molecular orbitals that reflect much better the relative reactivity of oxetene and its analogues (phosphetene, thiete and azete). Jayaprakash has reported an ab initio molecular orbital and DFT study of the electrocyclic ring-opening of oxetene in the gas phase. 6 It was shown that this pseudo-pericyclic process was characterized by a more planar transition state and an inferior barrier of activation compared to the electrocyclic ring-opening of cyclobutene.

Ruthenium-catalyzed decarbonylative couplings of aromatic aldehydes and terminal alkynes lead to alkenes, as demonstrated by Li in 2009. 7 An intramolecular variant was also reported by Saá (using protic acid additives) 8 and the mechanism of the latter transformation was explored through quantum mechanical calculations by Li. 9 Recently, Li and Moitessier studied in depth the mechanism of this reaction through computational chemistry and isotope-labeling experiments. Zhang's fused oxetene intermediate was ruled out in favor of an aldol-type reaction of a ruthenium-vinylidene complex. 10 <Scheme 3 near here> A detailed study of the directing role of hydroxy functions in the Paternò-Büchi [2+2] photocycloaddition was reported by Abe in 2011. 11 Through a combination of experimental and theoretical studies, it was shown that cooperative hydrogen-bonding stabilization and steric interactions were crucial.

Fused oxetenes are intermediates in the Fe(III)-catalyzed cyclization of yne-acetals. 12 Yu and Li have reported a DFT investigation of the reaction mechanism using the B3LYP method and the 6-311+G** basis set, excluding [2+2] cycloaddition of yne-oxonium 13 in favor of cyclization of the vinylic carbocation 14 to the fused oxetenium 15 with an activation barrier of 17.2 kcal/mol. These alkyneoxononium reactions are discussed in Section 2.06. 6 A parametric/DFT hybrid computational method DU8+ was recently applied to the analysis of published NMR data of naturally occurring oxetanes. 13 A high rate of structure misassignment was found with more than 30 proposed structures requiring revisions, selected examples being highlighted in Section 2.06.11.

Experimental Structural Methods

Electron momentum spectroscopy

Electron momentum spectroscopy of oxetane was reported by Chen, leading to the binding energy spectra and the electron momentum distribution for the outer-valence molecular orbitals. 14,15 It was shown that the ring-puckering motion of oxetane has a dramatic influence on the electron density distribution of the highest occupied molecular orbital.

Electronic and vibrational absorption spectroscopies

Ashfold reported the use of broadband transient electronic and vibrational absorption spectroscopies for an insightful study of the Paternò-Büchi reaction between benzaldehyde and cyclohexene. 16 Quantum yields and rates constants for competing pathways were determined.

IR spectroscopy

Benzoxetone 19 was formed quite easily from 6-oxocyclohexa-2,4-dienylideneketene 18 by irradiation at 340 nm, 18 being prepared by photolysis of 17 at 315 nm. 19 can reverse to 18 upon photolysis at 315 nm. No differences were observed in the IR spectra of 18 and 19 for at least 6 photolysis cycles. 17 <Scheme 5 near here> Benzoxete chemistry has been reviewed by Meier and the reader is redirected to this excellent article for details related to their isolation and reactivity. 18 Many erroneous or questionable benzoxetes are also discussed. Other reviews were also recently published. [START_REF] Balaban | Advances in Heterocyclic Chemistry[END_REF] 2H-Benz[b]oxetes were first isolated by Tomioka and Matsushita in 1997, by irradiation of orthohydroxyphenyldiazomethane (at l>300 nm) or benzofuranone (at l>254 nm) in an argon matrix at 10 K. [START_REF] Hideo | [END_REF] These highly strained compounds (38 kcal/mol) 21 are in valence equilibrium with their orthoquinoid structures, the latter being much more favored from a thermodynamic point of view. Theoretical and experimental IR spectrum of benzoxetes were compared and found in very good agreement. Benzoxete was also detected by irradiation of a vapor deposition prepared sample of 2- hydroxymethylphenol at 254 nm and 14 K. The characteristic signal at n = 1668 cm -1 (scissoring vibration of the CH2 motif) was clearly observed. 22

X-ray crystallography

X-ray crystallographic studies on a range of fused or spiro-oxetanes and b-lactones have been reported over the period of interest to this chapter, as in the cases of the natural products hawaiinolide E, 23 hawaiinolide A and C, 24 aphanalide A and C, 25 and rubesanolide A. 26 X-ray structures of various synthetic compounds have also been reported 27 including fused oxetanes resulting from the photocycloaddition of electronically excited carbonyl to isoxazoles (e.g. 20), 28 a planar oxetane arising from the Paternò-Büchi reaction of triplet benzophenone with 4-aryl-4H-pyrans, 29 an unprecedented tricyclic 3H-oxeto [2,3,4-ij]isochromene featuring a rare oxetane acetal reported in 2017 during the course of synthetic investigations of sigillin A, an insect repellent natural product isolated from springtails. 30 Spirocyclohexene-oxetanes were prepared through catalytic enantioselective cycloaddition to a,b,b-trisubstituted nitroalkenes, and a single structure 21 was reported, allowing the assignment of absolute configuration. 31 Spiro-oxetane embedded in heterocyclic structures have been disclosed recently. 32 Structures of spiro-b-lactones prepared via the [2+2] cycloaddition of disubstituted ketenes with isatins, such as 22, were also characterized by X-ray crystallography.

Thermodynamic Aspects

Oxetanes are prone to ring-opening 34 under acidic conditions at similar rates than epoxides, 35 an experimental observation that can be rationalized by their similar ring-strain (106 kJ/mol for oxetanes vs. 112 kJ/mol for epoxides). 36,37 On the other hand, oxetanes are hydrolyzed at slower rates than epoxides under basic conditions. Theoretical studies have addressed this reduction in hydrolysis rates. [38][39][40][41][42] Strain energies and equilibrium geometries of 2,4-oxytwistane 23 were computed ab initio using the MP2(fc)/6-31G(d) theoretical model and compared with oxetane. 43 The ring-strain energies (RSEs) of spiroepoxy-b-lactone 24 and spiroepoxy-oxetane 25 have been calculated from the energies of fully-optimized structures at the MP2 + ZP/cc-pVDZ level. The stability/reactivity of spiroepoxy-b-lactone 24 has been discussed and found to not correlate with RSE. 44 Gas-phase enthalpies of formation of oxetane 26 (-80 kJ/mol) and oxetene 10 (-52.8 kJ/mol) were determined by high-level quantum chemical calculations at the G3(MP2)//B3LYP level by Taskinen in 2009. 45 Closely related results were reported by Green in 2012 using an application of a QCISD(T) quantum chemistry strategy. 46 <Scheme 7 near here>

Reactivity of Fully Conjugated Rings

No examples of this class of compounds have been found in the literature in the period covered.

Reactivity of Nonconjugated Rings

Reaction with Nucleophiles

As shown by Carreira and Roger-Evans, [47][48][49][50] oxetanes are relevant bioisosters for gem-dimethyl substituents or carbonyl groups. a-Functionalization of oxetan-3-ones can be conveniently performed using Enders' SAMP/RAMP synthetic methodology 51 as shown by Shipman. 52 Hydrazone 28 was obtained in 68% yield and could be further hydrolyzed to the fused oxetane 29 in 60% yield and 84% enantiomeric excess. 31 The reaction proceeds through trienamine organocatalysis 59 and delivers a broad range of spirocyclic oxetanes, such as 41, containing two contiguous stereogenic centers including a quaternary one. It should be pointed out that the exact mechanism is still a matter of debate, whether being a pericyclic or a stepwise process. 59,60 <Scheme 12 near here> Intramolecular [4+2] cycloaddition reactions of sydnones 44 have been reported by Harrity as an efficient tool to prepare spiro-oxetane containing fused pyrazoles such as compound 45 obtained in 93% yield. Cycloaddition precursor 44 itself is prepared in a straightforward sequence from sydnone 42 and 3-oxetanone 43. 61 <Scheme 13 near here> 4-p-Photocyclization of 2-pyrone, a process reported for the first time in 1964, leads to a valuable bicyclic b-lactone 62 whose chemistry has been reviewed early 2020 by Coote. 63 Selected examples over the 2008-2020 period demonstrate the versatility of this building block for the straightforward synthesis of relevant cyclobutenes or cyclobutanes. [64][65][66][67][68][69][70][71][72]

Elimination

Elimination of spiro-oxetanes is a facile process that has been central to the synthesis of homoallylic alcohols. Catalytic processes have also been reported, such as the tris(pentafluorophenyl)boranecatalyzed isomerization of 2,2-disubstituted oxetanes 46 or 48. Under very mild conditions (0.5 mol% of Lewis acid, room temperature, short reaction times), good yields of the desired homoallylic products 47 and 49 were obtained.

Ring Expansion

Ghosh reported the synthesis of high-affinity P2 ligand for HIV-1 protease inhibitors such as (-)-52 using a short synthetic sequence relying on the Paternò-Büchi photoadduct (±)-50. Hydrogenation of the latter followed by an acid-catalyzed transacetalization using para-toluenesulfonic acid in warm methanol delivered (±)-51 in good yield. 74 <Scheme 15 near here> 3-Oxetanone 43 has been introduced as a versatile building block for medicinal chemistry. The corresponding aminals, hemiaminals and hemithioacetals 53 were found to react with trimethylsilyl cyanide and a catalytic amount of indium(III) triflate in acetonitrile at room temperature in a smooth ring-expansion process, going through postulated intermediate 54. The functional group tolerance of this cascade reaction is quite large and morpholines, thiomorpholines and piperazines 55 were obtained in moderate to excellent yields and diastereoselective ratios.

Reduction

Photoadduct 56, arising from a Paternò-Büchi reaction between benzaldehyde and trimethylsilyloxycyclopentene, was found to undergo clean reduction to the corresponding tertiary alcohol 57 or a regioselective rearrangement to cyclohexanone 58 via an aluminum(III)-mediated pinacol type process. 75 <Scheme 17 near here> One step reduction of b-lactones 59 to oxetanes 60 has not been reported in the literature, which prompted the development of a 2-step sequence implying reduction to the diol followed by cyclization under basic conditions. 76 The first step of the process turned out to be quite difficult, leading to decomposition via retro-aldol reactions. Finally, diisobutylaluminium hydride in dichloromethane proved to be the best reducing agent after an extensive screening. Cyclization reaction of the diol could be efficiently promoted by tosyl chloride and potassium tert-butoxide in refluxing tetrahydrofuran.

N X O R 1 R 2 TMSCN In(OTf) 3 (2-7.5 mol%) MeCN 20 °C 1-48 h O CN X N R 1 TMS R 2 N X R 1 OTMS CN R 2
<Scheme 18 near here>

Cycloreversion

Cycloreversion of oxetanes has been comprehensively reviewed in 2019. 77 Decarboxylation of b-lactone 78 is a fundamental process in organic synthesis that has been exploited by Chi in 2017. Fused b-lactone 61 could be transformed into the corresponding epoxide 62 without any loss in enantiopurity, via a decarboxylation in DMF at 110 °C followed by epoxidation with m-CPBA. 79 <Scheme 19 near here>

Alkyne-Carbonyl Metathesis

Carbonyl-alkyne metathesis reactions are powerful tools to build in a straightforward manner a,b-unsaturated carbonyls through the cycloreversion of oxetenes intermediates. [80][81][82][83][84][85] As evocated before, metal-catalyzed cyclization reactions of w-alkynyl carbonyls constitute efficient transformations for the synthesis of a,b-unsaturated ketones or aldehydes. As an example, Yamamoto 86 reported the gold-catalyzed carbocyclization 87 of a series of internal ketones 63 that delivered enones 66 in good to excellent yields. The fused carbocationic oxetane 65, arising from the p-activation of the alkyne followed by cyclization of the carbonyl, is postulated as an intermediate. Yu and Li reported a Fe(III)-catalyzed cyclisation of yne-acetal 67 that proceeds via fused oxetenium 70. 12 Ring opening of the latter followed by hydrolysis of the oxocarbenium leads to a,b-unsaturated ketones 68. A dramatic solvent effect was found, and nitromethane or acetone delivered the highest yields. The alkynyl motif could be substituted by aromatic, alkyl, vinyl or alkynyl groups with the same efficiency. Intermolecular versions are also possible as exemplified by the Fe(III)-catalyzed transformation of alkyne 72 into a,b-unsaturated ketone 74.

<Scheme 21 near here> Saá reported the easy preparation of hydroazulenones 76 and 77 from the corresponding acyclic enyne acetals 75 using tetrafluoroboric acid in dichloromethane at 0 °C. 88 The scope of the transformation is quite large and the reaction is postulated to go through formation of oxonium 78, followed by alkyne-oxonium metathesis. The fused O-substituted oxetenium 79 would then undergo an electrocyclic ring-opening to the divinyl ketone 80. Subsequent Nazarov electrocyclization and proton elimination would then deliver the observed products. A dramatic influence of the starting material alkene substitution on the regioselective course of the process was found. 1a,25-Dihydroxyvitamin D3, a seco-steroid having a key role in the regulation of calcium homeostasis, has been structurally edited to include a spiro-oxetane at C3 position. [95][96][97] The latter compound 115 is conveniently obtained via a Trost palladium-catalyzed cycloisomerisation 98 Spirocyclopropyl oxetanes 121 were reported to rearrange to 3-methylenetetrahydrofurans 122 upon treatment with a catalytic amount of Zeise's dimer and 20 mol% of tricyclohexylphosphine in dichloromethane at 45 °C. 102 The mechanism of this new platinum-catalyzed rearrangement was extensively studied (including with 13 C NMR-labeling) and it was found that oxidative addition of the substrate to platinum(II) was taking place at the least substituted carbon-carbon bond of the spirocyclopropyl unit. <Scheme 32 near here> Spirocyclic oxetanes 123, obtained by photocycloaddition of N-acylisatins and bicyclopropylidene, could be ring-opened by an excess of acid, such as hydrochloric or hydrobromic acid in acetonitrile at room temperature, yielding spirocyclopropyl butenolide 124 in good yield. 103 On the other hand, gbutyrolactones 125 possessing the two spirocyclopropane units were obtained upon action of hydroiodic acid and iodine. The Paternò-Büchi [2+2] photocycloaddition of oxindoles has also been reported using a visible-light microfluidic photoreactor. 107 Optimization of photocycloadditions under flow conditions can also be conducted in an automatic fashion using self-optimizing algorithms. Rueping demonstrated that the [2+2] photocyclization of benzophenone and furan in a capillary-type continuous photo-flow reactor could indeed be optimized without human intervention. 108

CH 2 Cl 2 45 °C, 1-2 h O PtL n O L n Pt R 1 R 1 R 2 R 2 R 1 R 2 -PtL n O R 2 R 1 O 122a, 34% 122b
Paternò-Büchi [2+2] photocycloaddition proceeds via direct carbonyl excitation or photosensitization followed by alkene interception as in Scheme 35. In this regard, alkyl substituted ketones are not the most reactive partners and require more energetic irradiation and specialized glassware. However, it was demonstrated in 2019 by Schmidt that a copper(I)-norbornene complex was able to catalyze the [2+2] carbonyl-olefin photocycloaddition with a broad range of alkyl ketones. 117,118 When applied to electron-rich ketones (such as 156), this DKR strategy is followed by a spontaneous decarboxylation process that afforded cyclopentene 158. 119 This concerted asynchronous retro-[2+2] reaction was studied using quantum mechanical computations and it was found that the anti-selective process was favored based on steric interactions between the carboxylate and the a-substituent in the syn-transition state. Researchers at Merck reported an optimized synthetic route towards spiro-oxetane containing piperidines and pyrrolidines via a simple intramolecular SN2-type displacement of a primary tosylate. 128,129 The latter is prepared in situ from diol 183 using n-butyllithium and tosyl chloride; the addition of a second equivalent of n-butyllithium then delivered the desired spiro-oxetane piperidine 185 in 66% yield on a 37 g scale. Similar strategy led to compounds 186 and 187 from ethyl nipecotate and methyl 1-benzylpyrrolidine-3-carboxylate (respectively). <Scheme 49 near here> 2.06.9.6 Dehydration of Hydroxy Acids Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOPCl) proved to be a reliable and efficient reagent for the synthesis of fused b-lactones within densely functionalized scaffolds. It has been applied to the

- R 2 R 2 + N N N t Bu O R 1 Bn X - 162 163 H O Ar R 1 O CO 2 R 2 CO 2 R 2 O O H R 1 Ar CO 2 R 2 CO 2 R 2 + cat. ( 5

(±)-Spongiolactone

Fused b-lactones are obtained through an elegant procedure reported by Romo starting from aldehydic acids. 153,154 Intramolecular Nucleophile-Catalyzed Aldol Lactonization (NCAL) reactions delivers b-lactones with excellent functional group tolerance; chiral amines were also shown to induce high enantioselectivity to the process 153 and applications to the total synthesis of natural products were disclosed, as in the (±)-spongiolactone case (Scheme 60). Extension to the dynamic kinetic resolution of keto-acids was also reported. Shen reported the isolation of phomopsidone A in 2014 from the mangrove endophytic fungus Phomopsis sp. A123. 166 The same year, artoheterophyllin E was isolated from the wood of Artocarpus heterophyllus, an evergreen fruit tree collected in Hainan province (PR China). 167 Although extensive NMR and mass spectrometry experiments were performed and discussed, the structure of these compounds are questionable in light of the well-known instability of benzoxetes that are in equilibrium with their thermodynamically favored ortho-quinoïds valence isomers. Indeed, phomopsidone A was recently suggested to possess excelsione's structure. 13 <Scheme 67 near here> Morusalbin B was extracted from the root bark of M. alba L. (Moraceae) in the Republic of Korea. Its quite complex structure is proposed to possess a fused benzoxete. 168 Recently, Zhang and Wang extracted from the fermentation broth of Fusarium oxysporum ZZP-R1 two new antimicrobial agents, among which Fusariumin D. 169 The structure of the latter was recently proposed to actually be a 4pyranone scaffold. 13 <Scheme 68 near here>

73

 73

  <Scheme 40 near here> Related NHC-catalyzed synthesis of fused b-lactones from enals and 2-enoylpyridines 115 or oxindolyl b,g-unsaturated a-ketoesters 116 were reported. Scheidt reported the first NHC-catalyzed dynamic kinetic resolution (DKR) of b-ketoesters 154 substituted in the a-position, leading to fused blactones.

  by Lupton were shown to catalyze the formation of b-lactones fused to cyclopentanes 161, starting from a,b-unsaturated acyl fluorides 159 and donor-acceptor cyclopropanes 160.

  2.06.11.4 (-)-Curcumanolide A and (-)-CurcumalactoneDesymmetrization of diones was shown to deliver fused b-lactone 222 with excellent enantiomeric excesses via an asymmetric nucleophile (Lewis base)-catalyzed aldol lactonization of 221. Baeyer-Villiger oxidation of the tricyclic lactone followed by dyotropic 1,2-acyl migration led to bislactone 224, a common intermediate in the synthesis of (-)-curcumanolide A and (-)-curcumalactone.[156][157][158] <Scheme 63 near here> A similar strategy was used by Romo for several other natural products, such as (±)-salinosporamide A, 159 (±)-cinnabaramide A 160 and (+)-omphadiol. .11.5 (+)-Dictyoxetane (+)-Dictyoxetane was isolated from a brown alga Dictyota dichotoma (Indian ocean) in 1985 and was found to possess a unique oxetane motif embedded in a 2,7-dioxatricyclo[4.2.1.0 3,8 ]nonane ring system.162 Magauer reported the very first synthesis of (+)-dictyoxetane in 2016.163,164 The challenging oxetane in 226 was constructed through the intramolecular displacement of an allylic mesylate, generated from 225, under basic conditions in 88% yield. <Scheme 65 near here>2.06.11.6 (+)-PleuromutilinCarbonyl-alkyne metathesis reactions[80][81][82][83][84][85] have found numerous applications in the context of total synthesis of natural products and only a few examples will be discussed here. During the course of investigations aiming at the total synthesis of (+)-pleuromutilin, Herzon reported the unexpected formation of the transient intermediate fused oxetene 230 from the oxidative cyclization/C-O reductive elimination of yne-aldehyde 227 using Ni(COD)2 (40 mol%), 4,5-dichloro-1,3-bis(2,6diisopropylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene (40 mol%) and triisopropylsilane as a reductant.

  .11.8 (-)-Mitrephorone AMitrephorone A is a member of the family of ent-trachylobanes which was isolated in 2005 from Mitrephora glabra, a Bornean shrub. Its structure features a highly congested hexacycle with an embedded oxetane. The central oxetane of mitrephorone A was prepared thanks to an impressive oxidative 4-exo-trig cyclization of a hydroxy enol ketone, derived from 231, activated with Koser's reagent.170 An alternative synthetic blueprint was reported a year later, 171 featuring a direct conversion of mitrephorone B to mitrephorone A via a bioinspired C-H oxidation with White-Chen catalyst. Electrochemical oxidation or photocatalyzed formation of the oxetane lead to lower yields.

  Fused oxetenium ions are intermediates in the synthesis of medium and large ring lactones from acetals and silylated ynol ethers. Sun reported that treating acetal 86 with a Lewis acid in the presence of 2,4,6-collidine led to the formation of oxocarbenium such as 91 that underwent a spontaneous electrocyclic ring-opening to 88.90 This unusual and elegant ring expansion strategy overcomes the current limitations encountered for the preparation of medium to large ring lactones, whether entropic or enthalpic by nature. Alder cycloadditions of the intermediate ortho-quinone methide similar to 104 are also possible as shown by Yoshida.93 When the aryne precursor 105 is reacted with active methylene derivatives such as 106 in the presence of potassium fluoride, coumarins 107 are obtained in moderate to good yields.Ortho-quinomethide 108 could also act as a nucleophile by delocalization of the lone pair of electrons of the nitrogen atom, and then react with an electrophile. Lu and Wang have demonstrated that benzoyl cyanide 110 was a relevant electrophile, leading to the formation of a-amino carbonitriles 112 via an elegant three-component reaction.94

	O	63	Ph	AuCl (2 mol%) AgSbF 6 (6 mol%) toluene 100 °C 2 h N Ts OMe CH 2 Cl 2 HBF 4 0 °C, 1 h OMe TIPSO + 86 O 87 OMe TMS OTf H O + N OMe 75 100	Bu	O + N Ts CsF, DMF Ph [Au] O H BF 3 •OEt 2 2,4,6-coll. CH 2 Cl 2 0 °C 20 °C, 15 min then Ph 2 Zn 20 °C, 12 h 64 80%, 76/77 = 2:1 O N + 88, 83 % [Au] + O O O Bu OMe Ph NMe 2 OH Ts Ph 65 77 76 101, 97%	Ph 66, 80 % O
		via		N MeO O 89	Ts	OMe Bu	MeO	O	O	Ts N	N	O	O 90 O •	BF 3 Bu MeO	O N	Ts	N 91 O Ph 2 Zn BF 3 O Bu O
				102	78			103					79	104	80
	O O Ph <Scheme 26 near here> OEt OEt OEt 72 O 69 82 Cl N O 94 105 108 67 + Ts O EtO OBn OBn OEt 73 N Ts 94 N Ts OMe H O N Ts Ph FeCl 3 •6H 2 O (5 mol%) O O OBn Et acetone 50 °C, 4 h 70 FeCl 3 •6H 2 O (5 mol%) DCE Ph N Ts O O protonation O OEt 71 68, 92% Ph O 74, 67% OBn OBn O then Ph NTs ring-opening Li 80 °C, 2 h OH Ha Bu Hb 76 (elimination of Hb) 77 (elimination of Ha) 81 and HN Ph O O 1. TMSOTf (120 mol%) CH 2 Cl 2 , 20 °C, 5 h 2. NaOH MeOH, 0 °C, 2 h O O O TMS 83, 62% [2+2] O O O H H 92 93, 70 % BF 3 •OEt 2 2,4,6-coll. CH 2 Cl 2 0 °C O Ph Ts N 3 Cl N O Ph NHTs O + + CuI (10 mol%) Et 4 NI (10 mol%) LiOH CH 2 Cl 2 / t BuOH (10:1) 30 °C, 3 h 97, 87% 96 95 TMS OTf N CO 2 Et O N O + NMe 2 N NMe 2 O OEt [4+2] KF DMF 80 °C 2.5 h 106 107, 95% 109 -EtOH Diels-<Scheme 27 near here> O O O OEt N -HNMe 2
		Ph	N	Li	84			Cl				N	85 O
				98											99
	<Scheme 25 near here> The inherent strain of benzoxetenes has been judiciously exploited by Miyabe who reported the synthesis of ortho-disubstituted aromatics such as 101 from an aryne precursor 100, a formamide TMS OTf CN O O t Bu O Ar CN O E CsF O MS 4 Å + O DMF t Bu CN NMe 2 60 °C NMe 2 3 h
	and diphenylzinc. 92 Transient formation of benzoxetene 103, arising from reaction of benzyne 102 NMe 2 112, 88 % 110 105 108 111
	and DMF, undergoes a ring opening reaction to 104 which is immediately trapped in situ by <Scheme 28 near here>
	diphenylzinc.
	2.06.6.7 Cycloisomerisation of Oxetane-Containing Enynes

<Scheme 22 near here> Silylated fused oxeteniums 85 were postulated as precursors to a,b-unsaturated ketones en route to 3,4-disubstitued pyrroles 83.

89

Initial semi-pinacol rearrangement leads to a ring-contracted intermediate 84 which undergoes an alkyne-ketone metathesis yielding the substituted pyrrole after spontaneous oxidation. <Scheme 23 near here> Ph <Scheme 24 near here> Spiro-oxetenes have been proposed as intermediates in the synthesis of 3-substituted oxindoles 97.

91

Indeed, copper(I)-catalyzed reaction of phenylacetylene 95, tosylazide 96 and isatin 94 led to a metal ynamidate 98 which could undergo a stepwise cyclization to spiro-oxetene intermediate 99. Ring opening of the latter finally afforded product 97 in excellent yield.

  of enyne 113 in the presence of vinyl bromide 114 in 66% yield. It was shown that the introduction of a spiro-oxetane modified the conformation of 115 compared to the parent 1a,25-dihydroxyvitamin D3, and thus favored the formation of ligand-vitamin D receptor complex.

	<Scheme 31 near here>
	O	+		H	OH	1. Pd(PPh 3 ) 4 (30 mol%) Et 3 N, toluene reflux, 1 h			H	H	OH
	OTBS		H			2. TBAF, THF 20 °C, 80 min
	113	Br		114		O	3	1	OH	115, 66%
	<Scheme 29 near here>
	2.06.7 Reactivity of Substituents Attached to Ring Carbon Atoms
		p-Cl-C 6 H 4	N	CO 2 Me
			116
	O 40b, R = CN R	CuBF 4 (5 mol%) Ligand (5.5 mol%) Cs 2 CO 3 , MS 4Å CH 2 Cl 2 -20 °C, 1 h	N H 117a, 86%, dr > 20:1, 99% ee p-Cl-C 6 H 4 O R CO 2 Me	Ligand:	O N Fe PPh 2	t Bu
	40c, R = CO 2 Me				117b, 91%, dr > 20:1, 94% ee
	<Scheme 30 near here>
	2.06.7.2 Exocyclic Epoxides and Cyclopropanes
	2-Methyleneoxetane 118 could be converted into the corresponding epoxide 119 with good
	diastereoselectivity (8:1) and quantitative yield using anhydrous dimethyldioxirane (DMDO). Epoxide-
	opening was best conducted with diisobutylaluminium hydride in dichloromethane, leading to 120 in
	56% yield as a 3:1 mixture of diastereomers. 101

2.06.7.1 Exocyclic Alkenes 1,3-Dipolar cycloaddition of azomethine ylide derived from 116 with exo-methylene oxetanes 40b,c leads to spiro-oxetanyl pyrrolidines 117a,b in excellent yields. This copper(I)-catalyzed process relies on a planar-chiral ferrocene P,N-ligand that allows for a perfect control of exo-selectivity and high enantiomeric excesses (94-99%).

99

Silver-catalyzed processes were also reported.

100

  Photocycloadditions of isoquinoline-1,3,4-trione with 5-methoxyoxazoles were reported by Zhang. Fused oxetanes were obtained in very good yields and could be rearranged to spiroisoquinolineoxazolines under acidic conditions. 1112.06.9.2 Intramolecular Paternò-Büchi CycloadditionsTransposed Paternò-Büchi [2+2] photocycloadditions of atropoisomeric enamides 141 have been reported by Sunoj and Sivaguru, during which a switch in excited states from n-p* of the carbonyl group to p-p* of the alkene occurs.112 After cycloaddition under sensitized conditions, chirally enriched oxetanes 142 were obtained.Photoreaction of 2-isopropoxy naphthoquinone 143 in the presence of bicyclopropylydene leads to the expected cyclobutane 144 after 3 hours. However, upon extended irradiation time, a g-hydrogen abstraction occurs from the triplet state of alkoxyketone 144 leading to the fused-oxetane 145 in 54% yield.113

	<Scheme 36 near here> Unprecedented angular tricyclic 4:4:4 oxetane-containing compounds were obtained by Aitken through a triple photochemical process, starting from 4-substituted cyclopentenones 136 and O H H HO OH 2 steps O O H H OAc D-isosorbide 128, 78% PhCHO Hg MPL (400 W) MeCN (0.03 M) 3-layer FEP flow reactor (1 mL/min) O O OAc H OAc H H O Ph H H O O H H H O Ph 129 130 129/130 = 1:2, 97% 42 g in a single 83 h run O O H H OH O Ph H H 131 K 2 CO 3 MeOH 20 °C, 16 h then separation 3 steps O O H H Ph HO HO (+)-goniofufurone 132, 22% + O O O O O H H 100 W Hg Lamp TpCu (10 mol%) Et 2 O 55 °C, 24 h + 133 134 (3 equiv) 135, 75% (NMR yield) > 95:5 exo/endo Cu N N N B N N N H TpCu(Norb) <Scheme 37 near here> <Scheme 38 near here> 2.06.9.3 Norrish II (Biradical) Cyclization O HO + hν (300 nm) CH 3 CN 20 °C 40 h O HO H O HO O HO H O HO H Norrish-I cleavage -H transfert Paternò-Büchi [2+2] 136 138 139 140 137, 24% N O O N O O 141 Hg MPL (400 W) Xanthone MeCN, 20 °C, 2 h 142, 73% (NMR yield) <Scheme 39 near here> alkenes. O 2.06.9.4 Cycloadditions of Ketene Equivalents

109

A relevant example is described in Scheme 36 with the reaction of ketone 133 and norbornene 134. In the presence of 10 mol% of copper(I) tri(1H-pyrazol-1-yl)hydroborate (TpCu) and under irradiation with a 100 W mercury lamp, a good yield of adduct 135 is obtained. The reaction pathway is mechanistically distinct from classical Paternò-Büchi reactions and relies on the excitation of an in situ formed TpCu(Norb) complex. 110 Under irradiation at 300 nm for 40 h in acetonitrile, 12 different angular tricyclic oxetanes were obtained with moderated to good diastereoselectivities. A plausible mechanism calls for an initial [2+2] photocycloaddition followed by a Norrish-I/g-H transfer. A final intramolecular Paternò-Büchi reaction would then deliver 137.

Bode reported in 2009 that chiral triazolium-derived NHCs were able to catalyze the formation of fused b-lactones with an excellent enantioselectivity via the intermolecular reaction of an enal 147 and an a-hydroxyenone 148.

114

Quite surprisingly, the use of chiral imidazolium-derived NHC led to a different fused lactone, also with excellent enantioselectivity. A detailed mechanistic discussion proposes that the different reactivities of the intermediate acylazolium species involved in the lactone-forming step, such as 153 derived from catalyst 150, might be responsible for this outcome.

  Unsaturated acylammonium intermediates are also competent partners as shown by Romo in 2013 123 who proposed a multicomponent organocascade reaction coined Nucleophile-Catalyzed Michael-Aldol-b-Lactonization (NCMAL), which led to the synthesis of b-lactone 174 in excellent yields and enantioselectivities. an elegant strategy that was applied to the total synthesis of (±)-vibralactone, a natural product isolated from the culture of Basidiomycete Borestereum vibrans.124 Palladium-catalyzed deprotection of b-mesyloxy ester 175 led to the desired b-lactone 176 in only 2 minutes in 95% yield. Although this reaction does not strictly fall into the area covered by this chapter focused on fused blactones, it would be interesting to study the formation of bicyclic b-lactones via this novel type of intramolecular cyclization.Bull reported the two-step preparation of diversely substituted oxetanes 180 from functionalized diazo-compounds 177 by rhodium-catalyzed O-H insertion followed by a base-mediated cyclization of intermediate 179. Several fused [3.2.0] ring systems 180 were obtained, in addition to an oxabicyclo[4.2.0]octane, in excellent yields.125,126 <Scheme 47 near here> Benzoxetes 182 were efficiently prepared by Qin through a sulfuryl fluoride-mediated conversion of 2-hydroxy acetophenones 181 in the presence of potassium carbonate in DMSO at 90 °C.

										Cat. (15 mol%)
	Ph	O	H	+	Ts	H N	O	Et	Cs 2 CO 3 , DQ 168 (10 mol%) MS 4Å 20°C CHCl 3	Ts Ph	N	H	O O Et		Cat.	O	N	N	N +	Mes BF 4 -
			147				167			20 h	169, 74 %, dr > 20:1
															er = 96:4
														gram scale		NO 2
																				iPr
		Ph	O	Cat		167		Ts Ph		O N	Et O	Cat	Ts Ph	N	O	Et O Cat		H 2 N	S O O	168 iPr	iPr
			170								171			172
	<Scheme 44 near here>
	MeO 2 C a,b-<Scheme 45 near here> LiHMDS MeO 2 C O THF -78 to 0 °C 10 min 173	MeO 2 C MeO 2 C -Li +	O	1. (S)-HBTM (20 mol%) EtNiPr 2 CH 2 Cl 2 0 °C, 10 min 2. acryloyl chloride MeO 2 C MeO 2 C dr > 19:1, 97% ee O H O 0 to 23 °C, 6 h Me 174, 72%	S N (S)-HBTM N	Ph
	mol%) DBU (1.2 equiv.) 2.06.9.5 Intramolecular Nucleophilic Displacements						Cat.	O	N	N	N +	Mes Cl -
											LiCl (0.5 equiv.)
											DQ (1.2 equiv.)
		164					165				CH 2 Cl 2 20 °C 1-72 h	166, 14 examples, 15-95%	DQ	t Bu
														dr > 99:1, 94-99% ee	O	O
	AllO	O	OMs OMe OMe		Pd(PPh 3 ) 4 (1 mol%) pyrrolidine CH 2 Cl 2 2 min	O	O	OMs OMe OMe			O	O	OMe OMe	3 steps	O	O	H	OH
		175															176, 95%	(±)-vibralactone, 59%
	<Scheme 46 near here>

t Bu t Bu t Bu Brown reported a novel palladium-catalyzed deallylative cyclization of 175 to synthesize b-lactone rings 176, 127 <Scheme 48 near here>

  closure of numerous b-hydroxyacids in the context of total synthesis of natural products, such as (+)-lactacystin, 130 a rocaglate-derived b-lactone, 131 (-)-7-methylomuralide, 132 (-)-omuralide,133 or salinosporamide A[134][135][136] to cite only a few. As an example, Burton reported a high-yielding sequence from b-hydroxyester 188 to salinosporamide A in 3 steps (61% overall), using BOPCl as the key coupling agent.137 As noted in previous versions of this chapter, this reaction leads to the fused blactone only and no traces of spiro-b-lactone (arising from the cyclization of the exocyclic secondary alcohol) were detected.Oxidative dearomatization of phenols is an interesting class of reactions that is relevant to the formation of fused oxetane, as shown for the first time by Li in 2014.139 Treatment of substituted phenol 194 by PhI(OAc)2 in aqueous acetonitrile led to the strained oxetane 195 in moderate yield.Moody reported a straightforward preparation of spiro-b-lactones 197 based on the selective 1,4-C-H insertion reactions of rhodium carbenes derived from a-diazoesters 196.140 The latter could be generated in flow or in classical batch chemistry. The obtained b-lactones could then be reduced to oxetanes using a two-steps protocol discussed in Section 2.06.6.3.Highly strained fused oxetane, such as the one embedded in merrilactone A 199, a natural product isolated from the pericarps of Illicium merrillianum in 2000, has been prepared by the Fukuyama procedure 142 involving a stereoselective epoxidation of the trisubstituted alkene of 198 followed by a homo-Payne rearrangement.143,144 This classical reaction has been used in several syntheses of the neurotrophic active natural product. 145 rare example of Wolff rearrangement of a rhodium carbene was reported by Chiu in 2017, leading to the fused b-lactone 205 in 39% yield alongside two other products. It was hypothesized that the conformation of the [4.4.0] bicyclic diazoketone 204 could be responsible for this specific outcome. 148 -Lactones can be transient intermediates en route to cyclopentenones via a tandem sequence of ynolate addition onto a ketone followed by intramolecular condensation onto an ester and decarboxylation. Such a sequence was used by Shindo in 2018 in the total synthesis of (-)stemoamine, an alkaloid isolated from the root of Stemona japonica Miq. that has been used in traditional Chinese medicine. 150 Addition of ynolate 208 (generated from 207 and t BuLi) to ketone 206 led to the formation of 211, that can undergo silica gel-assisted decarboxylation in refluxing benzene.Fully substituted oxetanes are found in natural products such as dichrocephone B 217. In 2018, the total synthesis and structure revision of this compound was reported by Christmann and Tantillo, featuring a Lewis acid-mediated cyclization of ent-dichrocephone A 216 into ent-dichrocephone B 217 in 88% yield. A low yield (12%) was obtained for the acid-mediated cyclization of 215.152

	2.06.9.8 Cationic Cyclizations		Et		THF	Et	Co(CO) 4	-
			Et Et	N N	Al +	N N	Et Et
	2.06.10 Ring Synthesis by Transformation of Another Ring Et Et THF
	OH 2.06.10.1 Rearrangement of Epoxides O cat. (3 mol%) CO (900 psi)	O	O	O
	NC <Scheme 58 near here> CO 2 Bn N 2 HO Br + H 177 H N OH CO 2 PMB O OH <Scheme 53 near here> HO 188 2.06.9.9 Intramolecular C-H Insertion NHBoc Rh 2 (OAc) 4 (0.5 mol%) CH 2 Cl 2 25 °C 17 h H X iPr 2 O BnO 2 C NC PhI(OAc) 2 O Br 22 °C, 24 h H TMSO MeCN/H 2 O (3:1) 0 to 20 °C, 1 h 202a, X = CH 2 202b, X = NBoc 178 179 H N CO 2 H OH HO O OH BCl 3 194 CH 2 Cl 2 0 °C, 30 min OH O 1. DMDO (0.06 M in acetone) CH 2 Cl 2 , 20 °C, 7 h 189 O O O 2. TsOH•H 2 O CH 2 Cl 2 , 20 °C, 24 h 2.06.10.3 Rearrangement of Diazoketones <Scheme 60 near here>	NHBoc H X 1. BOPCl (1.7 equiv.) NaH DMF 25 °C 16 h O 203a, 67% 203b, 91% CH 2 Cl 2 /pyridine (2:1) BnO 2 C O NC 180, 95% NHBoc H H H dr = 83:17 H N Cl O OH O O 20 °C, 3 h 2. Ph 3 PCl 2 (2 equiv.) CH 3 CN/pyridine (1:1) 20 °C, 2 h 195, 42 % salinosporamide A (61% over 3 steps) O H O HO O O O
	198						199, (±)-merrilactone A, 74%
	<Scheme 50 near here> <Scheme 56 near here> 2.06.11.2 ent-Dichrocephone B	OH O	R 2	SO 2 F 2 K 2 CO 3	O	R 2
	R 1 2.06.9.7 Electrophilic Cyclizations onto Alkenes DMSO 90 °C 20 h 181 Bromolactonization of b,g-unsaturated carboxylic acid is a reliable and simple method to synthesize R 1 182 19 examples 30-98 % b-lactones, especially on large scale. Millenium Pharmaceuticals has reported a practical synthesis of Pevonedistat, a NEDD8-activating enzyme (NAE) inhibitor using the bromolactonization of 190 to bicyclic intermediate 191 as one of the key steps. 138 ( ) n O O ( ) n Ar Rh 2 (oct) 4 (1 mol%) CH 2 Cl 2 reflux O O Ph O O Ph O O O O important class of reactions. 146 204 205, 39 % chiral chromium(III) complex and dicobalt octacarbonyl proved to be optimal to promote this 4-Br-C 6 H 4 O 4-Br-C 6 H 4 196 197 O O Ar 2.06.10.1 Carbonylation of Epoxides O N 2 O OH O H Rh(TFA) 4 (1.7 mol%) N 2 Enantioenriched fused b-lactones 201 can be prepared via an asymmetric ring-expansion/carbonylation reaction of meso-epoxides 200. Combination of catalytic amounts of a Br Br CH 2 Cl 2 EtOOC EtOOC 20 °C, 12 h
	CO 2 Et <Scheme 51 near here> 3 steps HO NHBoc HO 2 C DME/H 2 O (3:7) OH Br 2 pyridine 0 °C 197a, 61% 197b, 32% n-BuLi, THF 0 °C, 30 min O <Scheme 59 near here> CO (500 psi) O 190 191, 68 % NHBoc O X 197c, X = CH 2, 70% 7 steps H 2 N O S O O cat. 197d, X = NBoc, 58% Br cat. (5 mol%) O O H H Co 2 (CO) 8 (5 mol%) O <Scheme 54 near here> Metalloporphyrin catalysts are also efficient for C-H functionalization reactions, as shown by Che. For O N O H N O 197e, 68% HO N N • HCl pevenodistat, 24% N N example, the methylated iridium(III) porphyrin complex [Ir(TTP)Me(L)] (H2TTP = meso-tetrakis(p-t Bu O O Cr Cl O DME 2.06.11 Important Compounds and Applications 70°C O 16 h 201, 93%, 56% ee 200 Oxetanes are found in more than 600 naturally occurring structures produced by microorganisms or then TsCl 0 °C, 1.5 h tolyl)porphyrin; L = H2O or solvent) was able to catalyze the formation of spiro-b-lactone 197f from plants. 149
	N H 183 Acetal-substituted spiro-oxetanes 193 having a 1,5-dioxaspiro[3.4]octane scaffold were prepared N Boc then n-BuLi 0 °C, 30 min then 60 °C, 1 h N Boc N Cbz 184, 39-52% 185, 66 % 186, 15% (from ethyl nipecotate) 187, 33% (from methyl 1-benzylpyrrolidine-3-carboxylate) N Bn through a bromocation-induced cyclization of bis-homopropargylalcohols 192 with pyridinium O O H 23 C 11 NH BocN BocHN Br Br H 23 C 11 NH BocN BocHN OH OH PyHBr 3 CH 2 Cl 2 /H 2 O (1:1) 20 °C, 1 h 192 193, 27% cat. (1 mol%) L = H 2 O or solvent L O O O Ph the reaction was the epoxide ring-opening, leading to 203a,b as the final products. 147 O Ph octaethylporphyrin catalyst. Mechanistic investigations demonstrated that the rate-limiting step of p-Tol monoxide atmosphere (900 psi) in diisopropylether at room temperature in presence of an K 2 CO 3 N N N Ir p-Tol p-Tol Spiro-epoxides such as 202a and 202b were shown to react with high regioselectivity under carbon N tribromide. 104 a-diazoester 196f at room temperature in only 1 h. 141 Me p-Tol <Scheme 57 near here> 2.06.11.1 (-)-Stemoamine <Scheme 61 near here>
	<Scheme 52 near here>	N 2				CH 2 Cl 2 22 °C, 1 h
	196f						197f, 53%
	<Scheme 55 near here>

ring t Bu t Bu t Bu A b151

The very efficient sequence forms the A-ring of (-)-stemoamine.

Acknowledgments

The authors thank the Université de Strasbourg, the Université de Haute-Alsace, the CNRS and the Indo-French Centre for the Promotion of Advanced Research (IFCPAR, project 6105-1) for their financial support.


Ariane Biquand

Simone Spinozzi

Paola Tonino

Jérémie Cosette

Joshua Strom

Zaher Elbeck

Ralph Knöll

Henk Granzier

William Lostal

Isabelle Richard
email: richard@genethon.fr

Titin M-line insertion sequence 7 is required for proper cardiac function in mice

Keywords: Cardiomyopathy, Heart Failure, titin, Mex5, Is7, Alternative Splicing

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

Titin is the largest described protein with a molecular mass of 3 to 3.7 MDa, and is the third most abundant protein in striated muscle [START_REF] Jackel | Deletion in the Z-line region of the titin gene in a baby hamster kidney cell line, BHK-21-Bi[END_REF]. A single molecule of titin spans half a sarcomere from the Z-disc to the M-line, stretching over 1.2 µm [START_REF] Labeit | Titins: giant proteins in charge of muscle ultrastructure and elasticity[END_REF] and interacts with numerous protein partners through this length [START_REF] Chauveau | A rising titan: TTN review and mutation update[END_REF]. This giant protein plays a major role in sarcomere stability and organization, and also works as a molecular spring responsible for the passive elasticity of muscle [START_REF] Gregorio | The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity[END_REF][START_REF] Granzier | The giant protein titin: a major player in myocardial mechanics, signaling, and disease[END_REF]. In relation to the sarcomere, titin is organized into four distinct regions that differ in structure and function: the N-terminal segment that anchors titin to the Z-disc through binding to multiple proteins; the I-band, which constitutes the molecular spring region of titin; the A-band region, involved in thick filament length control; and a final C-terminal portion bound to thick filaments in the M-line, where two adjacent titin molecules overlap in an antiparallel orientation [START_REF] Chauveau | A rising titan: TTN review and mutation update[END_REF][START_REF] Martonfalvi | Low-force transitions in single titin molecules reflect a memory of contractile history[END_REF].

In humans, the titin gene TTN (MIM #188840) is located on chromosome 2q31 and consists of 294 kb containing 364 exons (the first non-coding plus 363 coding exons), which can theoretically generate over a million splice variants [START_REF] Bang | The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system[END_REF][START_REF] Guo | Titin diversity--alternative splicing gone wild[END_REF].

Traditionally, titin isoforms are divided in 3 main categories depending on presence of N2A and N2B elements within the I-band region of the protein. N2A isoforms (which contain only N2A elements) are mainly expressed in skeletal muscles, while N2B and N2BA isoforms (which contain only N2B elements, or both N2B and N2A, respectively) are specific to the heart [START_REF] Linke | I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure[END_REF][START_REF] Freiburg | Series of exon-skipping events in the elastic spring region of titin as the structural basis for myofibrillar elastic diversity[END_REF][START_REF] Labeit | Expression of distinct classes of titin isoforms in striated and smooth muscles by alternative splicing, and their conserved interaction with filamins[END_REF]. In addition to this "splicing hotspot", alternative splicing can also occur in the Z-disc and M-line regions of titin. The latter is encoded by exons 359 to 364 or Mex1 to Mex6. It is composed of a serine/threonine kinase (TK) domain followed by an alternation of immunoglobulin domains (M1 to M10) and unique insertion sequences (is1 to is7) (Fig. 1A). The TK domain is an inactive pseudokinase scaffold and acts as a strain sensor [START_REF] Puchner | Mechanoenzymatics of titin kinase[END_REF][START_REF] Bogomolovas | Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line[END_REF].

There are numerous binding partners for titin in the M-line: M-protein, FHL2 and Myomesin 1 [START_REF] Hu | The sarcomeric M-region: a molecular command center for diverse cellular processes[END_REF][START_REF] Lange | Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2[END_REF][START_REF] Van Der | Assembly of titin, myomesin and M-protein into the sarcomeric M band in differentiating human skeletal muscle cells in vitro[END_REF]. Alignment of the M4 domain with myomesin stabilizes the sarcomere during contraction [START_REF] Lange | Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum[END_REF]. In its most terminal region, M10, titin interacts with obscurin (Obscn), obscurin-like 1 (Obsl1), cardiomyopathy-associated protein 5 (Cmya5) and alpha-synemin (Synm) [START_REF] Fukuzawa | Interactions with titin and myomesin target obscurin and obscurinlike 1 to the M-band: implications for hereditary myopathies[END_REF][START_REF] Prudner | alpha-Synemin localizes to the M-band of the sarcomere through interaction with the M10 region of titin[END_REF] [START_REF] Sarparanta | Interactions with M-band titin and calpain 3 link myospryn (CMYA5) to tibial and limb-girdle muscular dystrophies[END_REF]. Interestingly, the only exon of this region that can be spliced out is Mex5 [START_REF] Kolmerer | Genomic organization of M line titin and its tissue-specific expression in two distinct isoforms[END_REF]. The fraction of fibres expressing a Mex5+ titin isoform is dependent on the muscle, with a higher Mex5+/Mex5-ratio in muscles undergoing aerobic exercises [START_REF] Guo | RBM20, a potential target for treatment of cardiomyopathy via titin isoform switching[END_REF]. The splicing of Mex5 exon depends on the species, the phase of development and the M-line structure of the muscle [START_REF] Kolmerer | Genomic organization of M line titin and its tissue-specific expression in two distinct isoforms[END_REF][START_REF] Pask | M-band structure, M-bridge interactions and contraction speed in vertebrate cardiac muscles[END_REF]. The Mex5 exon encodes for the insertion sequence 7 (is7), situated between two Ig domains, M9 and M10 (Fig. 1A). The is7, which is part of a binding site for calpain 3, an enzyme implicated in limb girdle muscular dystrophy R1 that is nearly absent in the heart [START_REF] Richard | Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A[END_REF], Charton et al., 2010). Fascinatingly, is7+ titin is the major isoform expressed in the heart (Charton et al., 2016, Guo and[START_REF] Guo | RBM20, a potential target for treatment of cardiomyopathy via titin isoform switching[END_REF].

Considering its huge size, it is not surprising that TTN is subject to numerous mutations. About one percent of the general population carries a heterozygous truncating variation in TTN with no consequences [START_REF] Schafer | Titin-truncating variants affect heart function in disease cohorts and the general population[END_REF]. When pathological, variations in TTN are responsible for titinopathies presenting with muscular and/or cardiac phenotypes.

Titinopathies have highly variable phenotype and prognosis depending on the nature and position of the mutations. Among them, TTN mutations are responsible for more than twentyfive percent of all dilated cardiomyopathies (DCMs) [START_REF] Tayal | Truncating Variants in Titin Independently Predict Early Arrhythmias in Patients With Dilated Cardiomyopathy[END_REF]. The majority of reported variants associated with DCM are frameshift-causing mutations in high percentage spliced in (PSI) exons located in the A-band region [START_REF] Roberts | Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease[END_REF][START_REF] Stohr | Stretch your heart-but not too far: The role of titin mutations in dilated cardiomyopathy[END_REF].

Animal models also confirmed the importance of several titin regions for the heart function. A canine naturally occurring missense N2B-TTN variant in Doberman pinscher dogs leads to DCM and sudden cardiac death [START_REF] Meurs | A missense variant in the titin gene in Doberman pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death[END_REF]. Several murine models presenting large Ttn deletions have been developed, all presenting with cardiac abnormalities: homozygous deletion of the PEVK domain [START_REF] Granzier | Truncation of titin's elastic PEVK region leads to cardiomyopathy with diastolic dysfunction[END_REF], of the N2B [START_REF] Radke | Targeted deletion of titin N2B region leads to diastolic dysfunction and cardiac atrophy[END_REF]; of the N2BA [START_REF] Nusayr | A Novel Mouse Model for Titin-Based Dilated Cardiomyopathy[END_REF]; in the A-band region [START_REF] Tonino | The giant protein titin regulates the length of the striated muscle thick filament[END_REF]; in the M-line segment [START_REF] Charton | Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin[END_REF][START_REF] Gotthardt | Conditional expression of mutant M-line titins results in cardiomyopathy with altered sarcomere structure[END_REF][START_REF] Radke | Deleting Full Length Titin Versus the Titin M-Band Region Leads to Differential Mechanosignaling and Cardiac Phenotypes[END_REF]. Notably, the M-line region of titin seems to play a determinant role in cardiac function; in fact, the loss of this protein segment is causative of cardiac lethality in mice. The absence of Mex1 and Mex2 exons reduces the mechanical stability of the sarcomere by preventing the formation of a continuous titin filament [START_REF] Weinert | M linedeficient titin causes cardiac lethality through impaired maturation of the sarcomere[END_REF]. In cardiomyocytes, the homozygous full deletion of Mband abrogates sarcomere formation [START_REF] Musa | Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation[END_REF]. In addition, while mutations in Mex5 are responsible of Tibial Muscular Dystrophies (TMD) in humans [START_REF] Hackman | Truncating mutations in C-terminal titin may cause more severe tibial muscular dystrophy (TMD)[END_REF] deletion of the C-terminal end of titin segment is causative of a myopathy with fatal cardiomyopathy [START_REF] Carmignac | C-terminal titin deletions cause a novel early-onset myopathy with fatal cardiomyopathy[END_REF].

Our laboratory previously reported that CRIPSR/Cas9-driven homozygous deletion of the Mex5 exon leads to a dystrophic phenotype of all the muscles that in non-mutant mice mainly express Mex5+ Ttn isoforms [START_REF] Charton | Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin[END_REF]. This mouse model (ΔMex5) also showed a progressive cardiac fibrosis, but previous publications were more focused on the skeletal muscle phenotype or the role of the is7 region in the interaction with calpain 3 [START_REF] Charton | Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin[END_REF][START_REF] Lostal | Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A[END_REF]. Here, we further characterized the cardiac phenotype in ΔMex5 mice, showing that absence of titin is7 region leads to DCM with decreased cardiac function, also confirming the presence of a progressive fibrosis. Our data showed sarcomere alterations and mislocalization of the C-terminal end of titin in ΔMex5 cardiomyocytes. In addition, molecular analyses revealed that the deletion of is7 caused a decreased expression of the M10-binding partners Obscn and Cmya5. A decreased expression of the two most important excitation-contraction coupling proteins (cardiac ryanodine and dihydropyridine receptors, or Ryr2 and Dhpr) was also observed. Altogether, these results explain how the deletion of the is7 region is responsible for the appearance of the cardiomyopathy in ΔMex5 mice, by destabilizing the sarcomeric organization in cardiomyocytes, suggesting also a possible alteration of the calcium-release machinery. To our knowledge, the results reported here are the first demonstration of the potential molecular mechanisms underlying DCM resulting from Mex5 alterations, providing useful insights for future therapeutic targets.

RESULTS

Mex5 homozygous deletion leads to dilated cardiomyopathy with altered cardiac function and fibrosis in mice.

To gain a better understanding of the is7 role in titin function, we previously generated a mouse model homozygous for a deletion of the Mex5 region (ΔMex5) (Fig. 1A) [START_REF] Charton | Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin[END_REF]. The ΔMex5 mice were reported to show a dystrophic phenotype in the skeletal muscle, with the soleus being the most affected muscle and with an involvement of the heart as observed by the presence of fibrotic tissue [START_REF] Charton | Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin[END_REF]. To better understand the role of is7 within the titin molecule, we studied the consequences of its deletion in the heart.

Of note, the cardiac tissue is the only muscle where titin isoforms containing this domain are expressed by the majority of the cells, suggesting the particular importance of is7 in this organ.

To evaluate whether the absence of the is7 impacts cardiac function, transthoracic echocardiographic measurements on wild type (WT) and ΔMex5 mice were performed at two, four and thirteen months of age (Fig. 1B-E; Table S1). Echocardiography in ΔMex5 mice showed a progressive increase in left ventricular dimension in both, diastole and systole compared to WT controls, with significant differences starting from four months of age (Fig. 1B,C; Table S1). The change in left ventricular volume is not associated with significant differences in wall thickness, or in the heart weight/body weight ratio (Table S1). Chamber dimension abnormalities were accompanied by decreased cardiac function, as shown by reduction in the fractional shortening percentage (Fig. 1D,E; Table S1).

In view of the significantly reduced fractional shortening of the left ventricular chamber in ΔMex5 mice starting at four months of age, we assessed systolic and diastolic ventricular properties via pressure-volume (PV) analysis at this time point. PV loops showed (Fig. 1F; Table S2) that, compared to WT mice, the end systolic pressure volume relationship (ESPVR) of the ΔMex5 mice LV chamber was shifted rightward, as a consequence of the increase in end systolic and end diastolic volumes (Table S2). Although with no significant difference, the End-systolic elastance (Ees), which provides an index of myocardial contractility, tends to be decreased in ΔMex5 mice (Fig. 1H) as well as the maximal left ventricular pressure (dP/dt max; mmHg/s) (Table S2). The end diastolic pressure volume relationship (EDPVR) and the relative coefficient β, which describes the passive properties of the myocardium, remain unchanged (Fig. 1I). In agreement with echography data, the ejection fraction was also significantly decreased in ΔMex5 hearts (Fig. 1G; Table S2).

Overall, the PV analysis revealed a decreasing trend in cardiac inotropy in ΔMex5 mice, as shown by the reduced Ees, consistent with the ventricular dilatation and dysfunction, but no change in diastolic stiffness, since no alterations in the coefficient-β were present.

To establish the impact of the Mex5 deletion on heart morphology, light sheet imaging, a technique that allows three-dimensional visualization of organs, was performed on hearts from four-month-old mice. Longitudinal, sagittal and transversal sections confirmed the left ventricular dilation in ΔMex5 hearts (Fig. 2A). Three-dimensional imaging allowed for a deeper analysis of the heart dimensions that, in contrast with the echocardiography measurements, revealed an observable difference in the LV free-wall between mutant and controls. The light sheet image quantification showed an increase in the volume of all the heart chambers also confirming the left ventricle dilation observed in echocardiography (Fig. 2B,C).

Histological and molecular characterizations of ΔMex5 and control hearts were performed at the same time point. Heart tissue sections from four-month-old mice were stained with hematoxylin phloxin saffron (HPS) and Sirius red (Fig. 2D). HPS-stained cross-sections of ΔMex5 hearts displayed large areas of damaged tissue with increased cellularity when compared to WT. Sirius red staining showed an extended presence of fibrotic tissue in ΔMex5 hearts, whereas normal connective tissue was found in WT hearts (Fig. 2D). Additional analyses, at different time points, indicated that the fibrotic tissue was already present in ΔMex5 hearts at 1 month of age (albeit with no significant increase in respect of WT controls), and that the fibrotic condition proportionally worsened with aging (Fig. 2E; Fig. S1A). Immunofluorescence analyses of Collagens 1a1 and 3a1 (Fig. S1B) and quantitative RT-PCR analysis of fibrosis markers (Collagen 1a1, Fibronectin, Collagen 3a1, Vimentin) (Fig. 2F,G; Fig. S1C,D) confirmed the observed fibrotic phenotype.

Different cardiac stress and inflammation markers, known to be associated with pathological cardiac conditions, were also measured by quantitative RT-PCR (Fig. 2H-J; Fig. S1E,F). In particular, the balance between the two Myosin heavy chains Myh7 and Myh6 was altered in ΔMex5 hearts as already reported in pathological heart remodeling [START_REF] Fatkin | An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy[END_REF][START_REF] Gupta | Factors controlling cardiac myosin-isoform shift during hypertrophy and heart failure[END_REF][START_REF] Vanderheyden | Myocardial gene expression in heart failure patients treated with cardiac resynchronization therapy responders versus nonresponders[END_REF][START_REF] Montgomery | Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure[END_REF]: Myh7 was increased, whereas Myh6 was decreased with respect to WT controls (Fig. 2H,I). Natriuretic peptide A (Nppa), whose expression is reactivated in cardiovascular pathologies [START_REF] Houweling | Expression and regulation of the atrial natriuretic factor encoding gene Nppa during development and disease[END_REF], transforming growth factor beta 1 (Tgf-β1), a key player in cardiac fibrosis and pathological remodeling and TIMP metallopeptidase inhibitor 1 (Timp1), known to be upregulated in deteriorating heart failure, were all upregulated, indicating a typical dilated cardiomyopathy (DCM) pattern in the mutant mice (Fig. 2J; Fig. S1E,F).

To identify the impact of the is7 deletion on sarcomere organization, electron microscopy on heart samples from four-month-old mice was performed (Fig. 2K). Ultrastructural analysis of ΔMex5 mice cardiac tissue revealed the presence of altered sarcomeres with irregular Z-disc, less defined A-and I-bands, and absence or disruption of the M-line, although occasional normal sarcomere structures were found (Fig. 2K i-ii). Abnormal condensed mitochondria (Mit), autophagic vacuoles, lipids, inflammatory infiltrate cells and collagen fibrils were also observed, further confirming the fibrotic condition (Fig. 2K iii-iv). Mitochondria quantitative analyses showed a statistically significant increased area and fractional area, and reduced perimeter in ΔMex5, while no significant differences on Feret's diameter was found. Shape parameters were significantly different in ΔMex5 mitochondria compared to WT, including the increase in roundness accompanied by the reduction of aspect ratio, and an increase in circularity and solidity, indicating a reduction in the elongation of ΔMex5 mitochondria (Fig.

2L).

We then compared ΔMex5 sarcomere length to WT controls. A huge variation in ΔMex5 sarcomere length was observed compared to controls, with a higher number of shorter sarcomeres in ΔMex5 hearts (Fig. 2M). These data demonstrated that the homozygous deletion of Mex5 in titin is causative of a DCM phenotype accompanied by cardiac fibrosis, inflammation and mitochondria morphological alterations, as well as altered sarcomere length.

Sarcomere alterations and titin "ultra-localization shift" in ΔMex5 hearts.

To investigate the underlying mechanisms leading to the cardiac abnormalities, a first characterization of the consequences of the Mex5 deletion on the expression of Titin at the RNA and protein levels was performed. Quantitative RT-PCR showed a slight (albeit not significant) reduction in Titin relative expression in ΔMex5 with respect to WT controls, suggesting that the deletion does not drastically affect titin mRNA level (Fig. 3A). At the protein level, Coomassie blue stained gels using lysates from WT controls and ΔMex5 hearts were performed to visualize the presence of the different titin isoforms. The results showed no increase in the degradation product T2 in ΔMex5 heart samples compared to controls, a slight decrease in N2BA/N2B ratio was observed, although not statistically significant (Fig. S2A,B). A more detailed analysis was conducted via Western Blot (WB) using a set of antibodies raised against all different domains of the C terminus titin (M1, is5, is6, M8M9, is7 and M10), while using an antibody specific for titin N-terminus (Z1Z2) as comparative control (Fig. 3B). No differences in Z1Z2, M1, is5 and is6 were observed between ΔMex5 and WT controls, indicating that the protein is present at similar levels with or without the is7. As expected, no specific labeling was detected for is7 on ΔMex5, confirming the deletion. Interestingly, a reduction of intensity was also observed for the two adjacent domains M8M9 and M10 in ΔMex5 compared to controls, suggesting that the deletion of the is7 affects the adjacent titin Ig domains (Fig. 3C).

Immunostainings of cardiac tissue from four-month-old mice were then performed to determine the presence of the different C-terminal end of titin domains and to assess the localization of titin in ΔMex5 mice (Fig. 3D). Is7 specific staining showed the complete loss of the domain, whereas M8M9 and M10 staining confirmed the presence of these domains on each side of the Mex5 deletion in WT tissues, although not all ΔMex5 sarcomeres were positive for M8M9 and M10 staining. Despite a reduced intensity of the staining on WB, immunofluorescence images showed a correct integration of titin in ΔMex5 cardiomyocytes, and no apparent change in localization with respect to WT controls (Fig. 3D).

Immunoelectron microscopy was performed with antibodies raised against is7 and M8M9. As expected, titin is7 was completely absent in ΔMex5 but present in WT sarcomeres, while titin M8M9 domain epitopes were labeled in a spotty pattern in both mutants and controls (Fig. 3E). However, the M8M9 staining is not present in all the sarcomeres and the pattern is less regular in ΔMex5 samples. Measurement of the distance between M8M9 epitopes across the M-line was increased in ΔMex5 compared to WT (88 ± 1 nm vs 94 ± 3 nm), indicating a modification in the organization of the domains and suggesting that the mutation alters titin localization at a sarcomeric level, shifting it more toward the Z-disc side (Fig. 3F).

These results suggest that the absence of Mex5 + titin in the heart causes sarcomere abnormalities.

Deletion of is7 leads to a classical molecular signature of a DCM.

To understand the molecular mechanisms underlying the cardiac phenotype in ΔMex5 mice, a RNAseq analysis on samples from WT and mutant hearts at four months of age was performed using Illumina sequencing. The reads were aligned onto the mm10 mouse genome and DESeq2 analysis was performed. A total of 3473 genes were identified as significantly dysregulated with a log2 fold change higher than 0.5 in ΔMex5 hearts compared to controls with 2116 up-regulated and 1356 down regulated (Fig. 4A). The top dysregulated genes at four months of age sorted by log2 fold change are presented in Tables S3 andS4. These included transcripts encoding for proteins implicated in cardiac fibrotic processes (e.g. Spp1, Cilp, Ltbp2, Sfrp2, Thbs4), fibrotic tissue structure (e.g. Tnc, Col12a1, Col8a2) and cardiac protection upon stress (e.g. Mpeg1, Gpnmb) for the up-regulated, and in mitochondria for the down regulated (e.g. mt-Ts1, mt-Ti). Gene Set Enrichment Analysis (GSEA) using the Gene Ontology Biological Process collection revealed that the most up-regulated genes are related to immune response activation (e.g. GO leukocytes migration, regulation of cell activation, leukocytes differentiation and adaptive immune response) (Fig. 4B,C; Tables S5 andS6).

The GSEA also confirmed that down-regulated genes were significantly enriched in the Gene Ontology Mitochondrial pathways (e.g. GO mitochondrial respiratory chain complex assembly, respiratory electron transport chain, mitochondrial electron transport NADH to ubiquinone) (Fig. 4B,D; Tables S5 andS6). Ingenuity Pathway Analysis (IPA) software was used to identify enriched pathways. We used the IPA toxicology analysis to obtain a picture of the negative effects of the is7 removal. Most of the significantly dysregulated genes are implicated in known cardiac diseases pathways as cardiac enlargement (86 genes), cardiac dysfunction (45 genes), cardiac dilatation (38 genes), cardiac fibrosis (27 genes), cardiac necrosis/cell death (35 genes), etc. (Fig. 4E). These results confirmed that deletion of Mex5 in titin is causative of DCM associated with severe inflammation, cardiac fibrosis and mitochondrial damage.

Interestingly, two genes, Cmya5 and Obscn, encoding for proteins that bind to titin at the M10 region were present among the down-regulated group with a high significance (adjusted pValues: 4.04e-4 and 0.001, respectively), although not the most dysregulated genes (Table S7) [START_REF] Benian | Titin and obscurin: giants holding hands and discovery of a new Ig domain subset[END_REF]Mayans, 2015, Sarparanta et al., 2010) . A close homolog of Obscn (i.e.

Obsl1) is also slightly dysregulated (Adjusted pValue 0.028). On the other hand, the two other proteins known to interact with titin in M10, alpha-synemin (Synm) and calpain 3 (Capn3), which are present at a low expression level, remained unchanged (Table S7). Cmya5 encodes for the cardiomyopathy-associated protein 5, a 500kDa tripartite motif (TRIM)-related protein, highly expressed in striated muscles and that serves as an anchoring protein that mediates subcellular compartmentation of intracellular signaling [START_REF] Benson | Myospryn is a novel binding partner for dysbindin in muscle[END_REF][START_REF] Benson | Ryanodine receptors are part of the myospryn complex in cardiac muscle[END_REF][START_REF] Sarparanta | Biology of myospryn: what's known?[END_REF]. Obscn is a 720 kDa sarcomeric protein with an essential structural role in positioning the longitudinal sarcoplasmic reticulum at the M-line [START_REF] Lange | Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum[END_REF]. We also noticed that two genes, Ryr2 and Atp2a2 encoding for Ryr2 and SERCA2, two proteins known to play a central role in Ca 2+ -handling, were down-regulated with high significance (Adjusted pValue 2.5e-05 and 1.6e-6, respectively; Table S8). RyR2 and SERCA2, are two of the main components of the cardiac Ca2 + -release/uptake machinery present at the level of the sarcomeric reticulum, and alterations in their expression levels are a hallmark of cardiac disease [START_REF] Liu | Nexilin Is a New Component of Junctional Membrane Complexes Required for Cardiac T-Tubule Formation[END_REF], Liu et al., 2020). Interestingly, Ryr2 clustering in striated muscle involves Cmya5 and these two proteins form an oligomeric protein complex in cardiac muscle [START_REF] Benson | Ryanodine receptors are part of the myospryn complex in cardiac muscle[END_REF]. Genes encoding for two additional proteins involved in Ca 2+ -handling were also slightly dysregulated: Jph2 and Pln (Table S8). Jph2 encodes for junctophilin2, which is a protein determinant in dyads formation between junctional SR and T-tubules [START_REF] Takeshima | Junctophilins: a novel family of junctional membrane complex proteins[END_REF], Spinozzi et al., 2020, Feng et al., 2020). Pln encodes for phospholamban, a protein that is involved in SERCA2 regulation [START_REF] Koss | The relative phospholamban and SERCA2 ratio: a critical determinant of myocardial contractility[END_REF].

Deletion of the is7 alters the expression of titin M10-binding partners and of junctional membrane complex proteins in ΔMex5 mice.

The data obtained with the RNAseq analysis guided us in further investigating the dysregulations in titin M10 binding partners and in assessing whether the Ca 2+ -release/uptake machinery was altered in the ΔMex5 mice. In agreement with RNAseq results, quantitative RT-PCR measurements showed a reduced mRNA expression for Cmya5, Obscn, RyR2, Atp2a2 and Pln, whereas Synm, Cacna1c (encoding for Dhpr) and Jph2 levels were not significantly changed (Fig. 5A-H, 4A and Table S8). At the protein level, WB analyses showed a decreased expression only for Cmya5, Obscn and Ryr2 in ΔMex5, in agreement with the reduction at the transcription level (Fig. 5I-L). On the other hand, no significant change in Jph2 or Serca2 was detected between mutant and WT hearts (Fig. 5M,N). Surprisingly, Dhpr protein levels were significantly reduced in ΔMex5 hearts, in contrast with transcriptomic and qPCR data (Fig. 5O). These results suggested a specific alteration in Ca 2+release, with abnormal expression of the two main proteins involved in this process, but no apparent alteration in the organization of the junctional membrane complex architecture (no change in Jph2 expression), or in the Ca 2+ -uptake machinery (no alterations in Serca2 protein levels were observed). Next, we investigated whether Cmya5 and/or Obscn localization was impacted by the destabilization of the M-line portion of titin in mutant mice.

Immunofluorescence analyses on heart sections from four-month-old ΔMex5 and WT mice showed no altered pattern for either Cmya5 or Obscn in mutant mice. α-actinin staining (Zdisc pattern) was used as sarcomeric localization control (Fig. 6A,B).

Abnormal Ryr2 and Dhpr levels are usually followed by alterations in Ca 2+ -release, one of the most common underlying mechanisms in heart failure. Often, DCM and Ca 2+ -handling defects are associated with T-tubules remodeling, which in turns reflects on the localization of Dhprs. This delocalization creates orphaned Ryr2 on the SR, impeding their activation [START_REF] Spinozzi | Nexilin Is Necessary for Maintaining the Transverse-Axial Tubular System in Adult Cardiomyocytes[END_REF][START_REF] Lipsett | Cardiomyocyte substructure reverts to an immature phenotype during heart failure[END_REF]. To understand if the reduction in the expression of

DISCUSSION

In this study, we characterized the cardiac phenotype induced by the deletion of the is7 in titin. Physiological and histological analyses revealed a progressive dilation of the left ventricle with decreased function and presence of extensive fibrosis. Morphological observations by electron microscopy showed alterations in the sarcomere and in mitochondrial shape and organization. Finally, analyses of the molecular mechanisms responsible for the cardiac defects in ΔMex5 demonstrated that the absence of the is7 alters the sarcomeric organization of the molecule and perturbs interactions with titin partners, ultimately leading to the abnormal expression of Excitation/Contraction (EC)-coupling proteins.

Heart contraction and relaxation cycles are possible owing to a perfect balance of mechanical force and elasticity. At the sarcomere level, the protein that most contributes to the elasticity of the cardiomyocyte is titin. Analysis of ΔMex5 hearts indicated no change in heart stiffness, consistent with the fact that the corresponding region is not involved in titin elastic properties compared to other regions of the molecule. The large sarcomere length inhomogeneity and the observed sarcomere alterations may be due to modifications of the spatial organization of the M-line segment of titin, known to have a role as a shock absorber [START_REF] Lange | The M-band: The underestimated part of the sarcomere[END_REF], making the sarcomere more susceptible to contraction-induced damages. The decrease in sarcomere length could also be due to fibrosis that makes sarcomeres resistant to stretch. In addition, there is evidence of reorganization of the topology of titin domains with respect to the sarcomere. It is unlikely that the removal of the is7 would be directly responsible for the observed reduction in sarcomere length because of its small size. It is more plausible that the presence or absence of the is7 determines the modality of interaction between titin and other proteins at the M-band [START_REF] Lange | The M-band: The underestimated part of the sarcomere[END_REF]. If this is the case, the altered interaction, when the is7 is absent, may change the anchoring of the titin molecules in the M-line, explaining the wider distance between M8M9 epitopes. Another possible explanation is that destabilization of the C-terminal part of titin, induced by the loss of the is7, bends the titin filament toward the N-terminal part, preventing it to span straight across the sarcomere.

WB analyses showed also that the absence of the is7 destabilizes the is7-adjacent domains.

We previously reported that a pathological cleavage can occur in the is4-is5 regions of titin in some titinopathies [START_REF] Charton | CAPN3-mediated processing of Cterminal titin replaced by pathological cleavage in titinopathy[END_REF], Sarparanta et al., 2010). WB analyses performed on different cells fractions did not show any pathological cleavage of titin C-terminus (data not shown), suggesting that a similar event is not happening here. Most likely, the destabilization might be due to partial degradation of the domains, rather than a "total" cleavage.

Intriguingly, molecular analyses showed that the is7 deletion impacts the expression of M10 partners of titin, Obscn and Cmya5, at both the RNA and protein levels, as demonstrated by their down-regulation in ΔMex5 hearts. Since the decrease happens at both levels, the defect could originate solely from a transcriptional dysregulation with no change in protein stability due to the lack of protein interactions. Therefore, these data suggest a transcriptional control that adapts the level of transcript to the level of the binding domain. Interestingly, Cmya5 was previously shown to be down-regulated in several muscle and cardiac disorders such as tibial muscular dystrophy, limb-girdle muscular dystrophy 2J and 2A [START_REF] Sarparanta | Interactions with M-band titin and calpain 3 link myospryn (CMYA5) to tibial and limb-girdle muscular dystrophies[END_REF] and Duchenne muscular dystrophy [START_REF] Tkatchenko | Identification of altered gene expression in skeletal muscles from Duchenne muscular dystrophy patients[END_REF]. Mutations in CMYA5 have also been associated with cardiac pathologies [START_REF] Xu | Investigation of Pathogenic Genes in Chinese sporadic Hypertrophic Cardiomyopathy Patients by Whole Exome Sequencing[END_REF][START_REF] Nakagami | Gene polymorphism of myospryn (cardiomyopathy-associated 5) is associated with left ventricular wall thickness in patients with hypertension[END_REF]. Different mutations in OBSCN were identified to be responsible for dilated cardiomyopathy [START_REF] Marston | OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency[END_REF]. Besides the sarcomere abnormalities, alterations in the expression levels of these proteins may also contribute to the phenotype, considering the role of the proteins and their implication in muscle diseases. In fact, it is well known that alterations in the interaction between Obscn and titin may results in cardiac defects [START_REF] Bang | The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system[END_REF][START_REF] Grogan | Deletion of obscurin immunoglobulin domains Ig58/59 leads to age-dependent cardiac remodeling and arrhythmia[END_REF], while alterations in Obscn expression are associated with defects in SR organization and dysregulated Ca 2+ -cycling [START_REF] Lange | Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum[END_REF][START_REF] Hu | Deregulated Ca(2+) cycling underlies the development of arrhythmia and heart disease due to mutant obscurin[END_REF]. In addition, Cmya5 interacts with Ryr2, also participating in the clustering of the channel in the dyads [START_REF] Benson | Ryanodine receptors are part of the myospryn complex in cardiac muscle[END_REF]. It is important to mention that Cmya5 is mainly, but not exclusively, localized in the Z-disc [START_REF] Sarparanta | Biology of myospryn: what's known?[END_REF]. However, since an interaction with titin M10 domain has been demonstrated [START_REF] Sarparanta | Interactions with M-band titin and calpain 3 link myospryn (CMYA5) to tibial and limb-girdle muscular dystrophies[END_REF], it cannot be excluded that variations in this interaction may reflect on the function in the Cmya5 localized in the Z-disc.

Interestingly, WB data showed that Ryr2 and Dhpr protein levels were decreased in ΔMex5 hearts. These two proteins are strictly associated to each other in the dyad and are the main ion channels responsible for Ca 2+ -release during EC-coupling. The loss of is7 + titin in cardiomyocytes decreases Obscn and Cmya5 expression, and we speculated that this alteration in turn induces a down-regulation of Ryr2 that sequentially alters Dhpr protein levels. Notably, it is known that reduction in Ryr2 and Dhpr protein levels can also be the result of their uncoupling, induced by abnormal organization of the dyads due to T-tubules remodeling [START_REF] Zhang | Ultrastructural uncoupling between Ttubules and sarcoplasmic reticulum in human heart failure[END_REF]. However, in our mouse model, this was not the case. In fact, additional molecular analyses of ΔMex5 cardiac tissue lysates at the protein level showed no change in the junctional SR protein Jph2 or in the longitudinal SR protein Serca2, two proteins that are well-known to be reduced in severe DCM where T-tubule remodeling is present [START_REF] Liu | Nexilin Is a New Component of Junctional Membrane Complexes Required for Cardiac T-Tubule Formation[END_REF], Spinozzi et al., 2020, Liu et al., 2020[START_REF] Wu | Ultrastructural remodelling of Ca(2+) signalling apparatus in failing heart cells[END_REF][START_REF] Takeshima | Junctophilins: a novel family of junctional membrane complex proteins[END_REF][START_REF] Lipskaia | Sarcoplasmic reticulum Ca(2+) ATPase as a therapeutic target for heart failure[END_REF]. Moreover, confocal microscopy on ΔMex5 heart samples showed no localization abnormalities of either Ryr2 or Dhpr, indirectly demonstrating that dyads and T-tubules organization was not altered. Altogether, these findings allow us to assume that Ryr2 and Dhpr reduced protein expression is most likely a direct consequence of Cmya5 and Obscn abnormal levels induced by the absence of the is7 in titin, rather than just a subsequent effect of the DCM phenotype.

Conclusion

In conclusion, preventing the cardiomyocytes from expressing their preferred titin isoforms (Mex5 + ) leads to a severe cardiomyopathy, confirming that the presence of the is7 is essential for titin function in the heart. Most likely, the absence of this part of the protein destabilizes the surrounding domains and leads to the described DCM phenotype. Our data are the first to suggest a possible involvement of titin M-line region in EC-coupling regulation. Further studies on this mouse model are necessary to gain a better understanding of the potential role of titin is7 in this physiological mechanism. Altogether, these results further stress how important the integrity of the M-line region of titin is for cardiac function and provides important insights for the identification of possible novel therapeutic targets.

MATERIAL AND METHODS

Animal

The ΔMex5 (C57BL/6N-Ttn gnt1 ) mouse model used in this study has been previously described [START_REF] Charton | Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin[END_REF]. This model was bred and housed in a barrier facility with 12h light, 12-h dark cycles and provided food and water ad libitum. C57BL/6N (WT) mice, strain-matched control, were obtained from Charles River laboratories. Males were included in the study at different ages according to the experiment. All mice were handled according to the European guidelines for the human care and use of experimental animals, and animal experimentations were approved by the Ethical Committee for Animal Experimentation C2AE-51 of Evry under number APAFIS#1720 and APAFIS#19736.

For histological and molecular analysis of mouse tissues, specimens were collected immediately after sacrifice, snap frozen in liquid-nitrogen-cooled isopentane and stored at -80°C.

Echocardiography

Conventional echocardiography was performed on anesthetised mice using a Vevo 770 (Visual sonics) with a 30 MHz cardiac probe (RMV707B). During the procedure, heart rate (HR) and temperature were monitored. For echocardiography recording, sweep speed, depth, focus and gain settings were optimized to obtain the most defined acquisitions. Two-D and M-mode echocardiography were obtained manually from long parasternal axis view at the level of the largest LV diameter (at the level of the papillary level). LV dimensions (LV enddiastolic diameter = LVEDD, LV end-systolic diameter = LVESD), posterior wall (PW) and interventricular septum (IVS) wall thickness were measured using the leading edge convention of the American Society of Echocardiography (ASE). SF and EF of the left ventricle, PW thickening (PWth) and LV mass were calculated from the above dimensions.

Pressure Volume loops

In vivo pressure-volume (PV) analysis was performed in four-month-old mice using a SciSense Advantage Admittance Derived Volume Measurement System and 1.2F catheters with 4.5 mm electrode spacing (SciSense, London, Ontario, Canada). Mice were anesthetized and ventilated with 1.5-3% isoflurane using a Physiosuite Rovent (Kent Scientific). Body temperature under anesthesia was maintained at 37°C using a Physiosuite RightTemp (Kent Scientific). A lateral incision through the skin and muscle was made below the ribcage and the diaphragm was cut in order to expose the apex of the heart. A small puncture was made in the apex of the left ventricle using a 28G needle and the 1.2F catheter was inserted into the LV. Baseline functional parameters were assessed during a pause in ventilation to avoid respiratory influences. For load-independent indices, including the end-systolic and enddiastolic pressure-volume relationships, the inferior vena cava was temporarily occluded to vary the preload conditions. Data acquisition and analysis was performed using LabScribe 3 (iWorx, Dover, NH) and curve fitting was performed with MATLAB (MathWorks, Natick, MA). Diastolic PV data was analyzed using a monoexponential fit with constant ( = + C) with the exponent (β) reported as the stiffness.

Electron Microscopy and Immunoelectron Microscopy

Intact LV papillary muscles from four-month-old male ΔMex5 mice (n= 4) and littermate WT controls (n= 4) were stretched from the slack length and processed for transmission electron microscopy (TEM). Briefly, fixation of cardiac tissue was performed with a mixture of 3.7 % paraformaldehyde, 3% glutaraldehyde and 0.2 % of tannic acid in 10 mM PBS, pH 7. Labeling of titin M-line epitopes by immunoelectron microscopy (IEM) was performed on bundles of skinned papillary muscle from WT (n= 4) and ΔMex5 (n= 4) mice, stretched to ~ 25 % in relaxing solution and processed by the pre-embedding technique previously described [START_REF] Tonino | The giant protein titin regulates the length of the striated muscle thick filament[END_REF]. Cardiac tissue bundles were fixed in 3.7 % paraformaldehyde in 10 mM PBS for 30 min at 4 C and after rinsed in PBS were incubated with glycine 50 mM in the same buffer and then blocked with 0.5 % bovine serum albumin (BSA) in PBS containing protease inhibitors and 0.05 % Tween 20, 1 h at 4 C. After blocking, muscle bundles were incubated 48 h at 4 C with the following polyclonal titin domains antibodies from rabbit:

anti-M8M9 (0.35 mg/mL, BioGenes, Germany) and anti-is7 (0.724 mg/mL, [START_REF] Charton | Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin[END_REF]), then followed by overnight incubation with secondary antibody AlexaFluor-568 goat anti-rabbit IgG (ab175471, Invitrogen, 2mg/mL). Controls were performed in muscle bundles by replacing each primary antibody with PBS/0.5% BSA solution containing protease inhibitors. Afterward, bundles of cardiac tissue were fixed with 3 % glutaraldehyde and processed for TEM as explained above. Digital images were obtained and M8M9 and is7 epitope distances were measured across the M-line from density plot profiles using ImageJ 1.49v (NIH-USA). These values were then corrected for shrinkage (caused by the TEM procedure) using the A-band width known value of 1.6 µm [START_REF] Sosa | Ultrastructure of skeletal muscle fibers studied by a plunge quick freezing method: myofilament lengths[END_REF].

filters were used except for a notch filter cutting the laser wavelengths. Voxel size: 2.5x2;5x2 µm.

The QTSPIM software (PhaseView, France) was used for image acquisition. To cover the full XY-surface of the heart, a 2x2 tile scan was manually performed for each sample. Tiles stiching was performed with IMARIS Stitcher software (Bitplane -Oxford Instruments, UK).

The reconstructed stacks for each heart were processed with IMARIS software (Bitplane -Oxford Instruments, UK), which automatically created a three-dimensional object for volume calculations.

RNA extraction

Total RNA extraction was performed from frozen tissues by the Trizol™ method (Thermo Fisher Scientific, Waltham, MA). Extracted RNA was dissolved in 20µl of RNase-free water and treated with Free DNA kit (Ambion) to remove residual DNA. Total RNA was quantified using a Nanodrop spectrophotometer (ND8000 Labtech, Wilmington Delaware).

RNA-sequencing

RNA from 3 WT mice and 3 ΔMex5 mice heart at four months were sequenced. RNA concentration was measured on a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA quality (RIN≥7) was controlled using an Agilent RNA 6000 Pico Kit on a 2100 Bioanalyzer instrument (Agilent Technologies). The sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced according to the Illumina protocol. The reads were paired using Fastq-pair and aligned onto the mouse genome (mm10) using STAR aligner. The analysis was performed using DESeq2 library on R software. Genes were considered as significantly dysregulated when absolute value of log2 fold change was higher than 0.5 and adjusted p-value smaller than 0.05, genes with low read count (<10) were filtred out. Gene Set Enrichement Analysis was performed using GSEABase library on R with Gene Ontology Biological Process (C5) gene sets. Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, Califorinia, USA) was used to investigate enriched toxicity pathways.

qRT-PCR analyses

One µg of RNA was reverse-transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and a mixture of random oligonucleotides and oligo-dT. Real-time PCR was performed using LightCycler480 (Roche, Basel, Switzerland) with 0.2mM of each primer and 0.1 mM of the probe according to the protocol Absolute QPCR Rox Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primers and Taqman probes (Eurogentec, Liege, Belgium) used are listed in Table S10. Data from the ubiquitous acidic ribosomal phosphoprotein (P0) was used to normalize the data across samples. The primer pairs and Taqman probes used for P0 amplification were: m181PO.F (5'-CTCCAAGCAGATGCAGCAGA-3'), m267PO.R (5'-ACCATGATGCG CAAGGCCAT-3') and m225PO.P (5' -CCGTGGTGCTGATGGGCAAGA A-3'). Each experiment was performed in duplicate. Quantification cycle (Cq) values were calculated with the LightCycler® 480 SW 1.5.1 using 2nd Derivative Max method.

Protein extraction, Coomassie Blue gel and Western Blot

Proteins were extracted from LV tissues pulverized in liquid nitrogen by solubilisation in urea buffer (in mol/L): 8 urea, 2 thiourea, 0.05 tris-HCl, 0.075 dithiothreitol with 3% SDS and 0.03% bromophenol blue, pH 6.8) and 50% glycerol with protease inhibitors ((in mmol/L): 0.04 E64, 0.16 leupeptin and 0.2 PMSF) at 60 °C for 10 min. Samples were centrifuged at 13,000 RPM for 5 min and stored at -80 °C.

The titin isoform visualization was performed by loading solubilized samples on agarose gels (1%). After electrophoresis at 15 mA per gel for 3h20, the gels were stained using Coomassie brilliant blue and scanned using a commercial scanner. For titin western blot, solubilized samples were run on a 0.8% agarose gel, then transferred onto polyvinylidene difluoride membranes using a semi-dry transfer unit (Trans-Blot Cell, Bio-Rad). Blots were stained with Ponceau S to visualize the total protein transferred. Blots were then probed with primary antibodies listed in Table S9 followed by secondary antibodies conjugated with infrared fluorescent dyes.

For the other proteins, the samples were prepared and separated following the NuPAGE 4 to 12% Bis-Tris or 3 to 8% Tris-Acetate Gel protocol (ThermoFisher) depending on the size of the protein, and transferred with iBlot 2 Dry Blotting System (ThermoFisher). Detection of proteins were performed using standard Odyssey protocol with primary specific antibodies listed in Table S9 followed by secondary antibodies conjugated with infrared fluorescent dyes. Blots were scanned using an Odyssey Infrared Imaging System (Li-COR Biosciences).

The quantification of each protein was performed in comparison to GAPDH protein quantity in the sample, using Image Studio software (Li-COR Biosciences).

Data and Statistical Analysis

The characterization parameters were compared between WT and ΔMex5 groups by nonparametric unpaired Mann-Whitney test. Data are expressed as mean ± SEM. Values of P < 0.05 were considered as statistically significant. Prism package (GraphPad Software) and R software were used for data analysis.

ACKNOWLEGMENTS

We are grateful to Histology team of Généthon for technical support. This work was supported by the Fundation Leducq project: Mechanical Triggers to Programmed Cell Death in Cardiomyocytesand how to prevent their Action in Failing Hearts (13CVD04), by

Figure 1 :Figure 2 :

 12 Figure 1: Cardiac functional characterization of ΔMex5 mice. A) Schematic

Figure 3 :Figure 4 :

 34 Figure 3: Sarcomeric characterization of ΔMex5 mice. A) qPCR results for titin relative

  2, for 1 h at 4  C. This step was followed by a postfixation in 1 % OsO 4 in the same buffer for 30 min at 4  C. Samples were then dehydrated in an ethanol graded series, infiltrated with propylene oxide, and transferred to a mix of propylene oxide/Araldite 502/Embed 812 resin polymerized for 48 h at 60  C. Ultrathin, 90 nm longitudinal sections were obtained with a Reichert-Jung

	ultramicrotome and contrasted with 1 % potassium permanganate and lead citrate. Specimens
	were observed in a TECNAI Spirit G2 transmission electron microscope (FEI, Hillsboro, OR)
	with a side-mounted AMT Image Capture Engine V6.02 (4Mpix) digital camera operated at
	100 kV. Digital images were stored for quantitative analyses of mitochondria morphology and
	measurements of sarcomere length with ImageJ 1.49v (NIH-USA).

these two proteins could be the result of a "feedback mechanism" due to a Ryr2/Dhpr uncoupling, immunofluorescence analyses using antibodies against Ryr2 and Dhpr were performed. The data showed no apparent mislocalization of either Ryr2, or Dhpr in ΔMex5 hearts when compared to WT (Fig.

6C, D), suggesting absence of remodeling in either the Ttubules or the SR. Therefore, these results demonstrated a specific decrease in Ryr2 and Dhpr 10 expression with no reorganization of either T-tubules or junctional SR.

European Commission MSCA-RISE-2014 Marie Slodowska Staff exchange project: Muscle Stress Relief (645648), and by NHLBI grant R35HL144998.

DATA AVAILABILITY

RNAseq raw data are available upon reasonable request by contacting the corresponding author.

Histology and Immunohistochemistry

Transverse cryosections (8 µm thickness) were prepared from frozen muscles, air dried, and stored at -80°C. Mouse sections were processed for Hematoxylin-Phloxine-Saffron (HPS) or Sirius red histological staining. Sections were visualized on an Axioscan Z1 automated slide scanner (Zeiss, Germany), using a Plan APO 10X / 0.45 NA objective. Quantification was performed by using ImageJ software.

Sections were immunostained overnight at 4°C with primary antibodies listed in Table S9.

After washing three times with PBS, muscle sections were incubated with a goat -secondary antibody conjugated with Alexa Fluor 594 or 647 dye (Molecular probe, dilution 1:1000) for 1 hour at room temperature (RT). Sections were mounted with DAPI-fluoromount-G (SouthernBiotech) and visualized on a LEICA TCS-SP8 confocal microscope (Leica, Germany) using a 63X APO CS2 1.4NA objective.

Light Sheet Imaging

Clarification of heart samples was performed following iDISCO clearing protocol [START_REF] Renier | iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging[END_REF]. Images were acquired using an Alpha3 (α 3 ) Light Sheet Microscope (PhaseView, France) equipped with a XLFLUOR objective, 2X magnitude, wd 340mm, 0.14 NA (Olympus, France), and with an ORCA FLASH 4.0 SCMOS camera (Hamamatsu, Japan).

Samples were immerged in a 10mL sample chamber containing Dibenzyl Ether solvent (DBE). Sidewalls of the sample holder are glass coverslips (0,17mm). Illumination arms located at each side of the sample holder create the excitation light sheet using two Plan-Neofluar 2.5X magnitude objectives (Zeiss, Germany). The sample is laid down on a motorized sample-holder, within the DBE-filled sample chamber, allowing for the threedimensional scan of the sample. To assess heart tissue volume, heart autofluorescence was used: images were acquired using a 488nm Laser line and to maximize the signal, no emission plot representation of Gene Set Enrichment Analysis of GO biological process ontology. C), D) Gene Set Enrichment Analysis (GSEA) enrichment score curves of GO leukocyte migration and GO mitochondrial respiratory chain complex assembly. E) Toxicity functions significantly activated in the heart of the ΔMex5 model.