Exertional Heat Stroke (EHS) is a life-threatening disease defined by severe hyperthermia, with a body temperature above 40°C, associated with a sudden neurological dysfunction, observed in healthy individuals exposed to intensive physical exercise. EHS shares many clinical and biological similarities with Malignant Hyperthermia (MH), a pharmacogenetic disorder associated with mutations in the RYR1, CACNA1S and STAC3 genes involved in calcium release required for skeletal muscle contraction. Several pieces of evidence support the existence of inherited genetic mutations that predispose some patients to EHS, but to date, the genetic bases of EHS remain poorly documented. Whole Exome Sequencing on a cohort of soldiers presenting with an EHS episode led to the identification of variants in the CACNA2D4 gene, encoding the α2δ4 isoform of regulatory subunits of voltage-gated calcium channels. In vitro expression of these variants suggested they can affect the function of the protein, and modulate the properties of the Cav1.1 channel. A mouse model with a knock-in for the S299R variant showed EHS-like crisis with signs of rhabdomyolysis and an elevation in core body temperature when submitted to an intense exercise protocol. We show that a CACNA2D4 transcript is expressed in mouse skeletal muscle and that the presence of the S299R variant in α2δ4 induces a modification in the calcium flux triggered by muscle cells depolarization. Altogether, our data point to the involvement of the CACNA2D4 gene in skeletal muscle function and suggest that its genetic variants could be responsible for EHS susceptibility. ### Competing Interest Statement The authors have declared no competing interest. Université Grenoble Alpes, https://ror.org/02rx3b187 Inserm, https://ror.org/02vjkv261 AFM-Telethon

The RyR1 calcium release channel is a key player in skeletal muscle excitation–contraction coupling. Mutations in the RYR1 gene are associated with congenital myopathies. Recently, a role of RyR1 in myotubes differentiation has been proposed and attributed to its calcium channel function, which nonetheless remains to be clearly demonstrated. In order to clarify RyR1 role in myogenesis, we have developed an in vitro model, the so-called RyR1-Rec myotubes, which are mouse primary myotubes with an inducible decrease in RyR1 protein amount and in RyR1-mediated calcium release. Using this model, we showed that the RyR1 protein decrease was responsible for an increase in both differentiation and fusion, from the RNA level to the morphological level, without affecting the myogenic factors MyoD and MyoG. Although an increase in mTOR pathway was observed in RyR1-Rec myotubes, it did not seem to be responsible for the role of RyR1 in myogenesis. Additionally, even if modulation of intracellular calcium level affected RyR1-Rec myotubes differentiation, we have shown that the role of RyR1 in myogenesis was independent of its calcium channel function. Therefore, our findings indicate that, besides its pivotal role as a calcium channel responsible for muscle contraction, RyR1 fulfills a calcium-independent inhibitor function of myogenesis.

BACKGROUND:Although genetically-engineered mouse models are revolutionizing our understanding of numerous human diseases, some of them fail to reproduce or to mimic the human condition or even exhibit distinct disease features depending on the mouse genetic background, on the environment conditions, and/or on unknown parameters. OBJECTIVE:Experiments aimed at further characterizing the muscle defects associated with the I-T substitution at position 4898 of the human type 1 ryanodine receptor (RyR1) protein sequence, responsible for central core disease in affected patients, to use this model for therapeutic development. RyR1 is a cationic channel in the sarcoplasmic reticulum membrane that is responsible for the Ca2+ release flux that triggers muscle contraction. The above I-T change was previously described to alter RyR1 channel permeation so as to produce muscle weakness. METHODS:We used the corresponding I4895T mouse model, previously shown unviable in the homozygous form, and with heterozygous animals suffering from depressed RyR1-mediated Ca2+ flux and muscle force production. We performed a full characterization, at the molecular level of the RYR1 gene and transcript, and at the functional level at the isolated fiber or whole animal levels. RESULTS:We found no significant deficit in the heterozygous animals, from force and activity parameters at the whole organism level, to contraction of isolated muscles and Ca2+ release in single isolated muscle fibers. CONCLUSIONS:Our results prompt the need for caution when using this model, and point to its potential limited relevance for preclinical studies.

Le depistage genetique des maladies rares permet d'identifier le(s) gene(s) responsable(s) chez environ 50 % des patients. Les cas restants se trouvent dans une impasse diagnostique, car les connaissances actuelles ne permettent pas d'identifier le bon gene ou de determiner si le(s) variant(s) detecte(s) sur le gene est(sont) pathogene(s) ou benin(s). On parle alors de << variants de signification inconnue >> (VSI). Dans le cas des maladies neuromusculaires, le gene RYR1 est souvent mis en cause, mais la majorite de ses variants identifies sont classes comme VSI, ce qui met a mal le diagnostic precis des patients. Notre projet vise a creer un pipeline d'analyses en combinant differentes approches (l'intelligence artificielle, les donnees de biologies structurales et les analyses fonctionnelles), afin d'obtenir une classification des variants de RYR1 plus efficace et d'ameliorer le diagnostic genetique des maladies liees a ce gene. Genetic screening of rare diseases allows identification of the responsible gene(s) in about 50% of patients. The remaining cases are in a diagnostic deadlock as current knowledge fails to identify the correct gene or determine if the detected variant on the gene is pathogenic. These are named "variants of unknown significance" (VUS). In the case of neuromuscular diseases, the RYR1 gene is often implicated, with the majority of variants classified as VUS, requiring reliable classification to help patient diagnosis. Our project aims to create an efficient classification pipeline, integrating artificial intelligence, structural biology data, and functional analyses to enhance genetic diagnosis of RYR1-related diseases.

More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene causing various myopathies collectively known as “RYR1-related myopathies.” There is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of central core disease due to a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 allele by guiding CRISPR-Cas9 cleavages onto frequent single-nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant RYR1 allele and identified specific CRISPR-Cas9 guide RNAs. Lentiviruses encoding these guide RNAs and the SpCas9 nuclease were used to transduce immortalized patient myoblasts, inducing the specific deletion of the mutant RYR1 allele. The efficiency of the deletion was assessed at DNA and RNA levels, and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides in cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective, and could apply potentially to 20% of all patients with a RYR1 mutation.

Le dépistage génétique des maladies rares permet d’identifier le(s) gène(s) responsable(s) chez environ 50 % des patients. Les cas restants se trouvent dans une impasse diagnostique, car les connaissances actuelles ne permettent pas d’identifier le bon gène ou de déterminer si le(s) variant(s) détecté(s) sur le gène est(sont) pathogène(s) ou bénin(s). On parle alors de « variants de signification inconnue » (VSI). Dans le cas des maladies neuromusculaires, le gène RYR1 est souvent mis en cause, mais la majorité de ses variants identifiés sont classés comme VSI, ce qui met à mal le diagnostic précis des patients. Notre projet vise à créer un pipeline d’analyses en combinant différentes approches (l’intelligence artificielle, les données de biologies structurales et les analyses fonctionnelles), afin d’obtenir une classification des variants de RYR1 plus efficace et d’améliorer le diagnostic génétique des maladies liées à ce gène.

Type 1 ryanodine receptor (RyR1) is the intracellular calcium channel responsible for skeletal muscle contraction. Mutations in the RYR1 gene can lead to RyR1-related myopathies (RyR1-RM), a group of genetic disorders. They are characterised by abnormal calcium homeostasis and muscle weakness of variable severity. Currently, there is no treatment for these pathologies. This study aims to present proof of concept for gene therapy approaches. Immortalized skeletal muscle cells from patients affected by RyR1-Related myopathies have been edited using Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9). CRISPR guides targeting different part of the gene were design in order to adapt the strategy depending on the patient's mutation and its localisation. The consequences of the expected editing will be verified at the DNA, RNA and functional level. The objective is to determine if these therapeutic approaches could improve calcium release in patient's cells and skeletal muscle contraction.

The expression of the Huntingtin protein, well known for its involvement in the neurodegenerative Huntington's disease, has been confirmed in skeletal muscle. The impact of HTT deficiency was studied in human skeletal muscle cell lines and in a mouse model with inducible and muscle-specific HTT deletion. Characterization of calcium fluxes in the knock-out cell lines demonstrated a reduction in excitation-contraction (EC) coupling, related to an alteration in the coupling between the dihydropyridine receptor and the ryanodine receptor, and an increase in the amount of calcium stored within the sarcoplasmic reticulum, linked to the hyperactivity of store-operated calcium entry (SOCE). Immunoprecipitation experiments demonstrated an association of HTT with junctophilin 1 (JPH1) and stromal interaction molecule 1 (STIM1), both providing clues on the functional effects of HTT deletion on calcium fluxes. Characterization of muscle strength and muscle anatomy of the muscle-specific HTT-KO mice demonstrated that HTT deletion induced moderate muscle weakness and mild muscle atrophy associated with histological abnormalities, similar to the phenotype observed in tubular aggregate myopathy. Altogether, this study points toward the hypotheses of the involvement of HTT in EC coupling via its interaction with JPH1, and on SOCE via its interaction with JPH1 and/or STIM1.

Background Congenital nemaline myopathies are rare pathologies characterised by muscle weakness and rod-shaped inclusions in the muscle fibres. Methods Using next-generation sequencing, we identified three patients with pathogenic variants in the Troponin T type 1 ( TNNT1 ) gene, coding for the troponin T (TNT) skeletal muscle isoform. Results The clinical phenotype was similar in all patients, associating hypotonia, orthopaedic deformities and progressive chronic respiratory failure, leading to early death. The anatomopathological phenotype was characterised by a disproportion in the muscle fibre size, endomysial fibrosis and nemaline rods. Molecular analyses of TNNT1 revealed a homozygous deletion of exons 8 and 9 in patient 1; a heterozygous nonsense mutation in exon 9 and retention of part of intron 4 in muscle transcripts in patient 2; and a homozygous, very early nonsense mutation in patient 3. Western blot analyses confirmed the absence of the TNT protein resulting from these mutations. Discussion The clinical and anatomopathological presentations of our patients reinforce the homogeneous character of the phenotype associated with recessive TNNT1 mutations. Previous studies revealed an impact of recessive variants on the tropomyosin-binding affinity of TNT. We report in our patients a complete loss of TNT protein due to open reading frame disruption or to post-translational degradation of TNT.

Objectives Exertional Heat Stroke (EHS) is one of the top three causes of sudden death in athletes. Extrinsic and intrinsic risk factors have been identified but the genetic causes still remain unclear. Our aim was to identify genes responsible for EHS, which is a necessary step to identify patients at risk and prevent crises. Design Genetic and functional laboratory studies Methods Whole Exome Sequencing (WES) was performed to search for candidate genes in a cohort of 15 soldiers who had a documented EHS episode. In silico and in vitro functional studies were performed to evaluate the effect of mutations identified in the candidate gene TRPV1. Results WES led to the identification of two missense variations in the TRPV1 gene. These variations were very rare or unreported in control databases and located in critical domains of the protein. In vitro functional studies revealed that both variations induce a strong modification of the channel response to one of its natural agonist, the capsaicin. Conclusions We evidenced mutations altering channel properties of the TRPV1 gene and demonstrated that TRPV1, which is involved in thermoregulation and nociception, is a new candidate gene for EHS. Our data provide the bases to explore genetic causes and molecular mechanisms governing the pathophysiology of EHS.

AbstractSome mutations in theRYR1gene lead to congenital myopathies, through reduction in this calcium channel expression level, but the functional whole organism consequences of reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specificRYR1deletion. Recombination in theRYR1gene resulted in a progressive reduction in the protein amount and was associated with a progressive muscle weakness and atrophy. Calcium fluxes in isolated muscle fibers were accordingly reduced. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution, membrane remodeling, associated with increase in the expression level of many proteins and inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Central Core Disease patients, pointing to common pathophysiological mechanisms related to RyR1 reduction.

Mutations in the RYR1 gene, encoding the skeletal muscle calcium channel RyR1, lead to congenital myopathies, through expression of a channel with abnormal permeability and/or in reduced amount, but the direct functional whole organism consequences of exclusive reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Tamoxifen-induced recombination in the RYR1 gene at adult age resulted in a progressive reduction in the protein amount reaching a stable level of 50% of the initial amount, and was associated with a progressive muscle weakness and atrophy. Measurement of calcium fluxes in isolated muscle fibers demonstrated a reduction in the amplitude of RyR1-related calcium release mirroring the reduction in the protein amount. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution and membrane remodeling. An increase in the expression level of many proteins was observed, as well as an inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Dusty Core Disease patients (a subtype of Central Core Disease), pointing to common pathophysiological mechanisms related to RyR1 reduction.

Several morphological phenotypes have been associated to RYR1-recessive myopathies. We recharacterized the RYR1-recessive morphological spectrum by a large monocentric study performed on 54 muscle biopsies from a large cohort of 48 genetically confirmed patients, using histoenzymology, immunohistochemistry, and ultrastructural studies. We also analysed the level of RyR1 expression in patients' muscle biopsies. We defined "dusty cores" the irregular areas of myofibrillar disorganisation characterised by a reddish-purple granular material deposition with uneven oxidative stain and devoid of ATPase activity, which represent the characteristic lesion in muscle biopsy in 54% of patients. We named Dusty Core Disease (DuCD) the corresponding entity of congenital myopathy. Dusty cores had peculiar histological and ultrastructural characteristics compared to the other core diseases. DuCD muscle biopsies also showed nuclear centralization and type1 fibre predominance. Dusty cores were not observed in other core myopathies and centronuclear myopathies. The other morphological groups in our cohort of patients were: Central Core (CCD: 21%), Core-Rod (C&R:15%) and Type1 predominance "plus" (T1P+:10%). DuCD group was associated to an earlier disease onset, a more severe clinical phenotype and a lowest level of RyR1 expression in muscle, compared to the other groups. Variants located in the bridge solenoid and the pore domains were more frequent in DuCD patients. In conclusion, DuCD is the most frequent histopathological presentation of RYR1-recessive myopathies. Dusty cores represent the unifying morphological lesion among the DuCD pathology spectrum and are the morphological hallmark for the recessive form of disease.

Adult genetic disorders causing brain lesions have been mostly described as white matter vanishing diseases. We present here the investigations realized in patients referred for psychiatric disorder with magnetic resonance imaging showing atypical basal ganglia lesions. Genetic explorations of this family revealed a new hereditary disease linked to glutathione metabolism.

In skeletal muscle, proteins of the calcium release complex responsible for the excitation-contraction (EC) coupling are exclusively localized in specific reticulum-plasma membrane (ER-PM) contact points named triads. The CRC protein triadin (T95) is localized in the sarcoplasmic reticulum (SR) subdomain of triads where it forms large multimers. However, the mechanisms leading to the steady-state accumulation of T95 in these specific areas of SR are largely unknown. To visualize T95 dynamics, fluorescent chimeras were expressed in triadin knockout myotubes, and their mobility was compared with the mobility of Sec61β, a membrane protein of the SR unrelated to the EC coupling process. At all stages of skeletal muscle cells differentiation, we show a permanent flux of T95 diffusing in the SR membrane. Moreover, we find evidence that a longer residence time in the ER-PM contact point is due to the transmembrane domain of T95 resulting in an overall triad localization.

BACKGROUND:The skeletal muscle fiber has a specific and precise intracellular organization which is at the basis of an efficient muscle contraction. Microtubules are long known to play a major role in the function and organization of many cells, but in skeletal muscle, the contribution of the microtubule cytoskeleton to the efficiency of contraction has only recently been studied. The microtubule network is dynamic and is regulated by many microtubule-associated proteins (MAPs). In the present study, the role of the MAP6 protein in skeletal muscle organization and function has been studied using the MAP6 knockout mouse line.METHODS:The presence of MAP6 transcripts and proteins was shown in mouse muscle homogenates and primary culture using RT-PCR and western blot. The in vivo evaluation of muscle force of MAP6 knockout (KO) mice was performed on anesthetized animals using electrostimulation coupled to mechanical measurement and multimodal magnetic resonance. The impact of MAP6 deletion on microtubule organization and intracellular structures was studied using immunofluorescent labeling and electron microscopy, and on calcium release for muscle contraction using Fluo-4 calcium imaging on cultured myotubes. Statistical analysis was performed using Student's t test or the Mann-Whitney test.RESULTS:We demonstrate the presence of MAP6 transcripts and proteins in skeletal muscle. Deletion of MAP6 results in a large number of muscle modifications: muscle weakness associated with slight muscle atrophy, alterations of microtubule network and sarcoplasmic reticulum organization, and reduction in calcium release.CONCLUSION:Altogether, our results demonstrate that MAP6 is involved in skeletal muscle function. Its deletion results in alterations in skeletal muscle contraction which contribute to the global deleterious phenotype of the MAP6 KO mice. As MAP6 KO mouse line is a model for schizophrenia, our work points to a possible muscle weakness associated to some forms of schizophrenia.

Recessive forms of catecholaminergic polymorphic ventricular tachycardia (CPVT) are induced by mutations in genes encoding triadin or calsequestrin, two proteins that belong to the Ca2+ release complex, responsible for intracellular Ca2+ release triggering cardiac contractions. To better understand the mechanisms of triadin-induced CPVT and to assay multiple therapeutic interventions, we used a triadin knockout mouse model presenting a CPVT-like phenotype associated with a decrease in calsequestrin protein level. We assessed different approaches to rescue protein expression and to correct intracellular Ca2+ release and cardiac function: pharmacological treatment with kifunensine or a viral gene transfer-based approach, using adeno-associated virus serotype 2/9 (AAV2/9) encoding the triadin or calsequestrin. We observed that the levels of triadin and calsequestrin are intimately linked, and that reduction of both proteins contributes to the CPVT phenotype. Different combinations of triadin and calsequestrin expression level were obtained using these therapeutic approaches. A full expression of each is not necessary to correct the phenotype; a fine-tuning of the relative re-expression of both triadin and calsequestrin is required to correct the CPVT phenotype and rescue the cardiac function. AAV-mediated gene delivery of calsequestrin or triadin and treatment with kifunensine are potential treatments for recessive forms of CPVT due to triadin mutations.

Dent-2 disease and Lowe syndrome are two pathologies caused by mutations in inositol polyphosphate 5-phosphatase OCRL gene. Both conditions share proximal tubulopathy evolving to chronic kidney failure. Lowe syndrome is in addition defined by a bilateral congenital cataract, intellectual disability, and hypotonia. The pathology evolves in two decades to a severe condition with renal complications and a fatal issue. We describe here a proof of principle for a targeted gene therapy on a mutation of the OCRL gene that is associated with Lowe syndrome. The affected patient bears a deep intronic mutation inducing a pseudo-exon inclusion in the mRNA, leading to a OCRL-1 protein loss. An exon-skipping strategy was designed to correct the effect of the mutation in cultured cells. We show that a recombinant U7-modified small RNA efficiently triggered the restoration of normal OCRL expression at mRNA and protein levels in patient's fibroblasts. Moreover, the PI(4,5)P2 accumulation and cellular alterations that are hallmark of OCRL-1 dysfunction were also rescued. Altogether, we provide evidence that the restoration of OCRL-1 protein, even at a reduced level, through RNA-based therapy represents a potential therapeutic approach for patients with OCRL splice mutations.

Julie Brocard Oriana Sarrault Didier Grunwald Isabelle Marty Cellular Myology and Pathologies, Grenoble Institute of Neurosciences, Inserm U1216, Grenoble, France Eric Denarier Physiopathology of the Cytoskeleton, Grenoble Institute of Neurosciences, Inserm U1216, Grenoble, France Julien Faure Cellular Myology and Pathologies, Grenoble Institute of Neurosciences, Inserm U1216, Grenoble, France CHU Grenoble, Biochemistry and Molecular Genetics, Grenoble, France 13 JOURNEES DE LA SOCIETE FRANCAISE DE MYOLOGIE Communication orale

Background: Central Core Disease (CCD) is a congenital myopathy often resulting from a mutation in RYR1 gene. Mutations in RyR1 can increase or decrease channel activity, or induce a reduction in the amount of protein. The consequences of a single mutation are sometimes multiple and the analysis of the functional effects is complex. Objective: The consequences of the p.Y4864H mutation identified in a CCD patient have been studied regarding both RyR1 function and amount. Methods: The amount of RyR1 in human and mouse muscles was evaluated using qRT-PCR and quantitative Western blot, and calcium release was studied using calcium imaging on primary cultures. The results were compared between human and mouse. Results: The p.Y4864H mutation induced an alteration of calcium release, and in addition was associated to a reduction in the amount of RyR1 in the patient’s muscle. This suggests two possible pathophysiological mechanisms: the alteration of calcium release could result from a modification of the channel properties of RyR1 or from a RyR1 reduction. In order to discriminate between the two hypotheses, we used the heterozygous RyR1 knockout (RyR1+/–) mouse model showing a comparable RyR1 protein reduction. No reduction in calcium release was observed in primary muscle culture from these mice, and no muscle weakness was measured. Conclusions: Because the reduction in the amount of RyR1 protein has no functional consequences in the murine model, the muscle weakness observed in the patient is most likely the result of a modification of the calcium channel function of RyR1 due to the p.Y4864H mutation.