Molecular basis of impaired extraocular muscle function in a mouse model of congenital myopathy due to compound heterozygous Ryr1 mutations.

Mutations in the RYR1 gene are the most common cause of human congenital myopathies, and patients with recessive mutations are severely affected and often display ptosis and/or ophthalmoplegia. In order to gain insight into the mechanism leading to extraocular muscle (EOM) involvement, we investigated the biochemical, structural and physiological properties of eye muscles from mouse models we created knocked-in for Ryr1 mutations. Ex vivo force production in EOMs from compound heterozygous RyR1p.Q1970fsX16+p.A4329D mutant mice was significantly reduced compared with that observed in wild-type, single heterozygous mutant carriers or homozygous RyR1p.A4329D mice. The decrease in muscle force was also accompanied by approximately a 40% reduction in RyR1 protein content, a decrease in electrically evoked calcium transients, disorganization of the muscle ultrastructure and a decrease in the number of calcium release units. Unexpectedly, the superfast and ocular-muscle-specific myosin heavy chain-EO isoform was almost undetectable in RyR1p.Q1970fsX16+p.A4329D mutant mice. The results of this study show for the first time that the EOM phenotype caused by the RyR1p.Q1970fsX16+p.A4329D compound heterozygous Ryr1 mutations is complex and due to a combination of modifications including a direct effect on the macromolecular complex involved in calcium release and indirect effects on the expression of myosin heavy chain isoforms.

Bi-allelic expression of the RyR1 p.A4329D mutation decreases muscle strength in slow-twitch muscles in mice.

Mutations in the ryanodine receptor 1 (RYR1) gene are associated with several human congenital myopathies, including the dominantly inherited central core disease and exercise-induced rhabdomyolysis, and the more severe recessive phenotypes, including multiminicore disease, centronuclear myopathy, and congenital fiber type disproportion. Within the latter group, those carrying a hypomorphic mutation in one allele and a missense mutation in the other are the most severely affected. Because of nonsense-mediated decay, most hypomorphic alleles are not expressed, resulting in homozygous expression of the missense mutation allele. This should result in 50% reduced expression of the ryanodine receptor in skeletal muscle, but its observed content is even lower. To study in more detail the biochemistry and pathophysiology of recessive RYR1 myopathies, here we investigated a mouse model we recently generated by analyzing the effect of bi-allelic versus mono-allelic expression of the RyR1 p.A4329D mutation. Our results revealed that the expression of two alleles carrying the same mutation or of one allele with the mutation in combination with a hypomorphic allele does not result in functionally equal outcomes and impacts skeletal muscles differently. In particular, the bi-allelic RyR1 p.A4329D mutation caused a milder phenotype than its mono-allelic expression, leading to changes in the biochemical properties and physiological function only of slow-twitch muscles and largely sparing fast-twitch muscles. In summary, bi-allelic expression of the RyR1 p.A4329D mutation phenotypically differs from mono-allelic expression of this mutation in a compound heterozygous carrier.

Quantitative reduction of RyR1 protein caused by a single-allele frameshift mutation in RYR1 ex36 impairs the strength of adult skeletal muscle fibres.

Here we characterized a mouse model knocked-in for a frameshift mutation in RYR1 exon 36 (p.Gln1970fsX16) that is isogenic to that identified in one parent of a severely affected patient with recessively inherited multiminicore disease. This individual carrying the RYR1 frameshifting mutation complained of mild muscle weakness and fatigability. Analysis of the RyR1 protein content in a muscle biopsy from this individual showed a content of only 20% of that present in a control individual. The biochemical and physiological characteristics of skeletal muscles from RyR1Q1970fsX16 heterozygous mice recapitulates that of the heterozygous parent. RyR1 protein content in the muscles of mutant mice reached 38% and 58% of that present in total muscle homogenates of fast and slow muscles from wild-type (WT) littermates. The decrease of RyR1 protein content in total homogenates is not accompanied by a decrease of Cav1.1 content, whereby the Cav1.1/RyR1 stoichiometry ratio in skeletal muscles from RyR1Q1970fsX16 heterozygous mice is lower compared to that from WT mice. Electron microscopy (EM) revealed a 36% reduction in the number/area of calcium release units accompanied by a 2.5-fold increase of dyads (triads that have lost one junctional sarcoplasmic reticulum element); both results suggest a reduction of the RyR1 arrays. Compared to WT, muscle strength and depolarization-induced calcium transients in RyR1Q1970fsX16 heterozygous mice muscles were decreased by 20% and 15%, respectively. The RyR1Q1970fsX16 mouse model provides mechanistic insight concerning the phenotype of the parent carrying the RYR1 ex36 mutation and suggests that in skeletal muscle fibres there is a functional reserve of RyR1.

STIM1 over-activation generates a multi-systemic phenotype affecting the skeletal muscle, spleen, eye, skin, bones and immune system in mice.

Strict regulation of Ca2+ homeostasis is essential for normal cellular physiology. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling basal Ca2+ levels and intracellular Ca2+ store refilling, and abnormal SOCE severely impacts on human health. Overactive SOCE results in excessive extracellular Ca2+ entry due to dominant STIM1 or ORAI1 mutations and has been associated with tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK). Both disorders are spectra of the same disease and involve muscle weakness, myalgia and cramps, and additional multi-systemic signs including miosis, bleeding diathesis, hyposplenism, dyslexia, short stature and ichthyosis. To elucidate the physiological consequences of STIM1 over-activation, we generated a murine model harboring the most common TAM/STRMK mutation and characterized the phenotype at the histological, ultrastructural, metabolic, physiological and functional level. In accordance with the clinical picture of TAM/STRMK, the Stim1R304W/+ mice manifested muscle weakness, thrombocytopenia, skin and eye anomalies and spleen dysfunction, as well as additional features not yet observed in patients such as abnormal bone architecture and immune system dysregulation. The murine muscles exhibited contraction and relaxation defects as well as dystrophic features, and functional investigations unraveled increased Ca2+ influx in myotubes. In conclusion, we provide insight into the pathophysiological effect of the STIM1 R304W mutation in different cells, tissues and organs and thereby significantly contribute to a deeper understanding of the pathomechanisms underlying TAM/STRMK and other human disorders involving aberrant Ca2+ homeostasis and affecting muscle, bones, platelets or the immune system.

Quantitative RyR1 reduction and loss of calcium sensitivity of RyR1Q1970fsX16+A4329D cause cores and loss of muscle strength.

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20 weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained 'cores' characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects.

Clinical, morphological and genetic characterization of Brody disease: an international study of 40 patients.

Brody disease is an autosomal recessive myopathy characterized by exercise-induced muscle stiffness due to mutations in the ATP2A1 gene. Almost 50 years after the initial case presentation, only 18 patients have been reported and many questions regarding the clinical phenotype and results of ancillary investigations remain unanswered, likely leading to incomplete recognition and consequently under-diagnosis. Additionally, little is known about the natural history of the disorder, genotype-phenotype correlations, and the effects of symptomatic treatment. We studied the largest cohort of Brody disease patients to date (n = 40), consisting of 22 new patients (19 novel mutations) and all 18 previously published patients. This observational study shows that the main feature of Brody disease is an exercise-induced muscle stiffness of the limbs, and often of the eyelids. Onset begins in childhood and there was no or only mild progression of symptoms over time. Four patients had episodes resembling malignant hyperthermia. The key finding at physical examination was delayed relaxation after repetitive contractions. Additionally, no atrophy was seen, muscle strength was generally preserved, and some patients had a remarkable athletic build. Symptomatic treatment was mostly ineffective or produced unacceptable side effects. EMG showed silent contractures in approximately half of the patients and no myotonia. Creatine kinase was normal or mildly elevated, and muscle biopsy showed mild myopathic changes with selective type II atrophy. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity was reduced and western blot analysis showed decreased or absent SERCA1 protein. Based on this cohort, we conclude that Brody disease should be considered in cases of exercise-induced muscle stiffness. When physical examination shows delayed relaxation, and there are no myotonic discharges at electromyography, we recommend direct sequencing of the ATP2A1 gene or next generation sequencing with a myopathy panel. Aside from clinical features, SERCA activity measurement and SERCA1 western blot can assist in proving the pathogenicity of novel ATP2A1 mutations. Finally, patients with Brody disease may be at risk for malignant hyperthermia-like episodes, and therefore appropriate perioperative measures are recommended. This study will help improve understanding and recognition of Brody disease as a distinct myopathy in the broader field of calcium-related myopathies.

Aberrant regulation of epigenetic modifiers contributes to the pathogenesis in patients with selenoprotein N-related myopathies.

Congenital myopathies are early onset, slowly progressive neuromuscular disorders of variable severity. They are genetically and phenotypically heterogeneous and caused by pathogenic variants in several genes. Multi-minicore Disease, one of the more common congenital myopathies, is frequently caused by recessive variants in either SELENON, encoding the endoplasmic reticulum glycoprotein selenoprotein N or RYR1, encoding a protein involved in calcium homeostasis and excitation-contraction coupling. The mechanism by which recessive SELENON variants cause Multiminicore disease (MmD) is unclear. Here, we extensively investigated muscle physiological, biochemical and epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, to understand the pathomechanism of MmD. We identified biochemical changes that are common in patients harboring recessive RYR1 and SELENON variants, including depletion of transcripts encoding proteins involved in skeletal muscle calcium homeostasis, increased levels of Class II histone deacetylases (HDACs) and DNA methyltransferases. CpG methylation analysis of genomic DNA of patients with RYR1 and SELENON variants identified >3,500 common aberrantly methylated genes, many of which are involved in calcium signaling. These results provide the proof of concept for the potential use of drugs targeting HDACs and DNA methyltransferases to treat patients with specific forms of congenital myopathies.

Current and future therapeutic approaches to the congenital myopathies.

The congenital myopathies - including Central Core Disease (CCD), Multi-minicore Disease (MmD), Centronuclear Myopathy (CNM), Nemaline Myopathy (NM) and Congenital Fibre Type Disproportion (CFTD) - are a genetically heterogeneous group of early-onset neuromuscular conditions characterized by distinct histopathological features, and associated with a substantial individual and societal disease burden. Appropriate supportive management has substantially improved patient morbidity and mortality but there is currently no cure. Recent years have seen an exponential increase in the genetic and molecular understanding of these conditions, leading to the identification of underlying defects in proteins involved in calcium homeostasis and excitation-contraction coupling, thick/thin filament assembly and function, redox regulation, membrane trafficking and/or autophagic pathways. Based on these findings, specific therapies are currently being developed, or are already approaching the clinical trial stage. Despite undeniable progress, therapy development faces considerable challenges, considering the rarity and diversity of specific conditions, and the size and complexity of some of the genes and proteins involved. The present review will summarize the key genetic, histopathological and clinical features of specific congenital myopathies, and outline therapies already available or currently being developed in the context of known pathogenic mechanisms. The relevance of newly discovered molecular mechanisms and novel gene editing strategies for future therapy development will be discussed.

Ca2+ handling abnormalities in early-onset muscle diseases: Novel concepts and perspectives.

The physiological process by which Ca2+ is released from the sarcoplasmic reticulum is called excitation-contraction coupling; it is initiated by an action potential which travels deep into the muscle fiber where it is sensed by the dihydropyridine receptor, a voltage sensing L-type Ca2+channel localized on the transverse tubules. Voltage-induced conformational changes in the dihydropyridine receptor activate the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum. The released Ca2+ binds to troponin C, enabling contractile thick-thin filament interactions. The Ca2+ is subsequently transported back into the sarcoplasmic reticulum by specialized Ca2+ pumps (SERCA), preparing the muscle for a new cycle of contraction. Although other proteins are involved in excitation-contraction coupling, the mechanism described above emphasizes the unique role played by the two Ca2+ channels (the dihydropyridine receptor and the ryanodine receptor), the SERCA Ca2+ pumps and the exquisite spatial organization of the membrane compartments endowed with the proteins responsible for this mechanism to function rapidly and efficiently. Research over the past two decades has uncovered the fine details of excitation-contraction coupling under normal conditions while advances in genomics have helped to identify mutations in novel genes in patients with neuromuscular disorders. While it is now clear that many patients with congenital muscle diseases carry mutations in genes encoding proteins directly involved in Ca2+ homeostasis, it has become apparent that mutations are also present in genes encoding for proteins not thought to be directly involved in Ca2+ regulation. Ongoing research in the field now focuses on understanding the functional effect of individual mutations, as well as understanding the role of proteins not specifically located in the sarcoplasmic reticulum which nevertheless are involved in Ca2+ regulation or excitation-contraction coupling. The principal challenge for the future is the identification of drug targets that can be pharmacologically manipulated by small molecules, with the ultimate aim to improve muscle function and quality of life of patients with congenital muscle disorders. The aim of this review is to give an overview of the most recent findings concerning Ca2+ dysregulation and its impact on muscle function in patients with congenital muscle disorders due to mutations in proteins involved in excitation-contraction coupling and more broadly on Ca2+ homeostasis.

Cellular, biochemical and molecular changes in muscles from patients with X-linked myotubular myopathy due to MTM1 mutations.

Centronuclear myopathies are early-onset muscle diseases caused by mutations in several genes including MTM1, DNM2, BIN1, RYR1 and TTN. The most severe and often fatal X-linked form of myotubular myopathy (XLMTM) is caused by mutations in the gene encoding the ubiquitous lipid phosphatase myotubularin, an enzyme specifically dephosphorylating phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate. Because XLMTM patients have a predominantly muscle-specific phenotype a number of pathogenic mechanisms have been proposed, including a direct effect of the accumulated lipid on the skeletal muscle calcium channel ryanodine receptor 1, a negative effect on the structure of intracellular organelles and defective autophagy. Animal models knocked out for MTM1 show severe reduction of ryanodine receptor 1 mediated calcium release but, since knocking out genes in animal models does not necessarily replicate the human phenotype, we considered it important to study directly the effect of MTM1 mutations on patient muscle cells. The results of the present study show that at the level of myotubes MTM1 mutations do not dramatically affect calcium homeostasis and calcium release mediated through the ryanodine receptor 1, though they do affect myotube size and nuclear content. On the other hand, mature muscles such as those obtained from patient muscle biopsies exhibit a significant decrease in expression of the ryanodine receptor 1, a decrease in muscle-specific microRNAs and a considerable up-regulation of histone deacetylase-4. We hypothesize that the latter events consequent to the primary genetic mutation, are the cause of the severe decrease in muscle strength that characterizes these patients.

STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility.

SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

Role of the JP45-Calsequestrin Complex on Calcium Entry in Slow Twitch Skeletal Muscles.

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La(3+)- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca(2+) concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca(2+) concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies.

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy.

Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

Characterization of excitation-contraction coupling components in human extraocular muscles.

Excitation-contraction coupling (ECC) is the physiological mechanism whereby an electrical signal detected by the dihydropyridine receptor, is converted into an increase in [Ca2+], via activation of ryanodine receptors (RyRs). Mutations in RYR1, the gene encoding RyR1, are the underlying cause of various congenital myopathies including central core disease, multiminicore disease (MmD), some forms of centronuclear myopathy (CNM) and congenital fibre-type disproportion. Interestingly, patients with recessive, but not dominant, RYR1 mutations show a significant reduction in RyR protein in muscle biopsies as well as ophthalmoplegia. This specific involvement of the extraocular muscles (EOMs) indicates that this group of muscles may express different amounts of proteins involved in ECC compared with limb muscles. In the present paper, we report that this is indeed the case; in particular the transcripts encoding RyR3, cardiac calsequestrin (CSQ2) and the α1 subunit of the cardiac dihydropyridine receptor are up-regulated by at least 100-fold, whereas excitation-coupled Ca2+ entry is 3-fold higher. These findings support the hypothesis that EOMs have a unique mode of calcium handling.

Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation-contraction coupling supramolecular complex.

The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.

Gain of function in the immune system caused by a ryanodine receptor 1 mutation.

Mutations in RYR1, the gene encoding ryanodine receptor 1, are linked to a variety of neuromuscular disorders including malignant hyperthermia (MH), a pharmacogenetic hypermetabolic disease caused by dysregulation of Ca(2+) in skeletal muscle. RYR1 encodes a Ca(2+) channel that is predominantly expressed in skeletal muscle sarcoplasmic reticulum, where it is involved in releasing the Ca(2+) necessary for muscle contraction. Other tissues, however, including cells of the immune system, have been shown to express ryanodine receptor 1; in dendritic cells its activation leads to increased surface expression of major histocompatibility complex II molecules and provides synergistic signals leading to cell maturation. In the present study, we investigated the impact of an MH mutation on the immune system by studying the RYR1Y522S knock-in mouse. Our results show that there are subtle but significant differences both in resting 'non-challenged' mice as well as in mice treated with antigenic stimuli, in particular the knock-in mice: (i) have dendritic cells that are more efficient at stimulating T cell proliferation, (ii) have higher levels of natural IgG1 and IgE antibodies, and (iii) are faster and more efficient at mounting a specific immune response in the early phases of immunization. We suggest that some gain-of-function MH-linked RYR1 mutations might offer selective immune advantages to their carriers. Furthermore, our results raise the intriguing possibility that pharmacological activation of RyR1 might be exploited for the development of new classes of vaccines and adjuvants.

Establishment of a human skeletal muscle-derived cell line: biochemical, cellular and electrophysiological characterization.

Excitation-contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present study, we characterized for the first time the excitation-contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor, whereas 3D-SIM (super-resolution structured illumination microscopy) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in Ca2+ homoeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow-twitch than fast-twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition, they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in Ca2+ homoeostasis due to mutations in genes involved in Ca2+ regulation.

ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

A novel, patient-derived RyR1 mutation impairs muscle function and calcium homeostasis in mice.

RYR1 is the most commonly mutated gene associated with congenital myopathies, a group of early-onset neuromuscular conditions of variable severity. The functional effects of a number of dominant RYR1 mutations have been established; however, for recessive mutations, these effects may depend on multiple factors, such as the formation of a hypomorphic allele, or on whether they are homozygous or compound heterozygous. Here, we functionally characterize a new transgenic mouse model knocked-in for mutations identified in a severely affected child born preterm and presenting limited limb movement. The child carried the homozygous c.14928C>G RYR1 mutation, resulting in the p.F4976L substitution. In vivo and ex vivo assays revealed that homozygous mice fatigued sooner and their muscles generated significantly less force compared with their WT or heterozygous littermates. Electron microscopy, biochemical, and physiological analyses showed that muscles from RyR1 p.F4976L homozygous mice have the following properties: (1) contain fewer calcium release units and show areas of myofibrillar degeneration, (2) contain less RyR1 protein, (3) fibers show smaller electrically evoked calcium transients, and (4) their SR has smaller calcium stores. In addition, single-channel recordings indicate that RyR1 p.F4976L exhibits higher Po in the presence of 100 µM [Ca2+]. Our mouse model partly recapitulates the clinical picture of the homozygous human patient and provides significant insight into the functional impact of this mutation. These results will help understand the pathology of patients with similar RYR1 mutations.