Susan Treves

Ryanodine receptor 1 (RyR1) is a key intracellular Ca ²⁺ release channel primarily expressed in skeletal muscle, but also in some immune cells. Gain-of-function RYR1 mutations are the most common cause of the pharmacogenetic disorder malignant hyperthermia susceptibility. Compatibly, mice knocked in for gain of function Ryr1 mutations develop malignant hyperthermia but also show subtle changes in their immune function, supporting a potential but yet to be explored effect of RyR1 mutations on the human immune system. Healthy controls and subjects classified as Malignant Hyperthermia Susceptible were enrolled in an anonymized allergy questionnaire study. PBMC and Epstein Barr virus immortalized B cells from the two cohorts were characterized for surface markers by flow cytometry and for calcium homeostasis using the intracellular calcium indicator Fluo-4. Serum samples were investigated for circulating cytokines by ELISA and Ig quantification was carried out by a clinical laboratory. Malignant hyperthermia susceptible individuals report a higher frequency of allergies, show changes in circulating immunoglobulin and cytokine levels and their circulating B cells exhibit a pre-activated state, with increased surface expression of the co-stimulatory molecules CD86 and CD40. Stimulation of EBV-immortalized B lymphocytes with the RyR1 agonist caffeine results in a tetracaine-sensitive increased surface expression of CD86, which was more pronounced in cells from malignant hyperthermia susceptible individuals. Our results support a functional role of RyR1 in B-lymphocyte Ca ²⁺ signaling and show that in humans the presence of gain of function RYR1 mutations leads to the pre-activation of B cells.

The ryanodine receptor 1 plays an essential role in skeletal muscle by releasing the calcium necessary for contraction from the sarcoplasmic reticulum and mutations in RYR1 , the gene encoding the ryanodine receptor 1 are associated with a variety of neuromuscular phenotypes, ranging from core myopathies and centronuclear myopathy to the pharmacogenetic disorder malignant hyperthermia. The muscle phenotype of the patient mainly depends on whether the mutations are dominant or recessive and on their location within the gene. Gain of function RYR1 mutations usually affect the biophysical properties of the calcium channel and are associated with malignant hyperthermia susceptibility trait with or without a myopathy. From a functional point of view, these mutations render the calcium channel oversensitive thereby facilitating its opening and allowing more calcium to be released from the sarcoplasmic reticulum. More recently exertional rhabdomyolysis has been observed in particularly athletic individuals who also carry gain of function RYR1 variants, but so far no functional studies have been performed investigating if and how such variants affect ryanodine receptor 1 calcium channels. In the present report we used an immortalized muscle cell line from an individual with exertional rhabdomyolysis carrying the heterozygous RyR1 duplication p.T4288_A4290dup in exon 91 and show that the presence of this variant is associated with morphometric changes in myotubes, enhanced myotube maturation and enhanced calcium release following membrane depolarization and pharmacological RyR1 activation. The enhanced calcium release would lead to more efficient muscle contraction but would also result in increased activation of SERCA pumps, more ATP consumption and increased enzymatic activity of the calcium dependent protease calpain 3. We hypothesize that these events are potentially the underlying (co)causes of exercise‐induced rhabdomyolysis in individuals carrying this RyR1 variant.

Muscle spindles are stretch receptors lying deep within the muscle belly involved in detecting changes in muscle length and playing a fundamental role in motor control, posture and synchronized gait. They are made up of an external capsule surrounding three to five intrafusal muscle fibres and a nuclear bag complex. Dysfunction of muscle spindles leads to abnormal proprioceptor function, which has been linked to aberrant bone and cartilage development, scoliosis, kyphosis and joint contractures. RYR1 , the gene encoding the calcium release channel of the sarcoplasmic reticulum, is the most common target of mutations linked to human congenital myopathies, a condition often accompanied by skeleton alterations and joint contractures. So far, the link between RYR1 mutations, altered muscle spindles and skeletal defects has not been investigated. To this end, we investigated heterozygous mice carrying recessive Ryr1 mutations isogenic to those present in a severely affected child. Here, we show that: (i) the RyR1 protein localizes to the polar regions of intrafusal fibres and exhibits a doubled row distribution pattern, typical for junctional sarcoplasmic reticulum proteins; (ii) muscle spindles of compound heterozygous mice show structural defects; and (iii) in intrafusal muscle fibres from dHT mice, RyR1‐mediated Ca ²⁺ release is significantly impaired and RyR1 protein content is reduced by 54%. These results support the hypothesis that Ryr1 mutations not only affect the function of extrafusal muscles, but also might affect that of intrafusal muscles. The latter may be one of the underlying causes of skeletal abnormalities seen in patients affected by recessive RYR1 mutations. image Key points: Dysfunction of muscle spindles leads to abnormal proprioceptor function, which can cause abnormal bone and cartilage development, scoliosis, kyphosis and joint contractures. Muscle spindles contain intrafusal muscle fibres, which are made up of nuclear bag and nuclear chain fibres; the polar regions of intrafusal muscle fibres contain contractile filaments and sarcotubular membranes. Patients with RYR1 mutations often present skeleton alterations and joint contractures from birth. Using the dHT mouse model carrying recessive Ryr1 mutations, we investigated whether muscle spindles from dHT mice show structural defects and altered RyR1 content and function. Our results support the hypothesis that RYR1 mutations also affect the function of intrafusal muscles and this may be one of the underlying causes of the skeletal abnormalities seen in patients.

RYR1-related congenital myopathies are rare disorders that severely impair muscle function and the quality of life of patients and their families. To date no pharmacological therapies are available to treat the severe muscle weakness of affected patients. The most severe forms of RYR1-related congenital myopathies are caused by compound heterozygous mutations (nonsense/frameshift in one allele and a missense mutation in the other), leading to reduced RyR1 protein levels and altered biochemical composition of muscles. In this pre-clinical study, we treated a mouse model carrying the RyR1 p.Q1970fsX16 + p.A4329D compound heterozygous pathogenic variants (dHT mice) for 15 weeks with 0.05 mg/kg 5-aza-2′-deoxycytidine, an FDA-approved drug targeting DNA methyltransferases. We evaluated muscle strength, calcium homeostasis and muscle proteome and report that drug treatment improves all investigated parameters in dHT mice. Importantly, the beneficial effects were particularly significant in fast twitch muscles which are the first muscles to be impaired in patients. In conclusion, this study provides proof of concept for the pharmacological treatment of patients with recessive RYR1-related congenital myopathies with the FDA approved 5-aza-2′-deoxycytidine, supporting its use in a phase 1/2 clinical trial.

Muscle spindles are stretch receptors lying deep within the muscle belly involved in detecting changes in muscle length and playing a fundamental role in motor control, posture and synchronized gait. They are made up of an external capsule surrounding 3-5 intrafusal muscle fibers and a nuclear bag complex. Dysfunction of muscle spindles leads to abnormal proprioceptor function, which has been linked to aberrant bone and cartilage development, scoliosis, kyphosis and joint contractures. RYR1 , the gene encoding the calcium release channel of the sarcoplasmic reticulum, is the most common target of mutations linked to human congenital myopathies, a condition often accompanied by skeleton alterations and joint contractures. So far, the link between RYR1 mutations, altered muscle spindles and skeletal defects has not been investigated. To this end, we investigated heterozygous mice carrying recessive Ryr1 mutations isogenic to those present in a severely affected child. Here we show that: (i) the RyR1 protein localizes to the polar regions of intrafusal fibers and exhibits a doubled row distribution pattern, typical for junctional sarcoplasmic reticulum proteins; (ii) muscle spindles of compound heterozygous mice show structural defects; (iii) RyR1 content in intrafusal muscle fibers from dHT mice is reduced by 54%. Such a massive reduction of mutant RyR1 in intrafusal muscle fibers leads to altered expression of intrafusal fiber proteins, severe scoliosis, alteration of gait and inter limb coordination. These results support the hypothesis that RYR1 mutations not only affect the function of extrafusal muscles, but might also affect that of intrafusal muscles. The latter may be one of the underlying causes of skeletal abnormalities seen in patients affected by recessive RYR1 mutations.

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.

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 Ca ²⁺ sensor STIM1 or the Ca ²⁺ channel ORAI1, both of which regulate Ca ²⁺ homeostasis through the ubiquitous store-operated Ca ²⁺ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca ²⁺ . There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1 ^R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1 ^R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca ²⁺ influx. Compared with Stim1 ^R304W/+ littermates, Stim1 ^R304W/+ Orai1 ^R93W/+ 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 Stim1 ^R304W/+ muscle and revealed a major restoration of gene expression in Stim1 ^R304W/+ Orai1 ^R93W/+ 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.

Skeletal muscles are a highly structured tissue responsible for movement and metabolic regulation, which can be broadly subdivided into fast and slow twitch muscles with each type expressing common as well as specific sets of proteins. Congenital myopathies are a group of muscle diseases leading to a weak muscle phenotype caused by mutations in a number of genes including RYR1. Patients carrying recessive RYR1 mutations usually present from birth and are generally more severely affected, showing preferential involvement of fast twitch muscles as well as extraocular and facial muscles. In order to gain more insight into the pathophysiology of recessive RYR1-congential myopathies, we performed relative and absolute quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying p.Q1970fsX16 and p.A4329D RyR1 mutations which were identified in a child with a severe congenital myopathy. Our in-depth proteomic analysis shows that recessive RYR1 mutations not only decrease the content of RyR1 protein in muscle, but change the expression of 1130, 753, and 967 proteins EDL, soleus and extraocular muscles, respectively. Specifically, recessive RYR1 mutations affect the expression level of proteins involved in calcium signaling, extracellular matrix, metabolism and ER protein quality control. This study also reveals the stoichiometry of major proteins involved in excitation contraction coupling and identifies novel potential pharmacological targets to treat RyR1-related congenital myopathies.