Skeletal Muscle Ryanodine Receptor Research Articles

Kinetics and mapping of Ca-driven calmodulin conformations on skeletal and cardiac muscle ryanodine receptors

Calmodulin transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during contraction and relaxation in cardiac and skeletal muscle. However, the structural dynamics by which calmodulin modulates the sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor, at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime FRET, we resolve different structural states of calmodulin and Ca2+-driven shifts in the conformation of calmodulin bound to ryanodine receptor. Skeletal and cardiac ryanodine receptor isoforms show different calmodulin-ryanodine receptor conformations, as well as binding and structural kinetics with 0.2-ms resolution, which reflect different functional roles of calmodulin. These FRET methods provide insight into the physiological calmodulin-ryanodine receptor structural states, revealing additional distinct structural states that complement cryo-EM models that are based on less physiological conditions. This technology will drive future studies on pathological calmodulin-ryanodine receptor interactions and dynamics with other important ryanodine receptor bound modulators.

Propagation of conformational instability in FK506-binding protein FKBP12

FKBP12 is the archetype of the FK506 binding domains that define the family of FKBP proteins which participate in the regulation of various distinct physiological signaling processes. As the drugs FK506 and rapamycin inhibit many of these FKBP proteins, there is need to develop therapeutics which exhibit selectivity within this family. The long β4-β5 loop of the FKBP domain is known to regulate transcriptional activity for the steroid hormone receptors and appears to participate in regulating calcium channel activity for the cardiac and skeletal muscle ryanodine receptors. The β4-β5 loop of FKBP12 has been shown to undergo extensive conformational dynamics, and here we report hydrogen exchange measurements for a series of mutational variants in that loop which indicate deviations from a two-state kinetics for those dynamics. In addition to a previously characterized local transition near the tip of this loop, evidence is presented for a second site of conformational dynamics in the stem of this loop. These mutation-dependent hydrogen exchange effects extend beyond the β4-β5 loop, primarily by disrupting the hydrogen bond between the Gly 58 amide and the Tyr 80 carbonyl oxygen which links the two halves of the structural rim that surrounds the active site cleft. Mutationally-induced opening of the cleft between Gly 58 and Tyr 80 not only modulates the global stability of the protein, it promotes a conformational transition in the distant β2-β3a hairpin that modulates the binding affinity for a FKBP51-selective inhibitor previously designed to exploit a localized conformational transition at the homologous site.

VP417 Obstetric and gynaecological features in females carrying mutations in the skeletal muscle ryanodine receptor (RYR1) gene: a questionnaire study

VP417 Obstetric and gynaecological features in females carrying mutations in the skeletal muscle ryanodine receptor (RYR1) gene: a questionnaire study

Abnormal myosin post-translational modifications and ATP turnover time associated with human congenital myopathy-related RYR1 mutations.

Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin. We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations. LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the β/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered-relaxed conformation. Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.

Risk of malignant hyperthermia in patients carrying a variant in the skeletal muscle ryanodine receptor 1 gene

Malignant hyperthermia is a life-threatening disorder, which can be prevented by avoiding certain anesthetic agents. Pathogenic variants in the skeletal muscle ryanodine receptor 1-gene are linked to malignant hyperthermia. We retrospectively studied 15 patients who presented to our clinic with symptoms of muscle dysfunction (weakness, myalgia or cramps) and were later found to have a variant in the skeletal muscle ryanodine receptor 1-gene. Symptoms, creatine kinase levels, electromyography, muscle biopsy and in vitro contracture test results were reviewed. Six out of the eleven patients, with a variant of unknown significance in the skeletal muscle ryanodine receptor 1-gene, had a positive in vitro contracture test, indicating malignant hyperthermia susceptibility. In one patient, with two variants of unknown significance, both variants were required to express the malignant hyperthermia-susceptibility trait. Neurologists should consider screening the skeletal muscle ryanodine receptor 1-gene in patients with myalgia or cramps, even when few to no abnormalities on ancillary testing.

P.08 Phase 1 open-label trial of Rycal S48168 (ARM210) for RYR1-related myopathies

Pathogenic variants in the <i>RYR1</i> gene, encoding the skeletal muscle ryanodine receptor (RyR1), are the most common cause of non-dystrophic neuromuscular disorders. Pathogenic <i>RYR1</i> variants exhibit downstream consequences, including aberrant sarcoplasmic reticulum (SR) Ca²⁺ leak, hypersensitivity to RyR1 agonists, diminished SR Ca²⁺ release, and/or decreased RyR1 protein expression. Decreased RyR1-calstabin1 association, due to <i>RYR1</i> variants and/or post-translational modifications of the RyR1 channel, exacerbates SR Ca²⁺ leak with detrimental downstream effects on muscle function. Rycal compounds have been shown to restore RyR1-calstabin1 association, stabilize the <i>RyR1</i> closed state, and mitigate aberrant Ca²⁺ leak in <i>RYR1</i>-related myopathy (<i>RYR1</i>-RM) muscle biopsies. We conducted a phase 1 trial of S48168 (ARM210), a Rycal compound, in ambulatory adults with <i>RYR1</i>-RM (NCT04141670). The primary endpoint was safety and tolerability over a one-month dosing period. Pharmacokinetic (PK) studies, muscle biopsies, motor/muscle function tests, and a patient-reported fatigue questionnaire were also included. Seven eligible individuals were enrolled and received either the low dose (120 mg, n=3) or high dose (200 mg, n=4) of S48168 (ARM210) daily. Three treatment emergent adverse effects grade 2 or above were reported, all unrelated to S48168 (ARM210) and there were no serious adverse events. S48168 (ARM210) showed a dose-dependent PK profile and C_max remained within pre-defined safety margins. At one month, self-reported fatigue scores decreased and shoulder abduction strength trended higher in the high dose group, while handgrip strength increased in both dose groups, though these exploratory comparisons were descriptive. Other preliminary efficacy results were mixed. Given the favorable safety profile and efficacy trends, further clinical development of S48168 (ARM210) is warranted for <i>RYR1</i>-RM.

Pre-operative exercise and pyrexia as modifying factors in malignant hyperthermia (MH)

Malignant hyperthermia (MH) is a life-threatening reaction triggered by volatile anesthetics and succinylcholine. MH is caused by mutations in the skeletal muscle ryanodine receptor (RYR1) gene, as is rhabdomyolysis triggered by exertion and/or pyrexia. The discrepancy between the prevalence of risk genotypes and actual MH incidence remains unexplained. We investigated the role of pre-operative exercise and pyrexia as potential MH modifying factors. We included cases from 5 MH referral centers with 1) clinical features suggestive of MH, 2) confirmation of MH susceptibility on Contracture Testing (IVCT or CHCT) and/or RYR1 genetic testing, and a history of 3) strenuous exercise within 72 h and/or pyrexia >37.5 °C prior to the triggering anesthetic. Characteristics of MH-triggering agents, surgery and succinylcholine use were collected. We identified 41 cases with general anesthesias resulting in an MH event (GA+MH, n = 41) within 72 h of strenuous exercise and/or pyrexia. We also identified previous general anesthesias without MH events (GA-MH, n = 51) in the index cases and their MH susceptible relatives. Apart from pre-operative exercise and/or pyrexia, trauma and acute abdomen as surgery indications, emergency surgery and succinylcholine use were also more common with GA+MH events. These observations suggest a link between pre-operative exercise, pyrexia and MH.

This Month in Anesthesiology

This Month in Anesthesiology

Understanding calmodulin regulation of RyR1 through steady state and stopped-flow fluorescence detection of Ca2+ binding kinetics to calmodulin bound to purified full-length RyR1

Calmodulin (CaM) undergoes unique structural changes induced by Ca2+ binding, and thereby activates the skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) at nanomolar [Ca2+] and inhibits it at micromolar [Ca2+]. CaM conversion from RyR1 activator to inhibitor is due to Ca2+ -binding at two EF-hand motifs in each of the two lobes of CaM. However, how CaM’s lobes contribute to this conversion is not fully understood. Here, we introduced single-cysteines for fluorescence labeling in WT and mutant CaMs (Ca2+ binding at N- or C-lobe ablated), attached the environment-sensitive fluorophore acrylodan to either lobe of these CaMs, and monitored fluorescence responses to Ca2+ association/dissociation to CaM free vs.

High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor

The type 1 ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, composed of four 565-kDa protomers. Cryogenic electron microscopy (cryo-EM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryo-EM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously. This allowed us to resolve the structure of the channel in the primed and open states at 3.4 and 4.0Å, respectively, with a single dataset. This method offers validation for detergent-based structures of the RyR and offers a starting point for utilizing a chemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

Ryanodine receptor type 1 (RyR1) releases Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca2+, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca2+, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [3H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca2+-binding site to sensitize RyR1 to Ca2+. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca2+. However, enhanced RyR1 activation occurred in the presence of Ca2+, AMPPCP, and Caf. In the absence of Ca2+, Na+ inhibited [3H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca2+-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca2+, Na+ is predicted to inhibit RyR1 by interacting with the Ca2+-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca2+-binding site, and conversely, Ca2+ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca2+, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca2+-, ATP-, and Caf-binding sites.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

High Resolution Structure of the Membrane Embedded Skeletal Muscle Ryanodine Receptor

The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, improving the incorporation rate by >20-fold compared to a dialysis-based approach. This allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 A, respectively. This method offers validation for detergent-based structures of the RyR and offers a method for utilizing an electrochemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.

Beneficial effects of dantrolene in the treatment of rhabdomyolysis as a potential late complication associated with COVID-19: a case report

BackgroundPatients with severe COVID-19 have disorders of the respiratory, cardiovascular, coagulation, skeletal muscle, and central nervous systems. These systemic failures may be associated with cytokine release syndrome, characterized by hyperpyrexia, thrombocytopenia, hyperferritinemia, and the elevation of other inflammatory markers. Rhabdomyolysis with high fever is a complication that is rarely found in COVID-19. The exact relations of these clinical conditions in patients with COVID-19 remain unknown.Case presentationWe present the case of a 36-year-old man with severe COVID-19 complicated by rhabdomyolysis and high fever. After admission, his condition continued to deteriorate, with a high body temperature. On day 9, the patient had elevated creatine kinase and myoglobin levels consistent with rhabdomyolysis (26,046 U/L and 3668 ng/mL, respectively). In addition to viral therapy, he was immediately treated with hydration. However, the patient had persistent fever and elevated creatine kinase levels. The patient was diagnosed with malignant hyperthermia as a late complication of COVID-19, although he had no hereditary predisposition to malignant hyperthermia or neuroleptic malignant syndrome. The administration of dantrolene with muscle relaxation and anti-inflammatory function showed potential efficacy for rhabdomyolysis, high fever, and increased plasma inflammatory markers.ConclusionsMalignant hyperthermia is triggered by not only anesthetic agents but also viral infections. A possible mechanism of malignant hyperthermia is hypersensitivity of calcium release from the sarcoplasmic reticulum. These include mutations in or the activation of the skeletal muscle ryanodine receptor calcium release channel. Dantrolene is a ryanodine receptor antagonist and is used as an anti-inflammatory agent. The administration of dantrolene showed potential efficacy for rhabdomyolysis, high body temperature due to inflammation, and increased inflammatory markers. The underlying mechanism of the association of rhabdomyolysis and high fever in COVID-19 might be similar to the pathogenesis of malignant hyperthermia.

Functional and Structural Interactions between Ca2+, ATP and Caffeine Binding Sites of Skeletal Muscle Ryanodine Receptor (RyR1)

Functional and Structural Interactions between Ca2+, ATP and Caffeine Binding Sites of Skeletal Muscle Ryanodine Receptor (RyR1)

Pre-Operative Exercise and Pyrexia as Modifying Factors in Malignant Hyperthermia

Background: Malignant Hyperthermia (MH) is a life-threatening reaction triggered by volatile anaesthetics and succinylcholine characterized by hypercapnia, hypermetabolism and muscle breakdown. MH is most commonly caused by mutations in the skeletal muscle ryanodine receptor (RYR1) gene, as is rhabdomyolysis triggered by exertion and/or pyrexia. The discrepancy between risk genotypes and actual MH incidence remains unexplained. We hypothesized that MH is a compound event, reflecting a synergistic effect of predisposing genotype, triggering anaesthetic, and exercise and/or pyrexia as modifying factors. Methods: We performed a retrospective study and identified 41 patients with a history of exercise and/or pyrexia within 72 hours prior to an MH event. We compared the frequency of a history of exercise and/or pyrexia within 72 hours of the triggering agent in “eventful anaesthesias” (n=41) and in “uneventful anaesthesias” (n=51) in the index cases and/or their MH susceptible relatives. Indications for and urgency of surgery, and use of succinylcholine were also recorded. Findings: In eventful anaesthesias, there was a history of exercise and/or pyrexia within 72 hours prior to the anaesthetic in 24(0) and 21(1) patients, compared to 0 and 1 in uneventful anesthesias. Trauma and acute abdomen as surgery indications, emergency surgery and succinylcholine use were also more common with eventful anaesthesias. Interpretation: Exercise and/or pyrexia prior to general anaesthesia are risk factors for MH. Rhabdomyolysis may mimic an acute abdomen, whilst trauma surgery may carry a specific risk due to muscle damage. These observations should inform perioperative guidelines with the potential to reduce fatal anaesthesia complications. Funding Statement: This work is not funded. Declaration of Interests: Sheila Riazi, Luuk van den Bersselaar, Gunilla Islander, Luc Heytens, Marc M. J. Snoeck, Andrew Bjorksten, Robyn Gillies, George Dranitsaris, Anna Hellblom, Susan Treves, Nicol C. Voermans, Heinz Jungbluth: These authors declare no financial interests. Ethics Approval Statement: The retrospective case study was performed with Research Ethics Board approval from the University of Toronto (REB Nr 19-5365), and in accordance with the ethics guidelines issued by other participating centres.

Efavirenz, atazanavir, and ritonavir disrupt sarcoplasmic reticulum Ca2+ homeostasis in skeletal muscles

While muscle fatigue, pain and weakness are common co-morbidities in HIV-1 infected people, their underlying cause remain poorly defined. To this end, we evaluated whether the common antiretroviral drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) could be a contributing factor by pertubating sarcoplasmic reticulum (SR) Ca2+ cycling. In live-cell imaging, EFV (6.0 μM), ATV (6.0 μM), and RTV (3.0 μM) elicited Ca2+ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca2+ release channel blocker ryanodine (50 μM), slowed the rate and amplitude of Ca2+ release from and reuptake of Ca2+ into the SR. EFV, ATV and RTV (1 nM - 20 μM) potentiated and then displaced [3H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca2+ release channel (RyR1). These drugs at concentrations 0.25–31.2 μM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤5 μM) potentiated and >5μM inhibited the ability of sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA1) to hydrolyze ATP and transport Ca2+. RTV (2.5–31.5 μM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca2+ transport. EFV (0.25–31.5 μM) had no measurable effect on SERCA1's ability to hydrolyze ATP and transport Ca2+. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca2+ homeostasis.

Bioenergetic defects in muscle fibers of RYR1 mutant knock-in mice associated with malignant hyperthermia

Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism, which has directed research interest into oxidative phosphorylation and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ∼16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression, and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA-Seq data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared with WT controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I-facilitated oxidative phosphorylation in mutant muscle. Furthermore, we observed a gene-dose-specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively, these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.

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.

New Compound Heterozygous Splice Site Mutations of the Skeletal Muscle Ryanodine Receptor (RYR1) Gene Manifest Fetal Akinesia: A Linkage with Congenital Myopathies

Mutations in the skeletal muscle ryanodine receptor (RYR1) gene have been linked to malignant hyperthermia susceptibility, central core disease, and minicore myopathy with external ophthalmoplegia. RYR1 is an intracellular calcium release channel and plays a crucial role in the sarcoplasmic reticulum and transverse tubule connection. Here, we report 2 fetuses from the same parents with compound heterozygous mutations in the RYR1 gene (c.10347+1G>A and c.10456-2Α>G) who presented with fetal akinesia and polyhydramnios at 27 and 19 weeks of gestation with intrauterine growth restriction in the third pregnancy. The prospective parents of the fetuses were heterozygous carriers for c.10456-2Α>G (mother) and c.10347+1G>A (father). Both mutations affect splice sites resulting in dysfunctional protein forms probably missing crucial domains of the C-terminus. Our findings reveal a new RYR1 splice site mutation (c.10456-2Α>G) that may be associated with the clinical features of myopathies, expanding the RYR1 spectrum related to these pathologies.