Central Core Disease Research Articles

Exon Skipping as a Therapeutic Strategy Applied to a RyR1 Mutation Causing Severe Core Myopathy

Central Core Disease is a myopathy resulting generally from a mutation in the RYR1 gene, encoding the skeletal muscle calcium release channel RyR1. No treatment is currently available for this disease. We studied a pathological situation in which an affected child harbors two recessive mutations, resulting in a massive reduction in the RyR1 amount. The paternal mutation inducing the inclusion of a new in frame exon in the mRNA of RyR1, resulted in the insertion of additional amino-acids and destabilization of the protein. We hypothesized that inducing the skipping of this exon would be sufficient to restore RyR1 expression and normalization of calcium releases. We developed U7-AON lentiviral vectors to induce exon-skipping on affected primary muscle cells. The efficiency of the exon skipping at the mRNA level, at the protein level and at the functional level using calcium imaging were evaluated. We observed in these affected primary muscle culture a reduction in the inclusion of the additional exon, an increase in the RyR1 protein expression, and a restoration of normal calcium releases. This study is the first demonstration of the potential of exon skipping for the therapy of Central Core Disease, from the molecular to the functional level.

Death in the Emergency Department

A healthy 6-year-old boy developed lower extremity rigidity, trismus, and fever after playing in a splash pool. On arrival in the emergency department, he appeared to be seizing. An endotracheal tube was emergently placed using succinylcholine. Cardiac arrest followed. He could not be resuscitated. Postmortem genetic analysis found a novel RYR1 variant. Family testing revealed the same variant in his father who also had muscle contracture testing diagnostic for susceptibility to malignant hyperthermia and central core disease diagnosed histologically. Because there was no exposure to volatile anesthetics before the onset of symptoms, this is a case of "awake" malignant hyperthermia worsened by succinylcholine.

Maximizing Functional Independence in Sporadic Late Onset Nemaline Myopathy

Sporadic late onset nemaline myopathy (SLONM) is a rare neuromuscular disorder of unknown pathogenesis. The incidence of nemaline myopathy is estimated to be 1:500,000 [1]; however, the late onset subtype accounts for only 4% of cases [2]. Nemaline rods, the characteristic feature of nemaline myopathy, arise from the z band of the sarcomere and are composed primarily of -actinin [1]. However, nemaline bodies may be present in a variety of conditions, including human immunodeficiency virus (HIV) infection, mitochondrial myopathy, and central core disease [1]. In SLONM, the diagnosis is based on characteristic findings of progressive proximal weakness, normal creatine kinase, myopathic motor unit (short duration, low amplitude) and fibrillation potentials on electromyography (EMG), and a muscle biopsy that demonstrates nemaline rods [3]. SLONM is associated with monoclonal gammopathy of undetermined significance (MGUS) [3]. The presence of a monoclonal gammopathy confers a high risk of mortality; 57% of individuals with this disease are reported to die of respiratory failure within 2 years of onset of muscle weakness. Alternatively, patients with SLONM and without a monoclonal gammopathy did not die of their disease when followed-up 4-23 years after symptom onset. Therefore, patients with SLONM may survive for many years, likely with functional impairment secondary to muscle weakness. To our knowledge, the rehabilitation process has never been described for a patient with SLONM. We theorize that structured rehabilitation therapy is an important component in maximizing functional ability for patients with SLONM.

Novel excitation-contraction uncoupled RYR1 mutations in patients with central core disease

Central core disease, one of the most common congenital myopathies in humans, has been linked to mutations in the RYR1 gene encoding the Ca(2+) release channel of the sarcoplasmic reticulum (RyR1). Functional analyses showed that disease-associated RYR1 mutations led to impairment of skeletal muscle Ca(2+) homeostasis; however, thorough understanding of the molecular mechanisms underlying central core disease and other RyR1-related conditions is still lacking. We screened by sequencing the complete RYR1 transcripts in ten unrelated patients with central core disease and identified five novel, p.M4640R, p.L4647P, p.F4808L, p.D4918N and p.F4941C, and four recurrent mutations. Four of the novel mutations involved amino acid residues that were positioned within putative transmembrane segments of the RyR1. The pathogenic character of the identified mutations was demonstrated by bioinformatic analyses and by the in vitro functional studies in HEK293 cells and RYR1-null (dyspedic) myotubes. Characterization of Ca(2+) channel properties of RyR1s carrying one recurrent and two novel mutations upholds the view that diminished intracellular Ca(2+) release caused by impaired Ca(2+) channel gating and/or Ca(2+) permeability is an important component of central core disease etiology. This study expands the list of functionally characterized disease-associated RyR1 mutations, increasing the value of genetic diagnosis for RyR1-related disorders.

Allele-Specific Gene Silencing in Two Mouse Models of Autosomal Dominant Skeletal Myopathy

We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4–6 month old heterozygous Ryr1Y524S/+ (YS/+) and Ryr1I4895T/+ (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively. Treatment of IT/+ mice resulted in a modest rescue of deficits in the maximum rate (∼38% rescue) and magnitude (∼78%) of ligand-induced Ca2+ release that occurred in the absence of a change in the magnitude of electrically-evoked Ca2+ release. Compared to the difference between the caffeine sensitivity of Ca2+ release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC50: 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC50: 2.8 mM) and 4 week (EC50: 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca2+ observed in FDB fibers from YS/+ mice was normalized to WT levels after 2 weeks of treatment with YS allele-specific siRNA. As determined by quantitative real time PCR, the degree of functional rescue in YS/+ and IT/+ mice correlated well with the relative increase in fractional WT allele expression.

Structural determination of the phosphorylation domain of the ryanodine receptor.

The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca(2+) cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca(2+)/calmodulin-dependent protein kinase β isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a β sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor.

Mutations in the Nebulin Gene in a Child with Nemaline (Rod) Myopathy

Nemaline myopathy, also called rod myopathy, is a relatively common congenital myopathy and probably second in incidence only to central core disease. The mainstay of diagnosis is histopathology, but detection of the causative mutation is mandatory for determining the mode of inheritance and for prenatal diagnosis. The authors report two siblings with nemaline myopathy caused by mutations in the nebulin gene.

C.P.1 Central core disease with a novel RYR1 mutation in a Korean family

Central core disease (CCD) is a form of myopathy inherited autosomal dominantly. It is caused by mutations in RYR1 and allelic to malignant hyperthermia. The diagnosis is established by characteristic muscle pathology and the identification of mutation. Here we report clinical and pathological features of a Korean family with CCD carrying a novel RYR1 mutation. A 15-year-old boy presented with slowly progressive weakness of bilateral legs since school age. The boy could not run and had difficulty in climbing stairs. His muscle weakness slowly progressed and he now has difficulty in rising up from a chair. His mother and sister had markedly milder proximal leg weakness. Diffuse muscle atrophy was noted especially in lower extremities. Muscle power examination disclosed predominantly proximal leg weakness. Serum creatine kinase was within normal limits, and needle electromyography showed active myopathic pattern. Muscle biopsy revealed multiple peripheral and central cores in muscle fibers devoid of oxidative enzyme activities. All the muscle fibers were of type 1. Sequence analysis of RYR1 disclosed a novel mutation in exon 101 (c.14590T>C[p.Tyr4864His]). None of the affected members showed facial muscle weakness or skeletal deformities, although it is said to be frequently accompanied in CCD. Marked difference in clinical severity is noted between proband and his mother; this kind of intrafamilial phenotypic variability had previously been reported in CCD. It has been known that mutations at C-terminal of RYR1 are associated with severe clinical phenotype and clearly demarcated central cores, while mutations outside the C-terminal show milder clinical phenotype and atypical cores in their muscle fiber. Interestingly, the mutation identified in this family is at C-terminal region, all the affected members showed mild phenotype of later-onset and atypical cores inexplicitly ovoid with indistinct borders in the subsarcolemmal areas.

C.P.2 In silico analysis of recessive RYR1 mutations identifies novel potential disease mechanisms

Mutations in RYR1 are responsible for malignant hyperthermia (MH) and several forms of congenital myopathy. Dominant mutations that give rise to central core disease (CCD) and MH cluster in three hotspot regions and the disease mechanisms associated with these diseases are well studied. In comparison, little is known about how recessive mutations, which occur throughout the gene, lead to disease. To address this issue, we reviewed the literature to map potentially functional domains within the ryanodine receptor type 1 (RyR1) and then analysed the nature and position of RYR1 mutations in 74 individuals with recessive disease, together with their clinical and histological phenotypes. Hypomorphic mutations, which abolish or markedly reduce RyR1 protein expression, were associated with more severe clinical phenotypes and with histological phenotypes other than CCD, such as multi-minicore disease, centronuclear myopathy and congenital fibre type disproportion. For non-hypomorphic mutations, CCD mutations were enriched in MH/CCD hotspot 3 and non-CCD core mutations in hotspot 2. Clustering of non-hypomorphic mutations in the triadin-binding domain (which lies within hotspot region 3) highlights abnormal triadin-binding as a potentially important disease mechanism. Non-hypomorphic mutations associated with ophthalmoplegia clustered in regions involved in ‘interdomain interactions’ which play roles in stabilising the closed conformation of the RyR1 channel. These results suggest that the variability seen in recessive RYR1 histological phenotypes may stem from differences in disease mechanism, determined in part by the location of non-hypomorphic mutations. Reduced overall RyR1 function, reduced stability of the closed channel conformation and abnormal triadin-RyR1 interactions are highlighted as potentially important disease mechanisms in various recessive RYR1-myopathies.

D.P.22 Muscle ultrasound as a consistent imaging marker for RYR1 related myopathies

Skeletal muscle ultrasound applications in neuromuscular medicine are increasing since high frequency transducers are capable of generating images of much improved resolution. Similar to MRI, diagnostic ultrasound imaging phenotypes are being identified, such as the finding of a “central cloud” in the rectus femoris of patients with collagen six myopathy. Here we describe an ultrasound imaging phenotype that is characteristic for RYR1 related myopathies: sparing of the rectus femoris next to a significantly increased echogenicity in the vastus lateralis. RYR1 related myopathies include the histological phenotypes of central core disease, multi-minicore disease, centro-nuclear myopathy. In addition to these established histological phenotypes muscle biopsy findings may be non-specific with a myopathic or dystrophic appearance and the presence of significant fibroadipose tissue. Clinically, autosomal dominant forms more commonly present with static or slowly progressive proximal weakness involving hip and axial muscles but can also be severe in infancy. Recessive forms typically present with more severe early hypotonia and weakness, and with extraocular muscle involvement in a number of patients. This clinical and histopathological variability can give rise to diagnostic confusion and delay. Muscle MRI has been shown to demonstrate relative sparing of the rectus femoris in patients with RYR1 mutations, but the acquisition of such an imaging study often requires sedation in this younger patient population that may also be at risk for malignant hyperthermia. Muscle ultrasound can demonstrate the same imaging phenotype via a quick, painless and highly tolerable bedside procedure. Here we present 15 patients with genetically proven RYR1 related myopathies with a variety of clinical, histological and genetic findings all demonstrating a consistent an ultrasound imaging phenotype. This easily obtained imaging finding may thus help to guide diagnostic considerations.

C.P.4 Congenital fiber type disproportion with myofibrillar disorganization and altered internal nuclei is caused by RYR1 mutation

Mutations in the gene encoding skeletal muscle ryanodine receptor (RYR1) have been implicated in various forms of neuromuscular diseases including congenital myopathy. There is a broad range of clinical and pathological features including central core disease, congenital neuromuscular disease with uniform type 1, multiminicore disease, core-rod myopathy, and malignant hyperthermia susceptibility. Here we describe a group of patients with RYR1 mutations showing a unique muscle pathology. We chose seven unrelated Japanese patients among 12,000 frozen muscle biopsies in NCNP, based on the characteristic pathological findings including fiber type disproportion), marked type 1 fiber predominance, and scattered fibers with abnormal internal nuclei. On NADH-TR stain, myofibrillar disorganization was commonly seen. Unique fibers characterized by darkly-stained central portion with multiple small vacuoles were seen in three patients. On electron microscopy, center of muscle fibers were frequently occupied by aggregations of enlarged, vacuolated sarcoplasmic reticulum and abnormal shaped nuclei. Clinically, all seven patients showed axial and proximal dominant muscle weakness of variable severity from neonatal period, and often required respiratory supports. Muscle weakness was stable or rather improved during childhood. In addition, some patients revealed ptosis and/or ophthalmoparesis. All patients had compound heterozygous mutations in the central portion of RYR1 distributed between 18 and 66. In conclusion, mutations in RYR1 gene are associated with a unique myopathy characterized as congenital fiber type disproportion associated with myofibrillar disorganization and altered internal nuclei. These pathological changes may be closely related to degeneration of sarcoplasmic reticulum.

C.P.3 Genetic and clinical heterogeneity of RYR1-related myopathies in a cohort of 60 Dutch families with identification of 40 novel mutations

RYR1-related myopathies have a very variable phenotype. Sequential analysis of RYR1 in the Netherlands has been performed since 2008. Our goal was to better define the phenotype or RYR1-related myopathies and to report several new mutations. We have therefore collected the results of genetic analysis clinical findings and in all patients in whom one or more RYR1 mutations were detected between 2008 and 2012. In this period, mutation analysis was performed in approximately 250 patients. In 79 patients belonging to 60 families one or more RYR1 mutations were found. In total, 55 mutations, of which 40 were novel mutations. The phenotype of these RYR1-related myopathies was very variable, including the well-known phenotypes central core disease, multiminicore disease, centronuclear myopathy, malignant hyperthermia, but also less well-defined presentations such as exercise-induced hyperCKemia, exercise-induced myalgia, heat-induced myalgia, statin-induced hyperCKemia, and viral-infection-induced hyperCKemia. Furthermore, cardiac and pulmonary manifestations were infrequently encountered while screening took only place in a minority of patients. Next, the muscle biopsies showed remarkable mitochondrial or inflammatory changes in few patients. In one patient, we could show the evolvement of pathological features in four subsequent biopsies in a period of 11years. Finally, complex inheritance patterns, with several mutations and AR and AD inheritance pattern of specific traits were encountered in some families. We conclude that RYR1-related myopathies are not rare in the Netherlands with many new mutations, some of which the pathogenicity remained unclear. In addition, RYR1-related myopathies are both clinically, histologically and genetically more diverse than previously reported. Future research should focus on functional studies of the ryanodine receptor to elucidate the pathogenicity of these mutations.

Abstract 370: Malignant Hyperthermia Mutation of RyR1 (Y522S) Increases Catecholamine-Induced Cardiac Arrhythmia Through Mitochondrial Injury

Introduction: Mutations in skeletal muscle type ryanodine receptor (RyR1) exhibit life-treating human disorders including malignant hyperthermia (MH) and central core disease, which features skeletal muscle rigidity triggered by anesthesia and body temperature elevation. However, cardiac arrhythmias and/or sudden cardiac death in these patients are also frequently reported during anesthesia or even under the conscious condition. Hypothesis: Chronic mitochondrial Ca 2+ overload through leaky mutant RyR1 expressed at cardiac mitochondria leads to mitochondria injury and cardiac arrhythmia. Methods: We determined the cardiac phenotype of knock-in mice carrying a leaky RyR1 MH mutation Y522S (YS). Results: WT- and YS - RyR1 were expressed at mitochondria but not at SR in heart. Ultra-structure of heterozygous YS hearts exhibited severe abnormal/disrupted mitochondrial morphology as well as myofibril contractures (Fig. A-E). YS cardiac mitochondria showed high susceptibility to Ca 2+ -induced opening of mitochondrial permeability transition pores and depolarized mitochondrial membrane potential compared to WT, suggesting compromised mitochondrial functions. Langendorff perfusion developed similar LV pressure in response to high dose bolus of Isoproterenol in WT and YS, but only YS heart frequently developed multiple ventricular extrasystoles after Isoproterenol (Fig. F). Conclusion: Chronic mitochondrial damage by Ca 2+ overload through leaky mutant RyR1 induces mitochondrial structural and functional disruption, which facilitates arrhythmogenic outbursts under acute catecholaminergic stress.

Mutations in MYH7 cause Multi-minicore Disease (MmD) with variable cardiac involvement

Central Core Disease (CCD) and Multi-minicore Disease (MmD) (the "core myopathies") have been mainly associated with mutations in the skeletal muscle ryanodine receptor (RYR1) and the selenoprotein N (SEPN1) gene. A proportion of cases remain unresolved. Mutations in MYH7 encoding the beta myosin heavy chain protein have been implicated in cardiac and, less frequently, skeletal muscle disorders. Here we report four patients from two families with a histopathological diagnosis of MmD, presenting in childhood with slowly progressive muscle weakness, more proximal in Family 1 and more distal in Family 2, and variable degrees of cardiorespiratory impairment evolving later in life. There was also a strong family history of sudden death in the first family. Muscle biopsies obtained in early childhood showed multiple minicores as the most prominent feature. Sequencing of the MYH7 gene revealed heterozygous missense mutations, c.4399C>G; p.Leu1467Val (exon 32) in Family 1 and c.4763G>C; p.Arg1588Pro (exon 34) in Family 2. These findings suggest MYH7 mutations as another cause of a myopathy with multiple cores, in particular if associated with dominant inheritance and cardiac involvement. However, clinical features previously associated with this genetic background, namely a more distal distribution of weakness and an associated cardiomyopathy, may only evolve over time.

Clinical Features and Ryanodine Receptor Type 1 Gene Mutation Analysis in a Chinese Family With Central Core Disease

Central core disease is a rare inherited neuromuscular disorder caused by mutations in ryanodine receptor type 1 gene. The clinical phenotype of the disease is highly variable. We report a Chinese pedigree with central core disease confirmed by the gene sequencing. All 3 patients in the family presented with mild proximal limb weakness. The serum level of creatine kinase was normal, and electromyography suggested myogenic changes. The histologic analysis of muscle biopsy showed identical central core lesions in almost all of the muscle fibers in the index case. Exon 90-106 in the C-terminal domain of the ryanodine receptor type 1 gene was amplified using polymerase chain reaction. One heterozygous missense mutation G14678A (Arg4893Gln) in exon 102 was identified in all 3 patients. This is the first report of a familial case of central core disease confirmed by molecular study in mainland China.

Structural and functional conservation of key domains in InsP3 and ryanodine receptors

Inositol 1,4,5-trisphosphate receptors (InsP3R) and ryanodine receptors (RyR) are tetrameric intracellular Ca2+ channels1. For each, the pore is formed by C-terminal transmembrane domains and regulated by signals detected by the large cytosolic structures. InsP3R gating is initiated by InsP3 binding to the InsP3-binding core (IBC, residues 224-604 of InsP3R1)2 and it requires the suppressor domain (SD, residues 1-223)2-8. We present structures of the N-terminal region (NT) of InsP3R1 with (3.6 Å) and without (3.0 Å) InsP3 bound. The arrangement of the three NT domains, the SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR19. The orientations of the three domains docked into a tetrameric structure of InsP3R10 and of the ABC domains in RyR9 are remarkably similar. The importance of the α-interface for activation of InsP3R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations9,11,12. InsP3 causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop (HS-loop) that is essential for InsP3R activation7. The loop is conserved in RyR and includes mutations associated with malignant hyperthermia and central core disease9,11,12. The HS-loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A-domain of RyR functionally replaced the SD in a full-length InsP3R, and an InsP3R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP3 and blocked by ryanodine. Activation mechanisms are conserved between InsP3R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit re-orients the first domain (SD or A-domain), allowing it, via interactions of the second domain of an adjacent subunit (IBC-β or B-domain), to gate the pore.

The Ryanodine Receptor N-Terminal Disease Hot Spot Intersubunit Interface is Disrupted by Channel Opening and Affected by Disease Mutations Acting via Long-Range Structural Changes

Ryanodine Receptors (RyRs) are intracellular calcium-release channels acting as one of the key regulatory elements in the excitation-contraction coupling. More than 350 mutations have been found in RyRs that are known to underlie severe genetic diseases. Mutations in the skeletal muscle isoform (RyR1) are associated with malignant hyperthermia (MH) and central core disease (CCD), while mutations in the cardiac isoform (RyR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD). Most mutations confer a gain of function, but the precise mechanisms that explain enhanced channel opening up to the molecular scale have remained elusive. Here we present pseudo-atomic models of the N-terminal disease hot spot in the open and closed states of the RyR, along with crystal structures of several disease mutants. The data show that the intersubunit interfaces formed by tetrameric N-terminal disease hot spots are disrupted upon channel opening in wild-type RyRs. This intersubunit interface harbors 19 disease mutations, the largest cluster within the N-terminal region, indicating the vulnerability of this interface in channel regulation. We present crystal structures and thermal stabilities of nine disease mutants located at other interfaces. The effect of most mutations are destabilizing to the protein, with decreases in melting temperatures as large as ∼10°C. Buried disease mutations cause structural changes to the intersubunit interface, while mutations affecting ionic pairing at the intra-subunit interface significantly alter relative domain orientations. Mutations far away from the intersubunit interface can thus affect these contacts via long-range conformational changes. These results illuminate the intersubunit interface between N-terminal disease hot spots as a prime target for disease mutations through direct or indirect conformational changes.

Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.

Core Myopathies

The core myopathies, Central Core Disease and Multiminicore Disease, are heterogeneous congenital myopathies with the common defining histopathological feature of focally reduced oxidative enzyme activity (central cores, multiminicores). Mutations in the gene encoding for the skeletal muscle ryanodine (RyR1) receptor are the most common cause. Mutations in the selenoprotein N (SEPN1) gene cause a less common variant. Pathogenic mechanisms underlying dominant RYR1 mutations have been extensively characterized, whereas those associated with recessive RYR1 and SEPN1 mutations are emerging. Identifying a specific genetic defect from the histopathological diagnosis of a core myopathy is complex and ought to be informed by a combined appraisal of histopathological, clinical, and, increasingly, muscle magnetic resonance imaging data. The present review aims at giving an overview of the main genetic and clinicopathological findings, with a major emphasis on features likely to inform the diagnostic process, as well as current treatments and perspectives for future research.

Defects in Ca2+ release associated with local expression of pathological ryanodine receptors in mouse muscle fibres

Mutations of the gene encoding the type 1 ryanodine receptor (RyR1) are associated with skeletal muscle disorders including malignant hyperthermia susceptibility (MHS) and central core disease (CCD). We used in vivo expression of EGFP-RyR1 constructs in fully differentiated mouse muscle fibres to characterize the function of several RyR1 mutants. Wild-type and Y523S, R615C, R2163H and I4897T mutants of RyR1 were separately expressed and found to be present within restricted regions of fibres with a pattern consistent with triadic localization. Confocal measurements of voltage-clamp-activated myoplasmic Ca(2+) transients demonstrated alterations of sarcoplasmic reticulum (SR) Ca(2+) release spatially correlated with the presence of exogenous RyR1s. The Y523S, R615C and R2163H RyR1 MHS-related mutants were associated with enhanced peak Ca(2+) release for low and moderate levels of depolarization, whereas the I4897T CCD mutant produced a chronic reduction of peak SR Ca(2+) release. For example, peak Ca(2+) release in response to a depolarization to -20 mV in regions of fibres expressing Y523S and I4897T was 2.0 ± 0.3 (n = 9) and 0.46 ± 0.1 (n = 5) times the corresponding value in adjacent, non-expressing regions of the same fibre, respectively. Interestingly no significant change in the estimated total amount of Ca(2+) released at the end of large depolarizing pulses was observed for any of the mutant RyR1 channels. Overall, results are consistent with an 'inherent' increase in RyR1 sensitivity to activation by the voltage sensor for the MHS-related RyR1 mutants and a partial failure of voltage-gated release for the CCD-related I4897T mutant, that occur with no sign of change in SR Ca(2+) content. Furthermore, the results indicate that RyR1 channel density is tightly regulated even under the present conditions of forced exogenous expression.