Mutations In Ryanodine Receptor Research Articles

A study of a family with the skeletal muscle RYR1 mutation (c.7354C>T) associated with central core myopathy and malignant hyperthermia susceptibility

Congenital myopathies are early onset hereditary muscle disorders. A sub-group of these is associated with malignant hyperthermia susceptibility. Mutations in the skeletal muscle ryanodine receptor ( RYR1) gene have been associated with various congenital myopathy phenotypes and may also cause malignant hyperthermia susceptibility. We describe nine affected members of an extended family presenting with a myopathy typically manifesting as upper eye lid ptosis, quadriceps atrophy and patellar dislocation. Three affected members underwent extensive genetic testing and have a RYR1 exon 46 c.7354C>T gene mutation; two of whom had muscle biopsies – both demonstrated central core myopathy. The only affected family member who underwent testing for malignant hyperthermia susceptibility was shown to be positive. The clinical phenotypes seen among affected family members varies widely in severity, and have features in common with those congenital myopathies associated with malignant hyperthermia susceptibility, raising the possibility that these conditions represent a spectrum of disease.

O.15 Mutations in the skeletal muscle ryanodine receptor (RYR1) gene presenting with exertional myalgia and rhabdomyolysis

O.15 Mutations in the skeletal muscle ryanodine receptor (RYR1) gene presenting with exertional myalgia and rhabdomyolysis

P3.41. Clinical and genetic findings in a large cohort of patients with congenital myopathies due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P3.41. Clinical and genetic findings in a large cohort of patients with congenital myopathies due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P3.43 An adult-onset, slowly progressive axial myopathy with cataracts due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P3.43 An adult-onset, slowly progressive axial myopathy with cataracts due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

Key role of the molecular autopsy in sudden unexpected death

Sudden Cardiac Death (SCD) is a major and tragic complication of a number of cardiovascular diseases. While in the older populations, SCD is most frequently caused by underlying coronary artery disease and heart failure, in those aged under 40 years, the causes of SCD commonly include genetic disorders, such as inherited cardiomyopathies and primary arrhythmogenic diseases. As part of the evaluation of families in which SCD has occurred, the role of genetic testing has evolved as an important feature in both establishing an underlying diagnosis and in screening at-risk family relatives. Specifically, in cases where no definitive cause is identified at postmortem, i.e. Sudden Unexpected Death (SUD), the "molecular autopsy" has emerged as a key process in the investigation of the cause of death. The combination of clinical and genetic evaluation of families in which SUD has occurred provides a platform for early initiation of therapeutic and prevention strategies, with the ultimate goal to reduce sudden death among the young in our communities.

Targeting intracellular calcium cycling in catecholaminergic polymorphic ventricular tachycardia: a theoretical investigation

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant arrhythmogenic disorder linked to mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin, predisposing the young to syncope and cardiac arrest. To define the role of β-adrenergic stimulation (BAS) and to identify potential therapeutic targeted sites relating to intracellular calcium cycling, we used a Luo-Rudy dynamic ventricular myocyte model incorporated with interacting Markov models of the L-type Ca(2+) channel (I(Ca,L)) and RyR2 to simulate the heterozygous state of mouse RyR2 R4496C mutation (RyR2(R4496C+/-)) comparable with CPVT patients with RyR2 R4497C mutation. Characteristically, in simulated cells, pacing at 4 Hz or faster or pacing at 2 Hz under BAS with effects equivalent to those of isoproterenol at ≥ 0.1 μM could readily induce delayed afterdepolarizations (DADs) and DAD-mediated triggered activity (TA) in RyR2(R4496C+/-) but not in the wild-type via enhancing both I(Ca,L) and sarcoplasmic reticulum (SR) Ca(2+) ATPase (I(UP)). Moreover, with the use of steady state values of isolated endocardial (Endo), mid-myocardial (M), and epicardial (Epi) cells as initial data for conducting single cell and one-dimensional strand studies, the M cell was more vulnerable for developing DADs and DAD-mediated TA than Endo and Epi cells, and the gap junction coupling represented by diffusion coefficient (D) of ≤ 0.000766*98 cm(2)/ms was required for generating DAD-mediated TA in RyR2(R4496C+/-). Whereas individual reduction of Ca(2+) release channel of SR and Na-Ca exchanger up to 50% was ineffective, 30% or more reduction of either I(Ca,L) or I(UP) could totally suppress the inducibility of arrhythmia under BAS. Of note, 15% reduction of both I(Ca,L) and I(UP) exerted a synergistic antiarrhythmic efficacy. Findings of this model study confirm that BAS facilitates induction of ventricular tachyarrhythmias via its action on intracellular Ca(2+) cycling and a pharmacological regimen capable of reducing I(Ca,L) could be an effective adjunctive to β-adrenergic blockers for suppressing ventricular tachyarrhythmias during CPVT.

The Deletion of Exon 3 in the Cardiac Ryanodine Receptor Is Rescued by β Strand Switching

Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the β trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca²⁺ release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.

Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value. This study presents a CPVT family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers. The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2. Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes. M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family.

1FC2.5 Recessive mutations in the skeletal muscle ryanodine receptor (RYR1) gene may mimick X-linked centronuclear (myotubular) myopathy (XLMTM)

of nighttime BiPAP use in 45 patients at an average age of 11.34±4.28 years. Patients at the mildest end of the phenotypic spectrum did not demonstrate a clear pattern of decline in respiratory function. Conclusions: Forced vital capacity is a viable outcome measure for patients at the moderate to severe end of the collagen VI myopathy phenotypic spectrum, a finding which should inform optimal surveillance and clinical trial planning.

King–Denborough syndrome with and without mutations in the skeletal muscle ryanodine receptor ( RYR1) gene

King–Denborough syndrome (KDS), first described in 1973, is a rare condition characterised by the triad of dysmorphic features, myopathy, and malignant hyperthermia susceptibility (MHS). Autosomal dominant inheritance with variable expressivity has been reported in several cases. Mutations in the skeletal muscle ryanodine receptor ( RYR1) gene have been implicated in a wide range of myopathies such as central core disease (CCD), the malignant hyperthermia (MH) susceptibility trait and one isolated patient with KDS. Here we report clinical, pathologic and genetic features of four unrelated patients with KDS. Patients had a relatively uniform clinical presentation but muscle biopsy findings were highly variable. Heterozygous missense mutations in RYR1 were uncovered in three out of four families, of which one mutation was novel and two have previously been reported in MH. Further RyR1 protein expression studies performed in two families showed marked reduction of the RyR1 protein, indicating the presence of allelic RYR1 mutations not detectable on routine sequencing and potentially explaining marked intrafamilial variability. Our findings support the hypothesis that RYR1 mutations are associated with King–Denborough syndrome but that further genetic heterogeneity is likely.

Increased Expression of Wild-Type or a Centronuclear Myopathy Mutant of Dynamin 2 in Skeletal Muscle of Adult Mice Leads to Structural Defects and Muscle Weakness

Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.

Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle characterized by disturbance of intracellular calcium homeostasis in the sarcoplasmic reticulum. Mutations of the ryanodine receptor 1 (RYR1) gene account for most cases, with some studies claiming up to 86% of mutations in this locus. However, RYR1 gene is large and variants are common even in the normal population. We examined 54 families with MH susceptibility and 21 diagnosed with equivocal MH. Thirty-five were selected for an anesthetic reaction, whereas the remainder for hyperCKemia. In these, we studied all 106 exons of the RYR1 gene. When no mutation was found, we also screened: sodium channel voltage-gated, type IV alpha subunit (SCN4A), calcium channel voltage-dependent, L type, alpha 1S subunit (CACNA1S), and L-type voltage-gated calcium channel alpha 2/delta-subunit (CACNL2A). Twenty-nine different RYR1 mutations were discovered in 40 families. Three other MH genes were tested in negative cases. Fourteen RYR1 amino acid changes were novel, of which 12 were located outside the mutational 'hot spots'. In two families, the known mutation p.R3903Q was also observed in malignant hyperthermia-nonsusceptible (MHN) individuals. Unexpectedly, four changes were also found in the same family and two in another. Our study confirms that MH is genetically heterogeneous and that a consistent number of cases are not due to RYR1 mutations. The discordance between in vitro contracture test status and the presence of a proven causative RYR1 mutation suggests that the penetrance may vary due to as yet unknown factors.

Long‐term high calcium decreases AMP‐activated protein kinase (AMPK) activity via activating protein phosphatase 2A

AMP‐activated protein kinase (AMPK) is activated by upstream kinases and negatively regulated by protein phosphatase 2A (PP2A). Calcium has recently been found to activate PP2A, which contains calcium binding sites. Our previous work has shown that chronic calcium exposure decreases AMPK activity. To define the specific molecular mechanism, activity of AMPK and PP2A was analyzed in C2C12 and skeletal muscles from mutant pigs possessing AMPKδ3 R225Q, ryanodine receptor mutation, or both. C2C12 myotubes treated with caffeine (calcium releasing agent) for 10 h decreased AICAR‐induced AMPK activity to control level and this negative effect has diminished by dantrolene (ryanodine receptor stabilizer). Interestingly, muscles from pigs with ryanodine receptor mutation and C2C12 cells administered with 10 h caffeine showed higher PP2A activity compared to control (P < 0.05). More importantly, the inhibitory effect of caffeine on AMPK activity was attenuated by PP2A inhibitor, okadaic acid or PP2A RNA interfering. These data show the inhibitory effect of chronic calcium on AMPK activity is exerted through the activation of PP2A.

P68 The pathological spectrum associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P68 The pathological spectrum associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene

Crystallographic Investigation of Several Malignant Hyperthermia and CPVT Mutations in Ryanodine Receptors

Mutations in the Ryanodine Receptor (RYR) are known to underlie many genetic diseases. In particular, the skeletal muscle isoform (RyR1) is involved in malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac isoform (RyR2) are known to cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD). Despite an extensive analysis of disease mutations on the functional level, very little is known about the structural changes induced by the mutations. Here we analyze the structures and stability of over 10 different disease mutants. We present crystal structures of mutant versions of the RyR1 N-terminal disease hot spot, encoding three domains, and of the RyR2 N-terminal domain, all solved between 2.0 and 3.0 Angstrom, and compare them to the wild type structures. The observed effects on structure and stability differ substantially among the mutants. Whereas some cause a major destabilization of the overall fold, others mainly cause relative domain-domain movements or confer large conformational changes within individual domains. We discuss the likely implications of the disease mutations on the overall structure and gating properties of the intact RyR.

Localization of the Dantrolene Target Sequence in the Cardiac Ryanodine Receptor by Combining cryo-EM and FRET Analysis

Mutations in ryanodine receptors (RyRs) cause calcium channel dysfunction in two skeletal muscle diseases, malignant hyperthermia (MH) and central core disease, and in polymorphic ventricular tachycardia in cardiac muscle. MH is an inherited condition that causes muscle rigidity and uncontrollable fever in reaction to administration of certain anesthetics. Dantrolene is a clinical drug that suppresses spontaneous Ca2+ release and is used as a therapeutic agent for individuals susceptible to MH. Recently, dantrolene has been shown to improve cardiomyocyte function in failing hearts. Biochemical studies suggest that the N-terminal region of RyR (amino acids 590-609 in RyR1 and 601-620 in RyR2) is the molecular target for dantrolene. We have attempted to localize the dantrolene target sequence by 3D cryo-electron microscopy. However, inserting a GFP after Arg-626 in RyR2 abolished the receptors’ binding to the GST-FKBP12.6 affinity column and prevented the purification of RyR2R626-GFP for structural studies. As an alternative approach, we designed several fluorescence resonance energy transfer (FRET) pairs based on our previous cryo-EM structures to map the location of Arg-626. We generated four pairs of dual insertions in RyR2 (RyR2S437-YFP/R626-CFP, RyR2R626-CFP/Y846-YFP, RyR2S437-YFP/Y846-CFP, and RyR2R626-YFP/S2367-CFP); three pairs between AF555-FKBP12.6 and RyR2-GFP (RyR2S437-GFP, RyR2R626-GFP, and RyR2Y846-GFP); and three pairs between CFP-FKBP12.6 and RyR2-YFP (RyR2S437-YFP, RyR2R626-YFP, and RyR2Y846-YFP). We estimated the 3D location of dantrolene binding sequence by measuring the FRET efficiencies in each of these pairs, and by correlating FRET efficiencies to the distance between donor and acceptor with known locations. Our results reveal the dantrolene target sequence is likely to be located in the clamp region close to the FKBP12.6 binding site.

RyR2 Channel Activity Determines the Potency of State-Dependent RyR2 Blockers for Suppressing Arrhythmogenic Calcium Waves

Mutations in ryanodine receptors (RyR2) or calsequestrin (casq2) cause catecholaminergic- polymorphic ventricular tachycardia (CPVT). We previously reported that the RyR2 open-channel blocker flecainide (FLEC) suppresses Ca2+ waves and prevents CPVT in mice and humans. Here we test the hypothesis that the open-state block by FLEC significantly contributes to FLEC efficacy in CPVT. We reasoned that FLEC would preferentially affect myocytes lacking casq2 (casq2-/-), which have higher rates of spontaneous RyR2 channel openings compared to WT channels. To test this hypothesis, we compared FLEC with tetracaine, a RyR2 channel blocker that has no state dependence and binds equally well to closed RyR2 channels. We found that FLEC reduced the incidence, amplitude and frequency of Ca2+ waves with significantly higher potency in casq2-/- myocytes compared to WT myocytes (Figure). In contrast, tetracaine did not suppress Ca2+ waves and had equal potency in WT and casq2-/- myocytes (Figure). Conclusion: RyR2 channel activity likely determines the potency of open-state RyR2 blockers such as FLEC for suppressing arrhythmogenic Ca2+ waves, a mechanism likely relevant to FLEC antiarrhythmic efficacy in CPVT. NIH HL88635 & HL71670.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The N-Terminal Disease Hot Spot of Ryanodine Receptors Forms a Cytoplasmic Vestibule

Ryanodine receptors (RyR) are ion channels that govern the release of Ca2+ from the endoplasmic reticulum. They thus regulate the contraction of skeletal and cardiac muscle. Mutations in RyR can lead to severe genetic conditions, including (but not limited to) malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT). Despite the detailed investigation of the functional effects of the mutations, locating their position in the full-length channel structure has traditionally proven to be difficult. Here we present the 2.5 Ångström resolution crystal structure of a region spanning most of the N-terminal disease hot spot (residues 1-559), containing over 50 disease mutations in RyR1 and RyR2. The hot spot consists of three domains that interact through a predominantly hydrophilic interface. We have been able to dock the position of this hot spot into various RyR1 cryo electron microscopy maps, allowing its unambiguous positioning in the cytoplasmic portion of the channel, forming a 240-kDa ring around the fourfold symmetry axis. The disease mutations can be grouped into three different categories, either destabilizing the interfaces between the three N-terminal domains, affecting the folding of individual domains, or affecting one of six interfaces with other RyR parts. We propose a model whereby the opening of RyR coincides with allosterically coupled motions within the N-terminal domains. This can be affected by mutations that target various interfaces within and across subunits. We also propose a mechanism whereby RyRs are activated by redox modification through the destabilization of observed domain-domain interfaces. The structure provides a framework to understand the many disease mutations that have been studied using functional methods.

Trans Complementation with the Common G1885E Polymorphism Attenuates Mutant RyR2 L433P Channel Dysfunction

A channelopathic Leu433Pro (L433P) mutation in the cardiac ryanodine receptor (RyR2) alters intracellular Ca2+ handling and is associated with malignant arrhythmia. This mutation is reported to exclusively co-inherit with a common single nucleotide polymorphism (SNP) Gly1885Glu (G1885E) present on the same allele (i.e. in cis). We investigated the functional impact of the G1885E SNP on L433P channels. RyR2-null HEK cells were used as the background for the systematic expression of homo- and heteromeric RyR2 channels composed of recombinant wild-type (WT) subunits, and those containing L433P and G1885E substitutions. Homomeric G1885E channels were functionally normal, but Ca2+ handling abnormalities associated with the L433P mutation were exacerbated by complementation with the G1885E in cis. At the cellular level, channel dysfunction manifested as reduced caffeine-evoked Ca2+ release and decreased ER Ca2+ store content. These findings suggest that although L433P is not a typical gain-of-function mutation it augments basal channel activity. Co-expression of L433P subunits with those containing G1885E (i.e. in trans complementation) attenuated L433P mutant channel dysfunction. Cells expressing heteromeric assemblies of L433P and G1885E subunits exhibited caffeine-activation profiles, homeostatic and post-activation Ca2+ fluxes and ER Ca2+ loads that were comparable to those typical of WT channels. Our data show that the genotype, allelic distribution and the resultant mode of L433P and G1885E channel assembly (i.e. cis or trans subunit complementation) has divergent effects on RyR2 channel (dys)function. The G1885E SNP may be a complex determinant of channel behaviour in situ and the potential therapeutic benefits of normalising mutant channel dysfunction via trans-complementation strategies should be further explored.

Novel Mechanisms of CPVT1 in a RyR2-V2475F Knock-In Mouse Model

Mutations in the cardiac ryanodine receptor (RyR2) have been implicated in Catecholaminergic Polymorphic Ventricular Tachicardia 1 (CPVT1), an arrhythmogenic syndrome triggered by emotional or physical stress in otherwise healthy individuals. At the cellular level, the adrenergic surge during stress presumably induces diastolic SR Ca2+ release, generating a favorable substrate for arrhythmias by the mechanism of delayed afterdepolarization (DAD). Increased sensitivity to luminal [Ca2+], unzipping of domain-domain interactions, and de-regulation by accessory proteins have been reported to contribute to RyR2 dysfunction in CPVT1. We generated a knock-in mouse model of CPVT1 (RyR2-V2475F) and characterized its mechanisms of arrhythmia. Expression of RyR2-V2475F in HEK293 cells induces a unique phenotype, namely, increased activity at diastolic [Ca2+] (pCa 7), which is not seen in other CPVT1 mutations. Mice homozygous for the V2475F mutation have not been detected in >500 mice, suggesting that the mutation causes catastrophic RyR2 dysfunction and embryonic lethality. Heterozygous mice, on the other hand, display increased propensity for premature ventricular complexes, bidirectional ventricular tachycardia and other forms of tachyarrythmias when injected a cocktail of norepinephrine and caffeine that is mostly innocuous in wild-type mice. Heterozygous Langendorff-perfused hearts subjected to adrenergic stimulation and tachypacing are also arrhythmogenic, indicating that the arrhythmias arise from mechanisms intrinsic to the heart and not necessarily from systemic factors. Despite this severe phenotype, ventricular cardiomyocytes isolated from RyR2-V2475F mice display only mild differences in the amplitude and kinetics of intracellular Ca2+ transients when compared to wild-type myocytes, regardless of stimulation frequency and β-adrenergic stimulation. Thus, cellular Ca2+ mishandling in ventricular myocytes of the RyR2-V2475F mice, although significant, does not appear sufficient to support the severe phenotype observed in whole hearts and intact mice. We are therefore investigating other mechanisms that may explain the arrhythmogenicity of heterozygous mice.