Mutations In Ryanodine Receptor Research Articles

Flecainide Specifically Targets the Monovalent Countercurrent Through the Cardiac Ryanodine Receptor, While a Dominant Opposing Ca2+/Ba2+ Current Is Present.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a highly arrhythmogenic syndrome triggered by stress, primarily linked to gain-of-function point mutations in the cardiac ryanodine receptor (RyR2). Flecainide, as an effective therapy for CPVT, is a known blocker of the surface-membrane Na+ channel, also affecting the intracellular RyR2 channel. The therapeutic relevance of the flecainide-RyR2 interaction remains controversial, as flecainide blocks only the RyR2 current flowing in the opposite direction to the physiological Ca2+ release from the sarcoplasmic reticulum (SR). However, it has been proposed that charge-compensating countercurrent from the cytosol to SR lumen plays a critical role, and its reduction may indeed suppress excessive diastolic SR Ca2+ release through RyR2 channels in CPVT. Monitoring single-channel properties, we examined whether flecainide can target intracellular pathways for charge-balancing currents carried by RyR2 and SR Cl- channels under cell-like conditions. Particularly, the Tris+ countercurrent flowed through the RyR2 channel simultaneously with a dominant reverse Ca2+/Ba2+ current. We demonstrate that flecainide blocked the RyR2-mediated countercurrent without affecting channel activity. In contrast, the SR Cl- channel was completely resistant to flecainide. Based on these findings, it is reasonable to propose that the primary intracellular target of flecainide in vivo is the RyR2-mediated countercurrent.

Disparate molecular mechanisms in cardiac ryanodine receptor channelopathies.

Mutations in the cardiac ryanodine receptor (RyR2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). This study investigates the underlying molecular mechanisms for CPVT mutations within the RyR2 N-terminus domain (NTD). We consulted the high-resolution RyR2 structure in both open and closed configuration to identify mutations G357S/R407I and A77T, which lie within the NTD intra- and inter-subunit interface with the Core Solenoid (CSol), respectively. Their structural and functional roles were compared to R169L, a mutation that lies within the NTD-NTD inter-subunit interface. Using chemical cross-linking and co-immunoprecipitation assays, we show that R169L disrupts NTD tetramerization, while it does not alter the NTD-CSol interaction. Single cell Ca2+ imaging revealed that R169L increases the number of spontaneous Ca2+ transients and the proportion of oscillating cells, while reducing the Ca2+ store content. G357S and R407I do not affect NTD tetramerization, but they also do not alter the NTD-CSol interaction. Functionally, RyR2G357S-expressing cells have Ca2+ handling properties similar to RyR2WT. A77T enhances the NTD-CSol interaction, while it does not affect NTD tetramerization. Like R169L, A77T also increases the number of spontaneous Ca2+ transients and the proportion of oscillating cells, and it reduces the Ca2+ store content. However, unlike R169L that displays Ca2+ transients of normal amplitude and shorter duration, Ca2+ transients for A77T are of smaller amplitude and normal duration. The NTD-CSol inter-subunit interface variant, A77T, produces a hyperactive channel by altering a different structure-function parameter to other CPVT mutations within the RyR2 NTD. Reduced NTD-NTD inter-subunit interaction and reinforced NTD inter-subunit interaction with CSol are distinct molecular mechanisms for gain-of-function RyR2 arrhythmogenic mutations.

Structural basis for ryanodine receptor type 2 leak in heart failure and arrhythmogenic disorders

Heart failure, the leading cause of mortality and morbidity in the developed world, is characterized by cardiac ryanodine receptor 2 channels that are hyperphosphorylated, oxidized, and depleted of the stabilizing subunit calstabin-2. This results in a diastolic sarcoplasmic reticulum Ca2+ leak that impairs cardiac contractility and triggers arrhythmias. Genetic mutations in ryanodine receptor 2 can also cause Ca2+ leak, leading to arrhythmias and sudden cardiac death. Here, we solved the cryogenic electron microscopy structures of ryanodine receptor 2 variants linked either to heart failure or inherited sudden cardiac death. All are in the primed state, part way between closed and open. Binding of Rycal drugs to ryanodine receptor 2 channels reverts the primed state back towards the closed state, decreasing Ca2+ leak, improving cardiac function, and preventing arrhythmias. We propose a structural-physiological mechanism whereby the ryanodine receptor 2 channel primed state underlies the arrhythmias in heart failure and arrhythmogenic disorders.

Loop-Mediated Isothermal Amplification for Detecting Gly-4891-Glu and Ile-4734 Multiple Mutations of Ryanodine Receptor in the Fall Armyworm, Spodoptera frugiperda.

The key mutations, such as the Gly-4891-Glu substitution and the Ile-4734 multiple substitutions within the ryanodine receptors (RyR), are linked to diamide resistance in fall armyworm (FAW), Spodoptera frugiperda. In this study, we found that FAW remained sensitive to cyantraniliprole and chlorantraniliprole, while its sensitivity to flubendiamide was reduced. Moreover, a low level of heterozygous mutation at I4743 was observed. To facilitate the detection procedure of these mutations, a simple and efficient loop-mediated isothermal amplification (LAMP) protocol was developed for operation. The reaction for detecting the G4891E and I4743 single or multiple mutations was carried out at 68 °C for 85 min and 68 °C for 85 min or 68 °C for 65 min, respectively. These LAMP reactions can be easily observed via visualization of the color change from pink to yellow. This assay provides a simple, convenient, and effective means of detecting mutations in the RyR of FAW for pest management purposes.

Disrupted Endoplasmic Reticulum Ca2+ Handling: A Harβinger of β-Cell Failure.

The β-cell workload increases in the setting of insulin resistance and reduced β-cell mass, which occurs in type 2 and type 1 diabetes, respectively. The prolonged elevation of insulin production and secretion during the pathogenesis of diabetes results in β-cell ER stress. The depletion of β-cell Ca2+ER during ER stress activates the unfolded protein response, leading to β-cell dysfunction. Ca2+ER is involved in many pathways that are critical to β-cell function, such as protein processing, tuning organelle and cytosolic Ca2+ handling, and modulating lipid homeostasis. Mutations that promote β-cell ER stress and deplete Ca2+ER stores are associated with or cause diabetes (e.g., mutations in ryanodine receptors and insulin). Thus, improving β-cell Ca2+ER handling and reducing ER stress under diabetogenic conditions could preserve β-cell function and delay or prevent the onset of diabetes. This review focuses on how mechanisms that control β-cell Ca2+ER are perturbed during the pathogenesis of diabetes and contribute to β-cell failure.

Assaying sarcoplasmic reticulum Ca2+-leak in mouse atrial myocytes.

More and more studies have suggested an essential role of sarcoplasmic reticulum (SR) Ca2+ leak of atrial myocytes in atrial diseases such as atrial fibrillation (AF). The increasing interest in atrial Ca2+ signaling makes it necessary to develop a more accurate approach for Ca2+ measurement in atrial myocytes due to obvious differences between atrial and ventricular Ca2+ handling. In the present study, we proposed a new approach for quantifying total SR Ca2+ leak in atrial myocytes with confocal line-scan Ca2+ images. With a very precious approximation of the histogram of normalized line-scan Ca2+ images by using a modified Gaussian distribution, we separated the signal pixel components from noisy pixels and extracted two new dimensionless parameters, F signals and R signals, to reflect the summation of signal pixels and their release components, respectively. In the presence of tetracaine blocking SR Ca2+ leak, the two parameters were very close to 0, and in atrial myocytes under normal conditions, the two parameters are well positive correlative with Ca2+ spark frequency and total signal mass, the two classic readouts for SR Ca2+ leak. Consistent with Ca2+ Spark readouts, the two parameters quantified a significant increase of SR Ca2+ leak in atrial myocytes from mice harboring a leaky type 2 ryanodine receptor mutation (RyR2-R2474S+/-) compared to the WT group. Collectively, this study proposed a simple and effective approach to quantify SR Ca2+ leak in atrial myocytes, which may benefit research on calcium signaling in atrial physiology and diseases.

Overexpression of cardiac calsequestrin as a novel gene-therapy approach to treat CPVT1: in silico and in vivo proves of principle

Abstract Background More than 200 gain-of-function mutations in the cardiac ryanodine receptor (RyR2) cause catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1). From a molecular point of view, 3 types of mutations exist [1]: Class 1 mutants enhance RyR2 opening in response to sarcoplasmic reticulum (SR) free Ca2+; Class 2 and Class 3 mutants enhance RyR2 opening in response to both cytosolic and SR free Ca2+. Purpose The study's objective was to evaluate in silico and in vivo, in CPVT1 knock-in mice carriers of the Class 1 mutation RyR2(R4496C/+), whether overexpression of cardiac calsequestrin (CASQ2) rescues the arrhythmic phenotype in CPVT1 mice. Rationale: Several studies performed in the 2000’s have collectively demonstrated that an increase in CASQ2 promotes the closure of RyR2 channels [2]: inspired by this evidence, here we test the hypothesis that CASQ2 gene therapy might be a novel treatment for CPVT1. Methods (1) In silico study: Properties of Class 1 and Class 2 RyR2s mutants (Figure, panels A-C) were introduced into an in silico model of the murine ventricular myocyte [3]. Virtual myocytes were paced either at baseline or during simulated delivery of 100 nM Isoproterenol at 1 Hz for 180 seconds. To simulate CASQ2 overexpression, protein levels were progressively increased, and the changes in the arrhythmic burden were documented. (2) In vivo study (performed in compliance with ethical regulations): an adeno-associated viral vector containing the cDNA sequence of CASQ2 was injected into RyR2(R4496C/+) mice (tail iv, 3×10^13 vg/kg) at 4 weeks of age. Eight weeks post-infection, animals were treated with an ip bolus of caffeine (120mg/kg) and epinephrine (2 mg/kg) to elicit arrhythmias (Epi/Caff Test). Results (1) In silico: dose-response curves were simulated to correlate CASQ2 levels with arrhythmia inducibility during virtual Isoproterenol delivery. For the simulated prototypical Class 1 mutation, 1.4-fold CASQ2 expression suppressed arrhythmias. For the prototypical Class 2 mutation, 1.8-fold CASQ2 expression was needed (Figure, panel D). (2) In vivo: At 12 weeks of age, both RyR2(R4496C/+) treated and non-treated littermates received the ip Epi / Caff bolus. Arrhythmias were observed in 17/32 (53%) untreated mice and in 0/12 treated animals (p=0.0012). Conclusion(s) We provide proof-of-principle demonstrating that in mice, the overexpression of CASQ2 may be a powerful and safe therapeutic strategy for CPVT1.

Risk assessment, fitness cost, cross-resistance, and mechanism of tetraniliprole resistance in the rice stem borer, Chilo suppressalis.

The rice stem borer (RSB), Chilo suppressalis, a notorious rice pest in China, has evolved a high resistance level to commonly used insecticides. Tetraniliprole, a new anthranilic diamide insecticide, effectively controls multiple pests, including RSB. However, the potential resistance risk of RSB to tetraniliprole is still unknown. In this study, the tetraniliprole-selection (Tet-R) strain was obtained through 10 continuous generations of selection with tetraniliprole 30% lethal concentration (LC30). The realized heritability (h2) of the Tet-R strain was 0.387, indicating that resistance of RSB to tetraniliprole developed rapidly under the continuous selection of tetraniliprole. The Tet-R strain had a high fitness cost (relative fitness=0.53). We established the susceptibility baseline of RSB to tetraniliprole (lethal concentration at LC50=0.727mg/L) and investigated the resistance level of 6 field populations to tetraniliprole. All tested strains that had resistance to chlorantraniliprole exhibited moderate- to high-level resistance to tetraniliprole (resistance ratio=27.7-806.8). Detection of ryanodine receptor (RyR) mutations showed that the Y4667C, Y4667D, I4758M, and Y4891F mutations were present in tested RSB field populations. RyR mutations were responsible for the cross-resistance between tetraniliprole and chlorantraniliprole. Further, the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-mediated genome-modified flies were used to study the contribution of RyR mutations to tetraniliprole resistance. The order of contribution of a single RyR mutation to tetraniliprole resistance was Y4667D>G4915E>Y4667C≈I4758M>Y4891F. In addition, the I4758M and Y4667C double mutations conferred higher tetraniliprole resistance than single Y4667C mutations. These results can guide resistance management practices for diamides in RSB and other arthropods.

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

Function of a mutant ryanodine receptor (T4709M) linked to congenital myopathy

Physiological muscle contraction requires an intact ligand gating mechanism of the ryanodine receptor 1 (RyR1), the Ca2+-release channel of the sarcoplasmic reticulum. Some mutations impair the gating and thus cause muscle disease. The RyR1 mutation T4706M is linked to a myopathy characterized by muscle weakness. Although, low expression of the T4706M RyR1 protein can explain in part the symptoms, little is known about the function RyR1 channels with this mutation. In order to learn whether this mutation alters channel function in a manner that can account for the observed symptoms, we examined RyR1 channels isolated from mice homozygous for the T4709M (TM) mutation at the single channel level. Ligands, including Ca2+, ATP, Mg2+ and the RyR inhibitor dantrolene were tested. The full conductance of the TM channel was the same as that of wild type (wt) channels and a population of partial open (subconductive) states were not observed. However, two unique sub-populations of TM RyRs were identified. One half of the TM channels exhibited high open probability at low (100 nM) and high (50 μM) cytoplasmic [Ca2+], resulting in Ca2+-insensitive, constitutively high Po channels. The rest of the TM channels exhibited significantly lower activity within the physiologically relevant range of cytoplasmic [Ca2+], compared to wt. TM channels retained normal Mg2+ block, modulation by ATP, and inhibition by dantrolene. Together, these results suggest that the TM mutation results in a combination of primary and secondary RyR1 dysfunctions that contribute to disease pathogenesis.

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.

Exaptation of I4760M mutation in ryanodine receptor of Spodoptera exigua (Lepidoptera: Noctuidae): Lessons from museum and field samples

Since 2007, diamide insecticides have been widely used in Korea to control various types of lepidopteran pests including Spodoptera exigua. For nearly a decade, diamide resistance in field populations of S. exigua across 18 localities has been monitored using bioassays. Despite their short history of use, resistance to diamide insecticides has emerged. Based on the LC50 values, some field populations showed a higher level of resistance to chlorantraniliprole, a diamide insecticide, compared to that of the susceptible strain, although regional and temporal variations were observed. To investigate resistance at a molecular level, we examined three mutations (Y4701C, I4790M, and G4946E) in the ryanodine receptor (RyR), which is the primary mechanism underlying diamide insecticide resistance. DNA sequencing showed that only the I4790M mutation was found in most field populations. As resistance levels varied significantly despite the uniform presence of the I4790M mutation, we considered the presence of another resistance factor. Further, the I4790M mutation was also found in S. exigua specimens collected prior to the commercialization of diamide insecticides in Korea as well as in other countries, such as the USA. This finding led us to hypothesize that the I4790M mutation were predisposed in field populations owing to selection factors other than diamide use. For further clarification, we conducted whole-genome sequencing of S. exigua (449.83 Mb) and re-sequencing of 18 individual whole genomes. However, no additional non-synonymous mutations were detected in the RyR-coding region. Therefore, we concluded that the high level of diamide insecticide resistance in Korean S. exigua is not caused by mutations at the target site, RyR, but is attributed to other factors that need to be investigated in future studies.

Arrhythmogenic mechanism of a novel ryanodine receptor mutation underlying sudden cardiac death.

The ryanodine receptor 2 (RyR2) is essential for cardiac muscle excitation-contraction coupling; dysfunctional RyR2 participates in the development of inherited arrhythmogenic cardiac disease. In this study, a novel RyR2 mutation A690E is identified from a patient with family inheritance of sudden cardiac death, and we aimed to investigate the pathogenic basis of the mutation. We generated a mouse model that carried the A690E mutation. Mice were characterized by adrenergic-induced ventricular arrhythmias similar to clinical manifestation of the patient. Optical mapping studies revealed that isolated A690E hearts were prone to arrhythmogenesis and displayed frequency-dependence calcium transient alternans. Upon β-adrenoceptor challenge, the concordant alternans was shifted towards discordant alternans that favour triggering ectopic beats and Ca2+ re-entry; similar phenomenon was also found in the A690E cardiomyocytes. In addition, we found that A690E cardiomyocytes manifested abnormal Ca2+ release and electrophysiological disorders, including an increased sensitivity to cytosolic Ca2+, an elevated diastolic RyR2-mediated Ca2+ leak, and an imbalance between Ca2+ leak and reuptake. Structural analyses reveal that the mutation directly impacts RyR2-FK506 binding protein interaction. In this study, we have identified a novel mutation in RyR2 that is associated with sudden cardiac death. By characterizing the function defects of mutant RyR2 in animal, whole heat, and cardiomyocytes, we demonstrated the pathogenic basis of the disease-causing mutation and provided a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

Increased Ca2+ Transient Underlies RyR2-Related Left Ventricular Noncompaction.

A loss-of-function cardiac ryanodine receptor (RyR2) mutation, I4855M+/-, has recently been linked to a new cardiac disorder termed RyR2 Ca2+ release deficiency syndrome (CRDS) as well as left ventricular noncompaction (LVNC). The mechanism by which RyR2 loss-of-function causes CRDS has been extensively studied, but the mechanism underlying RyR2 loss-of-function-associated LVNC is unknown. Here, we determined the impact of a CRDS-LVNC-associated RyR2-I4855M+/- loss-of-function mutation on cardiac structure and function. We generated a mouse model expressing the CRDS-LVNC-associated RyR2-I4855M+/- mutation. Histological analysis, echocardiography, ECG recording, and intact heart Ca2+ imaging were performed to characterize the structural and functional consequences of the RyR2-I4855M+/- mutation. As in humans, RyR2-I4855M+/- mice displayed LVNC characterized by cardiac hypertrabeculation and noncompaction. RyR2-I4855M+/- mice were highly susceptible to electrical stimulation-induced ventricular arrhythmias but protected from stress-induced ventricular arrhythmias. Unexpectedly, the RyR2-I4855M+/- mutation increased the peak Ca2+ transient but did not alter the L-type Ca2+ current, suggesting an increase in Ca2+-induced Ca2+ release gain. The RyR2-I4855M+/- mutation abolished sarcoplasmic reticulum store overload-induced Ca2+ release or Ca2+ leak, elevated sarcoplasmic reticulum Ca2+ load, prolonged Ca2+ transient decay, and elevated end-diastolic Ca2+ level upon rapid pacing. Immunoblotting revealed increased level of phosphorylated CaMKII (Ca2+-calmodulin dependent protein kinases II) but unchanged levels of CaMKII, calcineurin, and other Ca2+ handling proteins in the RyR2-I4855M+/- mutant compared with wild type. The RyR2-I4855M+/- mutant mice represent the first RyR2-associated LVNC animal model that recapitulates the CRDS-LVNC overlapping phenotype in humans. The RyR2-I4855M+/- mutation increases the peak Ca2+ transient by increasing the Ca2+-induced Ca2+ release gain and the end-diastolic Ca2+ level by prolonging Ca2+ transient decay. Our data suggest that the increased peak-systolic and end-diastolic Ca2+ levels may underlie RyR2-associated LVNC.

The ryanodine receptor mutation I4728M confers moderate-level resistance to diamide insecticides in Spodoptera litura.

The common cutworm, Spodoptera litura (Fabricius), is one of the most widespread and destructive polyphagous pests in tropical and subtropical Asia. S. litura has evolved resistance to different insecticides, including diamide insecticides. Here, we identified a ryanodine receptor (RyR) mutation (I4728M) associated with target site resistance to diamides in a field-collected population of S. litura. The contribution of this mutation to diamide resistance was investigated through establishing a near-isogenic resistant strain of S. litura. The ND21 population of S. litura, collected from Ningde, Fujian province of China in 2021, exhibited 130.6-fold resistance to chlorantraniliprole compared to the susceptible NJ-S strain. S. litura RyR mutation I4728M, corresponding to Plutella xylostella RyR I4790M, was identified in the ND21 population. SlRyR I4728M mutation of ND21 was introgressed into a susceptible background strain (NJ-S) with marker-assisted backcrossing. The introgressed strain named ND21-R, which was homozygous for the mutant 4728M allele, shared about 94% of the genetic background with the NJ-S strain. ND21-R strain showed moderate levels of resistance to two anthranilic diamides (19.1-fold to chlorantraniliprole, 19.7-fold to cyantraniliprole) and the phthalic diamide flubendiamide (23.4-fold). Genetic analysis showed that chlorantraniliprole resistance was autosomal, incompletely recessive and tightly linked with SlRyR I4728M mutation in the introgressed ND21-R strain of S. litura. Identification of the I4728M mutation and its contribution to diamide resistance in S. litura will help develop allelic discrimination assays for resistance monitoring and guide resistance management practices for diamides in S. litura. © 2023 Society of Chemical Industry.

Insecticide resistance monitoring of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) and its mechanism to chlorantraniliprole.

The rice leaffolder, Cnaphalocrocis medinalis (Guenée), has become an increasingly occurring pest in Asia in recent years. Chemical control remains the most efficient and primary tool for controlling this pest. In this study, we report the resistance status of C. medinalis in China to multiple insecticides including chlorantraniliprole and the main resistance mechanism. Significant variations among field populations of C. medinalis in their resistance to 10 insecticides were observed during 2019-2022. Most of the tested field populations have developed low-to-moderate levels of resistance to abamectin (RR = 2.4-22.2), emamectin benzoate (RR = 1.9-40.3) and spinetoram (RR = 4.2-24.8). Some field populations have developed low resistance to chlorpyrifos (RR = 0.9-6.8). Indoxacarb, metaflumizone, methoxenozide and Bacillus thuringiensis (Bt) potency against all tested populations remained similar. For diamides, significantly higher levels of resistance to chlorantraniliprole (RR = 64.9-113.7) were observed in 2022, whereas all tested field populations in 2019-2021 exhibited susceptible or moderate resistance level to chlorantraniliprole (RR = 1.3-22.1). Cross-resistance between chlorantraniliprole and tetraniliprole was significant. Analysis of ryanodine receptor (RyR) mutations showed that mutation of I4712M was present in resistant populations of C. medinalis with different levels of chlorantraniliprole resistance and was the main mechanism conferring diamide resistance. Mutation of Y4621D also was detected in one tested population. Resistance management strategies for the control of C. medinalis are discussed. C. medinalis has developed high level of resistance to chlorantraniliprole. RyR mutations were deemed as the mechanism. © 2023 Society of Chemical Industry.

Molecular and functional characterization of a novel mutation in the cardiac ryanodine receptor (RyR2) associated with long QT syndrome

Molecular and functional characterization of a novel mutation in the cardiac ryanodine receptor (RyR2) associated with long QT syndrome

Abstract 14182: A Missense Mutation in Ryanodine Receptor 2 Leads to Structural Remodeling of the Heart and Cardiac Arrhythmia in a Rabbit Model

Ryanodine receptor 2 (RyR2) is the Ca 2+ release channel of sarcoplasmic reticulum that provides the majority of Ca 2+ necessary for contractions of the heart. RyR2 mutations are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disorder with normal heart structure. There are reports suggesting that RyR2 mutations can also lead to structural heart disease, notably arrhythmogenic right ventricular cardiomyopathy (ARVC); however, additional research is required to support this association. We generated a rabbit model carrying the missense mutation RyR2-V2475F, linked to CPVT in humans. Heterozygous (V2475F +/- ) rabbits show a CPVT-like syndrome and display arrhythmia under pharmacological stress. Homozygotes (V2475F +/+ ) show a stronger phenotype, with 37% of the animals (13/35) experiencing sudden death at 22.5±3.8 weeks old during regular housing. Remarkably, gross examination of V2475F +/+ hearts showed significant structural remodeling, characterized by fibrous infiltrations and thinning of the right ventricle with or without left ventricle involvement. This phenotype is reminiscent of ARVC. Lead II ECG obtained in conscious rabbits by telemetry showed that V2475F +/+ animals have significant bradycardia (144.6±11.1 bpm) with prolonged QT interval (QTc: 180.4±8.2 ms, n=6) compared to WT (HR: 224.9±4.2 bpm, p=0.04; QTc: 157.4±5.3 ms, p=0.02, n=5). After subcutaneous injection of 0.5 mg/kg of isoproterenol, WT rabbits showed the expected positive chronotropic response (HR, 367±10 bpm, n=3) while V2475F +/+ animals developed bouts of ventricular tachycardia. Mild emotional stress applied by transporting the animal around the laboratory, also induced ventricular arrhythmia in 100% (5/5) V2475F +/+ rabbits, while WT littermates were unaffected. RyR2-V2475F expressed in a rabbit model triggers remarkably different phenotypes based on the zygosity of the animals, ranging from a purely arrhythmogenic syndrome (CPVT) in V2475F +/- animals to severe structural disease compatible with ARVC in V2475F +/+ rabbits. The RyR2-V2475F rabbit model is therefore an excellent model to dissect the intersection of arrhythmic and structural phenotypes due to RyR2 dysfunction.

A novel mutation in ryanodine receptor 2 (RYR2) genes at c.12670G>T associated with focal epilepsy in a 3-year-old child.

Ryanodine receptor 2 (RYR2) encodes a component of a calcium channel. RYR2 variants were well-reported to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), but rarely reported in epilepsy cases. Here, we present a novel heterozygous mutation of RYR2 in a child with focal epilepsy. At the age of 2 years and 7 months, the patient experienced seizures, such as eye closure, tooth clenching, clonic jerking and hemifacial spasm, as well as abnormal electroencephalogram (EEG). Then, he was analyzed by whole-exome sequencing (WES). The mutations of both the proband and his parents were further confirmed by Sanger sequencing. The pathogenicity of the variant was further assessed by population-based variant frequency screening, evolutionary conservation comparison, and American Association for Medical Genetics and Genomics (ACMG) scoring. WES sequencing revealed a novel heterozygous truncating mutation [c.12670G > T, p.(Glu4224*), NM_001035.3] in RYR2 gene of the proband. Sanger sequencing confirmed that this mutation was inherited from his mother. This novel variant was predicted to be damaging by different bioinformatics methods. Cardiac investigation showed that the proband had no structural abnormalities, but sinus tachycardia. We proposed that RYR2 is a potential candidate gene for focal epilepsy, and epilepsy patients carried with RYR2 variants should be given more attention, even if they do not show cardiac abnormalities.

Development of a water-soluble ryanodine receptor 1 inhibitor

Ryanodine receptor 1 (RyR1) is a Ca2+-release channel expressed on the sarcoplasmic reticulum (SR) membrane. RyR1 mediates release of Ca2+ from the SR to the cytoplasm to induce muscle contraction, and mutations associated with overactivation of RyR1 cause lethal muscle diseases. Dantrolene sodium salt (dantrolene Na) is the only approved RyR inhibitor to treat malignant hyperthermia patients with RyR1 mutations, but is poorly water-soluble. Our group recently developed a bioassay system and used it to identify quinoline derivatives such as 1 as potent RyR1 inhibitors. In the present study, we focused on modification of these inhibitors with the aim of increasing their water-solubility. First, we tried reducing the hydrophobicity by shortening the N-octyl chain at the quinolone ring of 1; the N-heptyl compound retained RyR1-inhibitory activity, but the N-hexyl compound showed decreased activity. Next, we introduced a more hydrophilic azaquinolone ring in place of quinolone; in this case, only the N-octyl compound retained activity. The sodium salt of N-octyl azaquinolone 7 showed similar inhibitory activity to dantrolene Na with approximately 1,000-fold greater solubility in saline.