Mutations In Ryanodine Receptor Research Articles

Congenital myopathies: not only a paediatric topic

This article reviews adult presentations of the major congenital myopathies - central core disease, multiminicore disease, centronuclear myopathy and nemaline myopathy - with an emphasis on common genetic backgrounds, typical clinicopathological features and differential diagnosis. The congenital myopathies are a genetically heterogeneous group of conditions with characteristic histopathological features. Although essentially considered paediatric conditions, some forms - in particular those due to dominant mutations in the skeletal muscle ryanodine receptor (RYR1), the dynamin 2 (DNM2), the amphiphysin 2 (BIN1) and the Kelch repeat-and BTB/POZ domain-containing protein 13 (KBTBD13) gene - may present late into adulthood. Moreover, dominant RYR1 mutations associated with the malignant hyperthermia susceptibility trait have been recently identified as a common cause of (exertional) rhabdomyolysis presenting throughout life. In addition, improved standards of care and development of new therapies will result in an increasing number of patients with early-onset presentations transitioning to the adult neuromuscular clinic. Lastly, if nemaline rods are the predominant histopathological feature, acquired treatable conditions have to be considered in the differential diagnosis. Recently identified genotypes and phenotypes indicate a spectrum of the congenital myopathies extending into late adulthood, with important implications for clinical practice.

Next generation sequencing reveals ryanodine receptor 1 mutations in a Chinese central core disease cohort

Ryanodine receptor 1 (RYR1), myosin heavy chain 7 (MYH7), and selenoprotein N1 (SEPN1) mutations are associated with core myopathies. RYR1 mutations cause most cases of central core disease (CCD). We screened 8 Chinese patients with clinicopathological diagnosis of CCD. Genetic analysis was carried out by targeted next generation sequencing (NGS) to identify causative genes. Variants were assessed for pathogenicity using bioinformatic approaches, and NGS results were confirmed by Sanger sequencing. One novel (p.L4578V) and heterozygous missense mutations in RYR1 were identified in 7 patients. Two patients carried a novel mutation, 1 had p.M4640R, 3 had p.R4861H, and 1 had p.R4861C. All patients had mild to moderate severity phenotypes. Histopathological findings demonstrated central cores and type I fiber predominance. NGS is an efficient strategy to identify variants in RYR1 in CCD. However, genetic results revealed by NGS must be combined with clinicopathologic features to validate the diagnosis. Muscle Nerve, 2016 Muscle Nerve 54: 432-438, 2016.

Exercise training prevents ventricular tachycardia in CPVT1 due to reduced CaMKII-dependent arrhythmogenic Ca2+ release.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is caused by mutations in the cardiac ryanodine receptor (RyR2) that lead to disrupted Ca(2+) handling in cardiomyocytes and ventricular tachycardia. The aim of this study was to test whether exercise training could reduce the propensity for arrhythmias in mice with the CPVT1-causative missense mutation Ryr2-R2474S by restoring normal Ca(2+) handling. Ryr2-R2474S mice (RyR-RS) performed a 2 week interval treadmill exercise training protocol. Each exercise session comprised five 8 min intervals at 80-90% of the running speed at maximal oxygen uptake (VO2max) and 2 min active rest periods at 60%. VO2max increased by 10 ± 2% in exercise trained RyR-RS (ET), while no changes were found in sedentary controls (SED). RyR-RS ET showed fewer episodes of ventricular tachycardia compared with RyR-RS SED, coinciding with fewer Ca(2+) sparks and waves, less diastolic Ca(2+) leak from the sarcoplasmic reticulum, and lower phosphorylation levels at RyR2 sites associated with Ca(2) (+)-calmodulin-dependent kinase type II (CaMKII) compared with RyR-RS SED. The CaMKII inhibitor autocamtide-2-related inhibitory peptide and also the antioxidant N-acetyl-l-cysteine reduced Ca(2+) wave frequency in RyR-RS equally to exercise training. Protein analysis as well as functional data indicated a mechanism depending on reduced levels of oxidized CaMKII after exercise training. Two weeks of detraining reversed the beneficial effects of the interval treadmill exercise training protocol in RyR-RS ET. Long-term effects of interval treadmill exercise training reduce ventricular tachycardia episodes in mice with a CPVT1-causative Ryr2 mutation through lower CaMKII-dependent phosphorylation of RyR2.

The effects of ryanodine receptor (RYR1) mutation on natural killer cell cytotoxicity, plasma cytokines and stress hormones during acute intermittent exercise in pigs

Stress susceptibility has been mapped to a single recessive gene, the ryanodine receptor 1 (RYR1) gene or halothane (Hal) gene. Homozygous (Halnn), mutated pigs are sensitive to halothane and susceptible to Porcine Stress Syndrome (PSS). Previous studies have shown that stress-susceptible RYR1 gene mutated homozygotes in response to restraint stress showed an increase in natural killer cell cytotoxicity (NKCC) accompanied by more pronounced stress-related hormone and anti-inflammatory cytokine changes. In order to determine the relationship of a RYR1 gene mutation with NKCC, plasma cytokines and stress-related hormones following a different stress model — exercise — 36 male pigs (representing different genotypes according to RYR1 gene mutation: NN, homozygous dominant; Nn, heterozygous; nn, homozygous recessive) were submitted to an intermittent treadmill walking. During the entire experiment the greatest level of NKCC and the greatest concentrations of interleukin (IL-) 6, IL-10, IL-12, interferon (IFN-)γ and tumor necrosis factor-α and stress-related hormones (adrenaline, prolactin, beta-endorphin) were observed in nn pigs, and the greatest concentration of IL-1 and growth hormone in NN pigs. Immunostimulatory effects of intermittent exercise on NKCC in nn pigs were concomitant with increases in IL-2, IL-12 and IFN-γ, the potent NKCC activators. Our findings suggest that stress-susceptible pigs RYR1 gene mutated pigs develop a greater level of NKCC and cytokine production in response to exercise stress. These results suggest that the heterogeneity of immunological and neuroendocrine response to exercise stress in pigs could be influenced by RYR1 gene mutation.

Congenital ptosis, scoliosis, and malignant hyperthermia susceptibility in siblings with recessive RYR1 mutations

Malignant hyperthermia susceptibility is a rare pharmacogenic disorder of skeletal muscle calcium regulation caused by mutations in the skeletal muscle ryanodine receptor 1 gene (RYR1). It is important to identify children who are candidates for ophthalmic surgery who might harbor RYR1 mutations because intraoperative malignant hyperthermia is potentially lethal. We report 2 siblings with congenital ptosis and scoliosis who were considered for ptosis surgery but were found to harbor underlying recessive RYR1 mutations.

The RyR2-P2328S mutation downregulates Nav1.5 producing arrhythmic substrate in murine ventricles

Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca2+ homeostasis and can arise from ryanodine receptor (RyR2) mutations including RyR2-P2328S. Previous reports established that homozygotic murine RyR2-P2328S (RyR2S/S) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2S/S hearts to connexin-43 (Cx43) and Nav1.5 expression and Na+ current (INa). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2S/S hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2S/S than WT, but comparable changes in AP durations (APD90) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2S/S compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced INa in RyR2S/S ventricles. We thus attribute arrhythmogenesis in RyR2S/S ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing INa, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak INa of the AP but nonlinear relationships between peak INa and maximum Na+ permeability.

Several Ryanodine Receptor Type 1 Gene Mutations of p.Arg2508 Are Potential Sources of Malignant Hyperthermia

Malignant hyperthermia (MH) is a pharmacogenetic disorder that occurs in predisposed individuals after exposure to volatile anesthetics or depolarizing muscle relaxants. Genetic mutations of ryanodine receptor 1 (RYR1), which are considered to cause MH, are found mainly in 3 regions called "hotspots." There are sometimes multiple mutations at the same site of RYR1. Although p.Arg2508 of RYR1 is located outside hotspots, several mutations or variants (including the known MH causative mutation p.Arg2508Cys) have been identified in this region. We hypothesized that any mutations or variants in RYR1 p.Arg2508 cause important changes in pathological conditions related to MH. In this study, we analyzed the functions of 4 different RYR1 variants containing mutations at p.Arg2508. We prepared and analyzed the functions of 4 mutated RYR1 genes: p.Arg2508His and p.Arg2508Gly are MH-related variants, whereas p.Arg2508Ser and p.Arg2508Lys have not been previously reported. Because the biochemical characteristics of lysine are similar to arginine, we assumed that p.Arg2508Lys RYR1 would have characteristics most similar to those of the wild-type RYR1. We introduced these 4 mutated RYR1 genes, p.Arg2508His, p.Arg2508Gly, p.Arg2508Ser, and p.Arg2508Lys into rabbit RYR1 cDNA and transfected the resultant clones into human embryonic kidney 293 cells. Using the ratiometric dye Fura-2 AM, we used the 340/380 nm ratio to analyze alterations in calcium homeostasis after stimulation with caffeine and 4-chloro-m-cresol (4CmC). We calculated the half-maximal activation concentrations (EC50) of cells transfected with each mutant and compared the EC50 value of cells expressing each mutant with that of cells expressing wild-type RYR1. Statistical significance between EC50 values were calculated using an unpaired 2-tailed t test. We used 300 different cells, by 30 cells in each of the wild type or mutant. Cells transfected with each of the 4 mutants, p.Arg2508His, p.Arg2508Gly, p.Arg2508Ser, or p.Arg2508Lys, were more sensitive to caffeine and 4CmC than cells transfected with the wild type (all 4 P ≤ 0.0004). Mean ± SD of EC50 values for caffeine of wild type, p.Arg2508His, p.Arg2508Gly, p.Arg2508Ser, and p.Arg2508Lys were 2.53 ± 0.89, 1.72 ± 0.72, 1.73 ± 0.79, 1.69 ± 0.80, and 1.61 ± 0.74 mM, respectively, and those for 4CmC were 125.92 ± 38.11, 70.42 ± 27.09, 79.30 ± 39.04, 73.03 ± 19.20, and 72.81 ± 28.44 mM, respectively. Any of these 4 mutations in RYR1 p.Arg2508 may cause important changes related to MH. Studying the effects of changes in amino acids at 2508 in RYR1 on the movement of this large protein may lead to a better understanding of the pathology of MH events.

MiRNAs regulated overexpression of ryanodine receptor is involved in chlorantraniliprole resistance in Plutella xylostella (L.).

The amino acid mutations in ryanodine receptor (RyR) and elevated activity of detoxification enzymes have been associated with the diamide insecticide resistance in the diamondback moth, Plutella xylostella (L.). The up-regulation of P. xylostella RyR mRNA (PxRyR) expression has also been reported in field populations of different graphical origin. However, whether the up-regulation of PxRyR is involved in diamide resistance remains unknown. In this paper, 2.28- to 4.14-fold higher expression of PxRyR was detected in five field collected resistant populations, compared to that in a susceptible population. The expression of PxRyR was up-regulated 5.0- and 7.2-fold, respectively, after P. xylostella was treated with LC50 and LC75 of chlorantraniliprole for 12 h. Suppression of PxRyR using RNA interference restored the toxicity of chlorantraniliprole against the fourth instar larvae from the resistant population. More importantly, the expression of PxRyR is regulated by two miRNAs, miR-7a and miR-8519. These findings provide an empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance, and shed light on the novel targets for the sustainable management of this devastating insect pest.

Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts.

AimsCardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav1.5 currents in homozygous RyR2-P2328S (RyR2S/S) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2S/S mice and their wild-type (WT) littermates.MethodsWe explored effects of 1 μm flecainide on WT and RyR2S/S atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na+ currents (INa) were recorded using loose patch clamping of superfused atria.ResultsRyR2S/S showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2S/S but pro-arrhythmic in WT. It increased INa in RyR2S/S atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2S/S, but reduced CV while sparing AERP in WT. Thus, RyR2S/S hearts have low λ relative to WT; flecainide then increases λ in RyR2S/S but decreases λ in WT.ConclusionsFlecainide (1 μm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca2+ release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.

0113 : Short-coupled polymorphic ventricular tachycardia at rest linked to a novel ryanodine receptor (RyR2) mutation: leaky RyR2 channels under non-stress conditions

The ryanodine receptor / Ca2+ release channel (RyR2) is one of the main actors of the excitation-contraction coupling in the heart. Single-point mutations on RyR2 have been associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) and sudden cardiac death. In this study, we characterized a novel heterozygous RyR2 mutation found in a 31-year-old female and her mother with syncope at rest and recurrent short-coupled premature ventricular contractions (PVCs) initiating polymorphic ventricular tachycardia (PMVT). Using site-directed mutagenesis, we expressed human RyR2-H29D mutant channels with its stabilizing protein calstabin2 (FKBP12.6). Single channel measurements of RyR2-H29D channels revealed significantly higher open probability (Po) and opening frequency (Fo) at diastolic levels of cytosolic Ca2+ when compared with RyR2-WT channels under non-stress conditions (i.e. in absence of PKA phosphorylation). This leaky phenotype at rest in RyR2-H29D channels was associated with a modest but significant depletion of calstabin2 binding from the RyR2 macromolecular complex when compared to RyR2-WT channels. Interestingly, under stress conditions, RyR2-H29D channels also exhibited a significant higher Po and Fo at diastolic concentrations of Ca2+ while no significant depletion of calstabin2 was observed. In conclusion, the RyR2-H29D mutation is associated with a clinical phenotype of short-coupled PMVT at rest. In contrast to CPVT-associated RyR2 mutations, RyR2-H29D causes a leaky channel at diastolic levels of Ca2+ under non-stress conditions. Leaky RyR2 may be an under-recognized mechanism for idiopathic PMVT at rest.

Potassium dependent rescue of a myopathy with core-like structures in mouse.

Myopathies decrease muscle functionality. Mutations in ryanodine receptor 1 (RyR1) are often associated with myopathies with microscopic core-like structures in the muscle fiber. In this study, we identify a mouse RyR1 model in which heterozygous animals display clinical and pathological hallmarks of myopathy with core-like structures. The RyR1 mutation decreases sensitivity to activated calcium release and myoplasmic calcium levels, subsequently affecting mitochondrial calcium and ATP production. Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis. Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology. We identify regulators of potassium homeostasis as biomarkers of disease that may reveal therapeutic targets in human patients with myopathy of central core disease (CCD). Altogether, our results suggest that amelioration of potassium leaks through potassium homeostasis mechanisms may minimize muscle damage of myopathies due to certain RyR1 mutations.

Flecainide: Current status and perspectives in arrhythmia management.

Flecainide acetate is a class IC antiarrhythmic agent and its clinical efficacy has been confirmed by the results of several clinical trials. Nowadays, flecainide is recommended as one of the first line therapies for pharmacological conversion as well as maintenance of sinus rhythm in patients with atrial fibrillation and/or supraventricular tachycardias. Based on the Cardiac Arrhythmia Suppression Trial study results, flecainide is not recommended in patients with structural heart disease due to high proarrhythmic risk. Recent data support the role of flecainide in preventing ventricular tachyarrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia associated both with ryanodine receptor and calsequestrin mutations. We herein review the current clinical data related to flecainide use in clinical practice and some concerns about its role in the management of patients with coronary artery disease.

Mitochondrial Abnormalities in a Mouse CPVT Model with RyR2 Loss-Of-Function Mutation

Mitochondria exchange matrix content in a unique and dynamic way by one of two mechanisms: a short-range direct interaction via closely spaced kissing junctions or long-range propagation of signal by the extrusion of extended nanotubular extensions (nanotunneling) (Huang et al 2013. Proc Natl Acad Sci U S A, 110(8): 2846-51). Both are normal, but infrequent features of cardiac myocytes. It is interesting to know whether this behavior may be affected by changes in Ca2+ homeostasis. We evaluated the cardiac ultrastructure in 7-month old heterozygous mice with a cardiac ryanodine receptor (RyR2) mutation (RyR2-A4860G) that depresses channel function and leads to Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) without hypertrophy (Zhao et al., submitted). The mutation is embryonic lethal in mice homozygous for the mutation. In ventricular myocytes of heterozygotes mice (RyR2-A4860G+/−) it has a dual effect on SR Ca2+ release: an initial reduction due to lower RyR2 activity, linked to random bursts due to SR Ca2+ overload. The most striking and only structural alteration in cardiac myocytes in the mutant left ventricle is a clear increase in the frequency of long thin nano-tunneling mitochondrial extensions. The frequency of nanotunneling was ∼3 fold higher in mutant than in WT mice. Numerous small mitochondrial profiles are more frequent in mutant mice resulting in an average surface area of 0.17± 0.15 µm2 in mutant versus 0.28 ± 0.22 µm2 in WT (p<0.00001). This decrease is mostly due to sections through the thin tunneling extensions. The increased nanotunneling frequency would indicate an enhanced rate of long range intermitochondrial signal transfer. Interestingly, no activation of nanotunneling has been reported by EM in other CPVT models and/or in other alterations affecting proteins of CRU and Ca2+ homeostasis, making this a unique mitochondrial response.

Gene expression of beta-adrenergic receptors and myosin heavy chain isoforms induced by ractopamine feeding duration in pigs not carrying the ryanodine receptor mutation

To evaluate the effects of ractopamine hydrochloride (RAC) feeding duration on mRNA abundance of beta-adrenergic receptors (β-AR) and myosin heavy chain (MyHC) isoforms in the Longissimus dorsi (LD) muscle of pigs not carrying the ryanodine receptor type I (RyR1) mutation, 80 finishing barrows (initial body weight=69.4±7.87kg) were randomly assigned to 1 of 5 treatments with 8 replicate pens per treatment and 2 pigs per pen. Pigs were fed a corn–soybean meal-based diet with no added RAC (control) or with 10mg/kg RAC fed for 7, 14, 21, or 28d before slaughter. On d 28, hair root samples were collected for RyR1 genotyping and LD muscle samples were collected to measure β-AR (β1- and β2-subtypes) and MyHC isoforms (Iβ, IIa, IIx/d, and IIb) gene expression using the quantitative real-time PCR method. Six heterozygous negative pigs for the RyR1 mutation were detected, and therefore not considered for statistical analysis. Increasing RAC feeding duration did not affect the abundance of β1-AR mRNA, but resulted in a tendency (P=0.073) towards a linear decrease in β2-AR mRNA abundance. Even though MyHC Iβ mRNA abundance was linearly decreased (P=0.002) with increasing RAC feeding duration, both MyHC IIa and IIx/d gene expressions were not affected by the RAC treatment. There was a linear increase (P=0.029) in MyHC IIb mRNA abundance as RAC feeding duration was increased. In conclusion, RAC feeding alters gene expression of β2-AR and MyHC isoforms in the LD muscle of pigs not carrying the RyR1 mutation in a time-dependent manner.

Short-coupled polymorphic ventricular tachycardia at rest linked to a novel ryanodine receptor (RyR2) mutation: Leaky RyR2 channels under non-stress conditions

BackgroundRyanodine receptor (RyR2) mutations have largely been associated with catecholaminergic polymorphic ventricular tachycardia (PMVT). The role of RyR2 mutations in the pathogenesis of arrhythmias and syncope at rest is unknown. We sought to characterize the clinical and functional characteristics associated with a novel RyR2 mutation found in a mother and daughter with PMVT at rest. Methods and resultsA 31-year-old female with syncope at rest and recurrent short-coupled premature ventricular contractions (PVCs) initiating PMVT was found to be heterozygous for a novel RyR2-H29D mutation. Her mother, who also had syncope at rest and short-coupled PMVT, was found to harbor the same mutation. Human RyR2-H29D mutant channels were generated using site-directed mutagenesis and heterologously expressed in HEK293 cells together with the stabilizing protein calstabin2 (FKPB12.6). Single channel measurements of RyR2-H29D mutant channels and wild type (WT) RyR2 channels were compared at varying concentrations of cytosolic Ca2+. Binding affinities of the RyR2-H29D channels and RyR2-WT channels to calstabin2 were compared. Functional characterization of the RyR2-H29D mutant channel revealed significantly higher open probability and opening frequency at diastolic levels of cytosolic Ca2+ under non-stress conditions without protein kinase A treatment. This was associated with a modest depletion of calstabin2 binding under resting conditions. ConclusionsThe RyR2-H29D mutation is associated with a clinical phenotype of short-coupled PMVT at rest. In contrast to catecholaminergic PMVT-associated RyR2 mutations, RyR2-H29D causes a leaky channel at diastolic levels of Ca2+ under non-stress conditions. Leaky RyR2 may be an under-recognized mechanism for idiopathic PMVT at rest.

Novel mutations and mutation combinations of ryanodine receptor in a chlorantraniliprole resistant population of Plutella xylostella (L.).

A previous study documented a glycine to glutamic acid mutation (G4946E) in ryanodine receptor (RyR) was highly correlated to diamide insecticide resistance in field populations of Plutella xylostella (Lepidoptera: Plutellidae). In this study, a field population collected in Yunnan province, China, exhibited a 2128-fold resistance to chlorantraniliprole. Sequence comparison between resistant and susceptible P. xylostella revealed three novel mutations including a glutamic acid to valine substitution (E1338D), a glutamine to leucine substitution (Q4594L) and an isoleucine to methionine substitution (I4790M) in highly conserved regions of RyR. Frequency analysis of all four mutations in this field population showed that the three new mutations showed a high frequency of 100%, while the G4946E had a frequency of 20%. Furthermore, the florescent ligand binding assay revealed that the RyR containing multiple mutations displayed a significantly lower affinity to the chlorantraniliprole. The combined results suggested that the co-existence of different combinations of the four mutations was involved in the chlorantraniliprole resistance. An allele-specific PCR based method was developed for the diagnosis of the four mutations in the field populations of P. xylostella.

Calcium in the heart: from physiology to disease

Contraction of the heart results from an increase of cytoplasmic Ca(2+) concentration ([Ca(2+)]i), the so-called systolic Ca(2+) transient. Most of this results from the release of Ca(2+) from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). In turn, the amplitude of this Ca(2+) transient determines the contractility of the heart. In this lecture, I consider the factors which govern the size and stability of this Ca(2+) release. The amplitude of the Ca(2+) transient is a steep function of SR Ca, resulting in a requirement for very precise beat-to-beat regulation of SR Ca content. This is achieved by a simple negative feedback mechanism, in which an increase of SR Ca content increases the size of the Ca(2+) transient, resulting in a decrease of Ca(2+) influx on the L-type Ca(2+) current and an increase of efflux through Na(+)-Ca(2+) exchange. Changing the activity of any of the Ca(2+)-cycling proteins will change the steady-state SR Ca content. This feedback mechanism has many consequences, including the fact that a change of RyR open probability has a only a temporary effect on the amplitude of the Ca(2+) transient due to a compensating change of SR Ca content. The remainder of the article considers the link between intracellular Ca(2+) waves and arrhythmias. This is done in the context of catecholaminergic polymorphic ventricular tachycardia, which is an inherited arrhythmia syndrome, in many cases due to a RyR mutation, where arrhythmias occur during exercise as a result of β-adrenergic stimulation. Calcium waves occur only when the SR Ca content exceeds a threshold level. Our data show that the threshold is reduced by the RyR mutation and that the adrenergic stimulation increases SR Ca content.

Multiple modes of ryanodine receptor 2 inhibition by flecainide.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca(2+)], [Mg(2+)], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca(2+)] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide.

Ryanodine receptor mutations presenting as idiopathic ventricular fibrillation: a report on two novel familial compound mutations, c.6224T>C and c.13781A>G, with the clinical presentation of idiopathic ventricular fibrillation.

Idiopathic ventricular fibrillation (IVF) is a rare genetically determined disease causing unexpected cardiac death in otherwise healthy individuals. This study identified two novel, functional heterozygous mutations in the ryanodine receptor 2 (RyR2) gene in a family with IVF. In the presented case all the patients received a thorough diagnostic workup to exclude structural heart disease. Blood was drawn from the patients, and genetic testing was performed including amplification and sequencing of splice locations in two exons of the RyR2 gene. The mutations were detected in five symptomatic family members. The genetic status of the five affected family members remains unclear. No clinically affected patient is without mutation. At this writing, one family member with confirmed mutation is asymptomatic. The differentiation between catecholaminergic polymorphic ventricular tachycardia (CPVT) and IVF remains a difficult issue, mainly based on clinical characteristics and gross genetic classification. In our case, the family history, exercise testing, and epinephrine stress testing do not suggest an association of arrhythmia and adrenergic triggers, which makes CPVT rather unlikely despite the fact that genetic testing showed RyR2 mutations. Currently, knowledge concerning the functional meaning of genetic mutations is growing. Future exploration of these functional aspects might give further impetus to allocation of these patients to a specific diagnosis.

Prevalence of exertional rhabdomyolysis in endurance horses in thePacificNorthwesternUnitedStates

Exertional rhabdomyolysis (ER) is a reported syndrome in competing endurance horses; however, the prevalence and cause of ER in this population have not been defined. To determine the prevalence of ER in a sample of endurance racing horses and investigate factors, including relevant genetic defects, contributing to the occurrence of rhabdomyolysis in this group. Prospective clinical study. Riders of 101 horses participating in one of four 50-mile (80.5 km) distance races completed a comprehensive questionnaire regarding the medical history, management and performance of their horse. Serum creatine kinase activity (CK) was measured before and 4 h after completion of exercise. Hair samples were analysed by PCR for the R309H mutation in the glycogen synthase gene (GYS1) responsible for type 1 polysaccharide storage myopathy (PSSM) and the C7360G mutation in the ryanodine receptor 1 (RYR1) gene causing malignant hyperthermia (MH). Samples were obtained from 68 Arabians, 20 half-Arabians and 13 horses of other breeds. Serum CK was above the resting reference interval (145-633 u/l) in 38 horses after racing (median 883 u/l, range 658-3739) but was compatible with values previously reported in apparently healthy endurance horses. Pathological ER was suspected to occur in 4 horses with serum CK activities exceeding 10,000 u/l 4 h after racing (median 84,825 u/l; range 10,846-381,790) including 3 Arabians and one half-Arabian horse. GYS1 and RYR1 mutations were not present in hair samples from any horses. Exertional rhabdomyolysis occurred at a prevalence of 4.0% in a sample of horses participating in 50 mile distance events and all affected horses were Arabian or half-Arabian. The cause of ER in the endurance horse population remains unknown; however, ER in competing Arabian endurance horses is unlikely to be due to type 1 PSSM or MH.