A ryanodine receptor 2 gene variant associated with left ventricular non-compaction, cardiac conduction disease, ventricular arrhythmias, and sudden cardiac death.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channel disorder in the heart, which is characterized by abnormal calcium handling, ventricular arrhythmias, and sudden cardiac death. This inherited disease is predominantly caused by mutations in the ryanodine receptor type 2 (RYR2). Most of the identified mutations are clustered into four distinct domains of the RYR2 channel. Although heterologous expression systems and animal models have brought important insights in the CPVT pathogenesis, the underlying electrophysiological mechanisms have not been completely understood. The aim of the study was to take cells from CPVT patients with specific RYR2 mutations, to create patient-specific induced pluripotent stem cells (iPSCs), to differentiate these cells into cardiomyocytes (CMs) and then to model the disease in vitro for a better understanding of the disease mechanism and for the investigation of novel therapeutic applications for CPVT patients. Somatic cells from skin biopsies of CPVT patients carrying the RYR2 mutation in domain a (R420W), domain b (A2254V), domain c (E4076K) or domain d (H4742Y) were isolated and reprogrammed into patient-specific iPSCs. The CPVT as well as healthy control (Ctrl) iPSC lines were differentiated into CMs. The CPVT- and Ctrl-iPSC-derived CMs were investigated for their biological, electrophysiological, and pharmacological differences between the RYR2 mutations in different domains and healthy controls. The differences in 3',5'-cyclic adenosine monophosphate (cAMP) dynamics were investigated as well. Electrophysiological analyses showed that the CPVT-CMs recapitulated the phenotype of CPVT both by patch-clamp assessment and by multielectrode array assessment. The single CPVTdCMs showed a unique early afterdepolarization (EAD) phenotype in basal condition and isoproterenol- (ISO-) challenged condition. However, CPVTa-, CPVTb- and CPVTc-CMs exhibited delayed afterdepolarization (DAD) and DAD-induced triggered activities (TAs), which were significantly enhanced after the ISO treatment. In the monolayer cultures, all CPVT-CMs revealed a significantly increased number of premature ventricular complex- (PVC-) like events and prolonged duration of ventricular tachycardia- (VT-) like events after the ISO treatment. In contrast, no increased appearance of arrhythmic events (DADs, EADs, DAD- or EAD-induced TAs, and PVC- and VT-like events) was observed in Ctrl-CMs after the ISO treatment. Four antiarrhythmic drugs (flecainide, dantrolene, rycal1 and rycal2) showed antiarrhythmic effects on CPVTa-, CPVTb- and CPVTc-CMs, but no or minor antiarrhythmic effect on CPVTd-CMs. FRET measurement revealed that the contribution of phosphodiesterase 2 (PDE2) to cAMP degradation in all CPVTa- (15.41%), b- (9.48%), c- (15.07%), d-CMs (7.9%) were significantly lower than in Ctrl-CMs (27.5%) in cytosol. The contribution of PDE2 to cAMP degradation at the RYR2 compartment in all CPVTa- (14.19%), b- (25.21%), c- (17.32%), d-CMs (8.6%) were also significantly lower than in Ctrl-CMs (39.98%). Similar to PDE2, the contribution of PDE3 to cAMP degradation in all CPVT-CMs were significantly lower than in Ctrl-CMs both in cytosol and at the RYR2 compartment. However, for PDE4, there were lower activities in cytosol in CPVTb- and CPVTc-CMs when compared to Ctrl-CMs, and lower activities at the RYR2 compartment in CPVTc- and CPVTd-CMs when compared to Ctrl-CMs. Furthermore, the data indicate that PDE4 is the major regulator of cAMP level in CPVT-CMs both in cytosol and at the RYR2 compartment after ISO stimulation and that PDE2 and PDE3 have a smaller contribution to regulate the cAMP level. Taken together, this study reveals that mutation-specific CPVT-iPSCs can be used to model the disease in vitro, to investigate the disease pathophysiological and molecular mechanisms and to optimize drug therapies.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

BackgroundCatecholaminergic polymorphic ventricular tachycardia is an inherited disease presenting with arrhythmic events during physical exercise or emotional stress. If untreated, catecholaminergic polymorphic ventricular tachycardia is a highly lethal condition: About 80% of affected individuals experience recurrent syncope, and 30% experience cardiac arrest. Catecholaminergic polymorphic ventricular tachycardia is caused by mutations in genes encoding ryanodine receptor type 2 (RyR2) and cardiac calsequestrin (CASQ2). In cases of sympathoadrenergic activation, both mutations result in a spontaneous Ca2+ release in cardiac cells, facilitating ventricular arrhythmias.Case presentationWe present a case of a 17-year-old Caucasian boy who survived sudden cardiac death caused by ventricular fibrillation while performing running exercise in a fitness center. The diagnostic workup included blood tests, coronary angiography, electrophysiological testing, and cardiac magnetic resonance imaging, but all results were normal. Because the patient’s medical history included recurrent syncope during physical and emotional stress, we strongly suspected catecholaminergic polymorphic ventricular tachycardia as the underlying disease. Genetic screening was performed and confirmed the diagnosis, revealing a new heterozygous point mutation in the gene for RyR2, c.12520T>A (p.F4174 l, exon 90, RyR2 gene). The patient was discharged from our hospital after undergoing implantation of an implantable cardioverter defibrillator for secondary prevention. Shortly after implantation, the implantable cardioverter defibrillator terminated a sustaining ventricular tachycardia episode by antitachycardic pacing. This episode occurred early in the morning while the patient was asleep.ConclusionsWe present a case of catecholaminergic polymorphic ventricular tachycardia associated with a novel single point mutation in the RyR2 gene, which, to the best of our knowledge, has not been described in the literature so far. Our patient experienced arrhythmic events under both resting conditions and physical activity, an uncommon finding in patients with catecholaminergic polymorphic ventricular tachycardia. This novel mutation may cause arrhythmias independent of sympathoadrenergic stimulation, but further evidence is needed to prove causality.

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

A rare case of tetralogy of Fallot with catecholaminergic polymorphic ventricular tachycardia

Chronic Dermatomyositis with Intermittent Trifascicular Block: An Electrophysiologic-Conduction System Correlation

Ryanodine receptors type 2 (RyR2) regulate excitation-contraction coupling. Their dysfunction leads to pathological states, such as arrhythmia, heart failure and sudden death. Our homology model detected comparable structural determinants in the pores of the K+ channel KcsA and RyR2. A Glycine hinge motif in the inner transmembrane helix of many K+ channels is also found in RyR2. We tested the hypothesis that the Glycine at this position (4864) was critical for RyR2 function, as it is in K+ channels. A single substitution by Alanine in the human sequence did not alter Ca2+ release in transfected HEK293 cells, [3H]ryanodine binding or ion translocation and gating properties of purified channels, indicating that Glycine is not necessary. However Valine and Proline substitutions led to significant changes. Though well expressed in HEK293 cells, Valine mutant channels were not active as Ca2+ release entities in cells or as isolated channels reconstituted in planar lipid bilayers, and did not bind ryanodine (P<0.001 vs wild-type). The Proline mutation preserved the responses to caffeine and single channel activity, albeit with dramatic modifications, but [3H]ryanodine binding was abolished (P<0.01 vs wild-type). Our data suggest that a lack of flexibility in the pore impairs proper gating. To go further, we examined the NH2-terminal parts of RyR2 and showed by co-immunoprecipitation, chemical crosslinking and gel filtration that these domains can oligomerize within the tetrameric channel due to the presence of disulphide bonds. We used constructs (GST-BT4L) corresponding to the first 906 amino acids of the human RyR2 protein to test their interaction with endogenous NH2-terminal domains in HEK293 microsomes expressing RyR2. At diastolic Ca2+ concentrations (≤ 250 nM), GST-BT4L significantly induced a twofold increase in [3H]ryanodine binding (P<0.05 vs control). Moreover GST-BT4L raised the open probability of single channels (P<0.05 vs GST alone), in low Ca2+ conditions and with a physiological ion flow. Our results provide evidence that GST-BT4L can disrupt the NH2 terminus self-association, which moderately opens RyR2 channels. Hence, we propose that while a general flexibility of the pore inner helix is required to allow the channel to open, the tetramerization of the NH2 terminus domains facilitates RyR2 closure and prevents Ca2+ leak at diastolic Ca2+ concentrations. Since these two regions are hot spots of RyR2 mutations in Catecholaminergic Polymorphic Ventricular Tachycardia 1, these insights may have implications in the understanding of the mechanisms underlying this Ca2+ leak-related channelopathy.

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

Key role of the molecular autopsy in sudden unexpected death

Ventricular arrhythmias and sudden cardiac death

Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca2+ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2-/-, RyR2-R4496C+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT.

case presentation: A 48-year-old woman presents with exertional dyspnea and recurrent syncope. One year earlier, a permanent pacemaker was placed after she complained of fatigue and was found to have high-grade atrioventricular block. Now, she has echocardiographic evidence of moderate to severe left ventricular (LV) systolic dysfunction with regional wall-motion abnormalities. Nuclear imaging is notable for heterogeneous myocardial uptake of technetium Tc99m sestamibi, and coronary angiography reveals widely patent epicardial vessels. Multiple episodes of nonsustained ventricular tachycardia (VT) are documented on continuous ECG monitoring. What are the diagnostic considerations for this patient, and what further evaluations are indicated? This patient presents with dilated cardiomyopathy (DCM) with electric instability (DCM+E), which we define as conduction disease and arrhythmia out of proportion to the severity of LV systolic dysfunction. Diverse causes can result in DCM+E and fall into general categories of inflammatory, infectious, hereditary, and infiltrative processes. Cardiac presentation associated with these conditions is distinct from more common causes of DCM such as ischemic heart disease, viral myocarditis, valvular dysfunction, pregnancy, or substance abuse. Clinical features that are suggestive of DCM+E include supraventricular arrhythmias or conduction disease that precedes cardiomyopathy, multiple VT morphologies, and features suggestive of ischemic heart disease (Q waves, regional wall-motion abnormalities, perfusion defects, ventricular aneurysm) in the absence of epicardial stenoses. In this Clinician Update, we focus on the diagnostic approach to patients with DCM+E. Emphasis is placed on diagnoses that are relatively common or for which the clinical management would be impacted significantly by recognition of the underlying cause. Ischemic heart disease may present with conduction disease and a high burden of arrhythmia, especially in the setting of acute myocardial ischemia/infarction. The exclusion of obstructive coronary artery disease is strongly recommended in patients with DCM+E because atherosclerosis is so prevalent, evidence-based treatment is readily available, and the …

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a rare inherited channelopathy, responsible for potentially lethal malignant arrhythmic episodes. Atypical non-juvenile form of CPVT may not mislead an alternative diagnosis of calcium release deficiency syndrome (CRDS). The index case was a 58 years-old woman who experienced aborted sudden cardiac arrest. The initial complete diagnostic workup (including norepinephrine challenge) was completely negative. She was implanted with an entirely subcutaneous defibrillator. During her follow-up, she received an appropriate electrical shock (ventricular fibrillation) despite β-blocker treatment. Three sisters (46, 40 and 18 years-old) as well as 2 cousins, one paternal uncle and one paternal aunt had sudden cardiac deaths (SCD) without etiology in the family history. There were no additional reports of pregnancy loss, neonatal death, seizures or SCD among the family members. The genetic analysis in this proband revealed a missense pathogenic variant c.13823 G>A, p.(Arg4608Gln) in the RYR2 gene, encoding the Ryanodine Receptor type 2. This c.13823 G>A, p.(Arg4608Gln), variant in the RYR2 gene was supposed to be a potential disease-causing variant in CPVT. Unfortunately, before the end of the proband's genetic analysis, her 20 years-old daughter experienced SCD, whilst being implanted with an insertable cardiac monitor. Familial segregation analysis confirmed the four symptomatic sisters harbor also the same variant confirming the pathogenic role of this variant. We also identified 7 carriers who were clinically negative for CPVT in the next generation. Whole were treated with Nadolol 80 mg per day, and the follow-up was uneventful after twenty-four months. The Ryanodine Receptor type 2 c.13823 G>A, p.(Arg4608Gln) pathogenic variant is described in a malignant familial form of CRDS.

Modelling CPVT in a dish: Characterization of two novel ryanodine receptor mutations using human induced pluripotent stem cell-derived cardiomyocytes