Cause Catecholaminergic Polymorphic Ventricular Tachycardia Research Articles

Diverse Phenotypic Manifestations in a Family with a Novel RYR2 E4107A Variant.

Cardiac ryanodine receptor (RyR2) gain-of-function mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Conversely, RyR2 loss-of-function mutations cause a new disease entity, termed calcium release deficiency syndrome (CRDS), which may include RYR2-related long QT syndrome (LQTS). Importantly, unlike CPVT, patients with CRDS do not always exhibit exercise- or epinephrine-induced ventricular arrhythmias, which precludes a diagnosis of CRDS. Here we report a boy and his father, who both experienced exercise-induced cardiac events and harbor the same RYR2 E4107A variant. In the boy, an exercise stress test (EST) and epinephrine provocation test (EPT) did not induce any ventricular arrhythmias. QTc was slightly prolonged (QTc: 474 ms), and an EPT induced QTc prolongation (QTc-baseline: 466 ms, peak: 532 ms, steady-state: 527 ms). In contrast, in his father, QTc was not prolonged (QTc: 417 ms), and neither an EST nor EPT induced QTc prolongation. However, an EST induced multifocal premature ventricular contraction (PVC) bigeminy and bidirectional PVC couplets. Thus, they exhibited distinct clinical phenotypes: the boy exhibited LQTS (or CRDS) phenotype, whereas his father exhibited CPVT phenotype. These findings suggest that, in addition to the altered RyR2 function, other unidentified factors, such as other genetic, epigenetic, and environmental factors, and aging, may be involved in the diverse phenotypic manifestations. Considering that a single RYR2 variant can cause both CPVT and LQTS (or CRDS) phenotypes, in cascade screening of patients with CPVT and CRDS, an EST and EPT are not sufficient and genetic analysis is required to identify individuals who are at increased risk for life-threatening arrhythmias.

One Heart, Two Problems: Cardiomyopathy and CPVT Secondary to RYR2 Mutation

<h3>Introduction</h3> RYR2 mutations are known to cause catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disorder with risk for life threatening atrial (AT) and ventricular (VT) arrhythmias. Individuals with RYR2 mutation less commonly exhibit arrhythmogenic right ventricular cardiomyopathy (ARVC). We report the first case of a pediatric patient with RYR2 mutation, manifesting with CPVT, ARVC, and severe dysfunction requiring heart transplant (HTx). <h3>Case Report</h3> A 4.5 year old male with developmental delay presented after multiple syncopal episodes both at rest and with activity. A Holter showed AT, and he was started on a beta adrenergic antagonist with decreased frequency of syncopal episodes. He then developed sinus bradycardia with marked sinus pauses, prompting dual chamber pacemaker placement at age 6 years. He later exhibited bidirectional VT and whole exome sequencing identified a de novo, likely pathogenic variant in RYR2. He was medically managed until 16 years of age when he had syncope with exercise and was found to have both frequent AT and moderately depressed ventricular function with an ejection fraction of 45%. Given recurrent syncope with exercise, he underwent uncomplicated sympathectomy and optimization of his medical antiarrhythmic management. Over the next few months, he developed progressive heart failure symptoms and was admitted with worsening biventricular dysfunction and tachyarrhythmias. During his admission, he had a cardiac arrest with initial monitoring demonstrating VT, and was placed on extracorporeal membrane oxygenation support. He was transitioned to a durable left ventricular assist device (VAD), and was eventually listed for HTx. His VAD course was complicated by RV dysfunction needing PDE5 inhibitor; his arrhythmia burden was managed on flecainide, sotalol, and nadolol. After 80 days of VAD support, he underwent HTx. Pathology of his explanted heart revealed biventricular fibrofatty myocardial replacement consistent with ARVC with prominent LV involvement. <h3>Summary</h3> This case highlights an expanded spectrum of RYR2 mutation related disease which may include prominent atrial arrhythmias, ARVC, and developmental delay. Patients with CPVT and RYR2 mutation warrant close monitoring for ventricular dysfunction, especially if there is a significant arrhythmia burden.

Characterization of the mechanism by which a nonsense variant in RYR2 leads to disordered calcium handling.

Heterozygous missense variants of the cardiac ryanodine receptor gene (RYR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These missense variants of RYR2 result in a gain of function of the ryanodine receptors, characterized by increased sensitivity to activation by calcium that results in an increased propensity to develop calcium waves and delayed afterdepolarizations. We have recently detected a nonsense variant in RYR2 in a young patient who suffered an unexplained cardiac arrest. To understand the mechanism by which this variant in RYR2, p.(Arg4790Ter), leads to ventricular arrhythmias, human induced pluripotent stem cells (hiPSCs) harboring the novel nonsense variant in RYR2 were generated and differentiated into cardiomyocytes (RYR2‐hiPSC‐CMs) and molecular and calcium handling properties were studied. RYR2‐hiPSC‐CMs displayed significant calcium handling abnormalities at baseline and following treatment with isoproterenol. Treatment with carvedilol and nebivolol resulted in a significant reduction in calcium handling abnormalities in the RYR2‐hiPSC‐CMs. Expression of the mutant RYR2 allele was confirmed at the mRNA level and partial silencing of the mutant allele resulted in a reduction in calcium handling abnormalities at baseline. The nonsense variant behaves similarly to other gain of function variants in RYR2. Carvedilol and nebivolol may be suitable treatments for patients with gain of function RYR2 variants.

Loss-of-function mutations in cardiac ryanodine receptor channel cause various types of arrhythmias including long QT syndrome.

Gain-of-function mutations in RYR2, encoding the cardiac ryanodine receptor channel (RyR2), cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Whereas, genotype-phenotype correlations of loss-of-function mutations remains unknown, due to a small number of analysed mutations. In this study, we aimed to investigate their genotype-phenotype correlations in patients with loss-of-function RYR2 mutations. We performed targeted gene sequencing for 710 probands younger than 16-year-old with inherited primary arrhythmia syndromes (IPAS). RYR2 mutations were identified in 63 probands, and 3 probands displayed clinical features different from CPVT. A proband with p.E4146D developed ventricular fibrillation (VF) and QT prolongation whereas that with p.S4168P showed QT prolongation and bradycardia. Another proband with p.S4938F showed short-coupled variant of torsade de pointes (scTdP). To evaluate the functional alterations in these three mutant RyR2s and p.K4594Q previously reported in a long QT syndrome (LQTS), we measured Ca2+ signals in HEK293 cells and HL-1 cardiomyocytes as well as Ca2+-dependent [3H]ryanodine binding. All mutant RyR2s demonstrated a reduced Ca2+ release, an increased endoplasmic reticulum Ca2+, and a reduced [3H]ryanodine binding, indicating loss-of-functions. In HL-1 cells, the exogenous expression of S4168P and K4594Q reduced amplitude of Ca2+ transients without inducing Ca2+ waves, whereas that of E4146D and S4938F evoked frequent localized Ca2+ waves. Loss-of-function RYR2 mutations may be implicated in various types of arrhythmias including LQTS, VF, and scTdP, depending on alteration of the channel activity. Search of RYR2 mutations in IPAS patients clinically different from CPVT will be a useful strategy to effectively discover loss-of-function RYR2 mutations.

Novel cases of pediatric sudden cardiac death secondary to TRDN mutations presenting as long QT syndrome at rest and catecholaminergic polymorphic ventricular tachycardia during exercise: The TRDN arrhythmia syndrome.

TRDN mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT) but may present with abnormal electrocardiogram (ECG) findings provoking a diagnosis of long QT syndrome (LQTS). We report two novel cases of sudden cardiac death in children due to mutations of TRDN, providing further insight into this rare and aggressive inherited arrhythmia syndrome. Whole exome sequencing (WES) was performed in two unrelated children who experienced cardiac arrest during exercise and were negative for targeted testing of LQTS. WES identified a novel homozygous splice-site mutation in both patients, denoted c.22+1G>T, absent from gnomAD and suggesting a founder variant in the Iranian population. We now summarize the genetic architecture of all reported TRDN-related patients, including 27 patients from 21 families. The average age-onset was 30 months (range 1-10) for all cases. Adrenergic-mediated cardiac events were common, occurring in 23 of 27 cases (85%). LQTS was diagnosed in 10 cases (37%), CPVT in 10 (37%) cases, and in 7 cases. No phenotypic diagnosis was provided. Five cases (15%) had evidence for associated skeletal myopathy. Four missense TRDN variants (24%) were observed in diseased cases, while the remaining variants reflect putative loss-of-function (LOF) mutations. No disease phenotype was reported in 26 heterozygous carriers. In conclusion, TRDN mutations cause a rare autosomal recessive arrhythmia syndrome presenting with adrenergic-mediated arrhythmic events, but with ECG abnormalities leading to a diagnosis of LQTS in a proportion of cases. Heterozygous carriers are free of disease manifestations.

Functional Analysis of a Novel Heterozygous Variant in CASQ2 as a Distinct Cause of Catecholaminergic Polymorphic Ventricular Tachycardia

Variants in RYR2 and CASQ2 cause catecholaminergic polymorphic ventricular tachycardia (CPVT) 1 and 2, respectively. Most cases of CPVT2 are homozygous for CASQ2 variants. We recently reported the first large family with CPVT caused by a heterozygous CASQ2 variant. Here, we assess the functional mechanisms of this pathogenic CASQ2 variant and compare it to a CPVT1 (RYR2 variant) phenotype using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM).

Alpha blockade potentiates CPVT therapy in calsequestrin-mutant mice.

Spontaneous calcium release evoking delayed afterdepolarization is believed to cause catecholaminergic polymorphic ventricular tachycardia (CPVT), a lethal human arrhythmia provoked by exercise or emotional stress. β-Adrenergic blockers are the drug of choice, but fail to achieve complete arrhythmia control in some patients. These individuals often require flecainide, device implantation, and/or sympathetic denervation. To optimize the arrhythmia therapy by pharmacological inhibition of the sympathetic nervous system in the homozygous calsequestrin knockout (CASQ2(Δ/Δ)) mouse model of CPVT2. A heart telemetry device was implanted for continuous electrocardiographic recording at rest and during provocation testing. Calcium transients and abnormal calcium release were studied in cardiomyocytes isolated from adult mice. Adrenergic receptor expression was determined by using Western blotting and confocal microscopy. Adult CASQ2(Δ/Δ) mice suffer from complex ventricular arrhythmia at rest and ventricular tachycardia during treadmill exercise and after epinephrine injection. β-Adrenergic blockers, propranolol and metoprolol, attenuated arrhythmia at rest but not after stress. Reserpine had no efficacy in controlling arrhythmia. Agents with α-blocking activity, phentolamine or labetalol, abolished both exercise- and epinephrine-induced arrhythmia. In contrast, injection of α-adrenergic agonist phenylephrine reproducibly provoked ventricular tachycardia. Isolated cardiomyocytes from CASQ2(Δ/Δ) mice had delayed calcium release waves upon exposure to sympathetic agonists, which were abolished by phentolamine. Hearts of calsequestrin-mutant mice expressed more α1-adrenergic receptor than did wild type control mice (P < .05). We identified a contribution of the α-adrenergic pathway to the pathogenesis of catecholamine-induced arrhythmia. α-Blockade emerges as an effective therapy in the murine model of CPVT2 and should be tried in humans resistant to β-blockers.

Exon 3 deletion of RYR2 encoding cardiac ryanodine receptor is associated with left ventricular non-compaction

Ryanodine receptor gene (RYR2) mutations are well known to cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, RYR2 exon 3 deletion has been identified in patients with dilated cardiomyopathy (DCM) and/or CPVT. This study aimed to screen for the RYR2 exon 3 deletion in CPVT probands, characterize its clinical pathology, and confirm the genomic rearrangement. Our cohort consisted of 24 CPVT probands. Polymerase chain reaction (PCR)-based conventional genetic analysis did not identify any mutations in coding exons of RYR2 in these probands. They were screened using multiplex ligation-dependent probe amplification (MLPA). In probands identified with RYR2 exon 3 deletion, the precise location of the deletion was identified by quantitative PCR and direct sequencing methods. We identified two CPVT probands from unrelated families who harboured a large deletion including exon 3. The probands were 9- and 17-year-old girls. Both probands had a history of syncope related to emotional stress or exercise, exhibited bradycardia, and were diagnosed with left ventricular non-compaction (LVNC). We examined 10 family members and identified six more RYR2 exon 3 deletion carriers. In total, there were eight carriers, of which seven were diagnosed with LVNC (87.5%). Two carriers under the age of 4 years remained asymptomatic, although they were diagnosed with LVNC. Using quantitative PCR and direct sequencing, we confirmed that the deletions were 1.1 and 37.7 kb in length. RYR2 exon 3 deletion is frequently associated with LVNC. Therefore, detection of the deletion offers a new modality for predicting the prognosis of patients with LVNC with ventricular/atrial arrhythmias, particularly in children.

Screening of whole RYR 2 gene in arrhythmogenic right ventricular cardiomyopahy/dysplasia

Introduction: Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia (ARVC/D) is predominantly caused by desmosomal gene mutations, that account for only ∼50% of ARVC/D cases. RYR2 gene mutations usually cause Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) but have been associated with atypical phenotype of ARVC/D. In addition RYR2 is a large gene, therefore usually not analysed in routine. We aim to determine the prevalence and phenotype associated with RYR2 mutations in a large ARVC/D population. Methods and results: We analyzed the whole RyR2 coding sequence (105 exons and related splice sites) in a population of 64 ARVC/D probands without desmosomal gene mutations. We identified six heterozygous putative missense mutations in six unrelated probands. Two (p.Pro1583Ser and p.Leu4670Val) were considered as disease-causing mutations and four (p.Ala2213Ser, p.Gly2367Arg, p.Tyr2932His, p.Val3219Me) were considered as genetic variants of unknown significance (GVUS). Phenotype associated with RYR2 variants was unremarkable, associating typical ECG abnormalities, frequent monomorphic ventricular tachycardia with only a minority developing polymorphic ventricular arrhythmias. All displayed typical right ventricular (RV) abnormalities associating RV dilatation and wall motion abnormalities. Fibro-fatty replacement was demonstrated when tissue available for analysis. Conclusion: In this first systematic screening of the whole RyR2 gene in a large ARVC/D cohort without mutation in desmosomal genes, we observed that (i) putative mutations were not infrequent (9% of ARVC/D probands) and (i) were predominantly associated with a conventional phenotype of ARVC/D, not overlapping with CPVT, in contrast with previous findings. Therefore, we propose that RYR2 gene should be added to the panel of genes analysed in patients with ARVC/D.

The Cardiac Ryanodine Receptor N-Terminal Region Contains an Anion Binding Site that Is Targeted by Disease Mutations

Ryanodine receptors (RyRs) are calcium release channels located in the membrane of the endoplasmic and sarcoplasmic reticulum and play a major role in muscle excitation-contraction coupling. The cardiac isoform (RyR2) is the target for >150 mutations that cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and other conditions. Here, we present the crystal structure of the N-terminal region of RyR2 (1-547), an area encompassing 29 distinct disease mutations. The protein folds up in three individual domains, which are held together via a central chloride anion that shields repulsive positive charges. Several disease mutant versions of the construct drastically destabilize the protein. The R420Q disease mutant causes CPVT and ablates chloride binding. The mutation results in reorientations of the first two domains relative to the third domain. These conformational changes likely activate the channel by destabilizing intersubunit interactions that are disrupted upon channel opening.

S4153R Is a Gain-of-Function Mutation in the Cardiac Ca2+ Release Channel Ryanodine Receptor Associated With Catecholaminergic Polymorphic Ventricular Tachycardia and Paroxysmal Atrial Fibrillation

Mutations in ryanodine receptor 2 (RYR2) gene can cause catecholaminergic polymorphic ventricular tachycardia (CPVT). The novel RYR2-S4153R mutation has been implicated as a cause of CPVT and atrial fibrillation. The mutation has been functionally characterized via store-overload-induced Ca2+ release (SOICR) and tritium-labelled ryanodine ([3H]ryanodine) binding assays. The S4153R mutation enhanced propensity for spontaneous Ca2+ release and reduced SOICR threshold but did not alter Ca2+ activation of [3H]ryanodine binding, a common feature of other CPVT gain-of-function RYR2 mutations. We conclude that the S4153R mutation is a gain-of-function RYR2 mutation associated with a clinical phenotype characterized by both CPVT and atrial fibrillation.

Ryanodine Receptor Use-Dependent Block Suppresses Ca2+ Waves in Permeabilized Casq2-/- and RyR2-R4496C Myocytes

Background-Casq2 and RyR2 mutations can both cause catecholaminergic-polymorphic ventricular tachycardia (CPVT). Leaky RyR2s (increased open probability) generate Ca2+ waves, delayed afterdepolarizations and ventricular arrhythmias. We hypothesize that the RyR2 open-state inhibitors flecainide (FLEC) and R-propafenone (RPROP) abolish this sequence of events in both CPVT genotypes.Methods and Results-Using confocal microscopy, we recorded Ca2+ waves in permeabilized myocytes from casq2-/-, RyR2-R4496C and wild-type (WT) mice. Casq2-/- myocytes showed the highest wave frequency and speed and the lowest amplitude, RyR2-R4496C exhibited intermediate values and WT had the lowest values (Figure). We next obtained FLEC and RPROP concentration-response curves and calculated IC50 and efficacy of wave inhibition. Both FLEC and RPROP reduced all wave parameters with higher potency (lower IC50) and efficacy in casq2-/- and RyR4496C compared to WT (FLEC: IC50 12μM vs 22μM vs 49μM; efficacy 43% vs 29% vs 21% for casq2-/-, RyR2-R4496C and WT respectively. RPROP: IC50 18μM vs 28μM; efficacy 36% vs 17% for RyR2-R4496C and WT respectively; n=10-60 cells per group).Conclusion-RyR2 activity determines the potency and efficacy of open-state blockers for suppressing arrhythmogenic Ca2+ waves in permeabilized myocytes from casq2-/- and RyR2-R4496C CPVT models.View Large Image | View Hi-Res Image | Download PowerPoint Slide

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

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

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

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.

In the RyR2 R4496C Mouse Model of CPVT, β-Adrenergic Stimulation Induces Ca Waves by Increasing SR Ca Content and Not by Decreasing the Threshold for Ca Waves

mutations of the ryanodine receptor (RyR) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These mutations predispose to the generation of Ca waves and delayed afterdepolarizations during adrenergic stimulation. Ca waves occur when either sarcoplasmic reticulum (SR) Ca content is elevated above a threshold or the threshold is decreased. Which of these occurs in cardiac myocytes expressing CPVT mutations is unknown. we tested whether the threshold SR Ca content is different between control and CPVT and how it relates to SR Ca content during β-adrenergic stimulation. ventricular myocytes from the RyR2 R4496C(+/-) mouse model of CPVT and wild-type (WT) controls were voltage-clamped; diastolic SR Ca content was measured and compared with the Ca wave threshold. The results showed the following. (1) In 1 mmol/L [Ca(2+)](o), β-adrenergic stimulation with isoproterenol (1μmol/L) caused Ca waves only in R4496C. (2) SR Ca content and Ca wave threshold in R4496C were lower than those in WT. (3) β-Adrenergic stimulation increased SR Ca content by a similar amount in both R4496C and WT. (4) β-Adrenergic stimulation increased the threshold for Ca waves. (5) During β-adrenergic stimulation in R4496C, but not WT, the increase of SR Ca was sufficient to reach threshold and produce Ca waves. in the R4496C CPVT model, the RyR is leaky, and this lowers both SR Ca content and the threshold for waves. β-Adrenergic stimulation produces Ca waves by increasing SR Ca content and not by lowering threshold.

Mutant Ryanodine Receptor-dependent Calcium Leak, RyR2 Open Probability, Calcium Sparks And Cardiac Arrhythmogenesis

Ca2+ leak from the sarcoplasmic reticulum (SR) represents a major mechanism underlying arrhythmic disease in the heart. Nevertheless the links between cardiac ryanodine receptor (RyR2) single channel behavior, Ca2+ sparks, and Ca2+ waves remains surprisingly enigmatic. Here we investigate the relationship between a known missense mutation in RyR2 (R2474S) and Ca2+ sparks. R2474S has been shown to cause catecholaminergic polymorphic ventricular tachycardia (CPVT) in humans and knockin mice under conditions of stress and catecholamine exposure. Heterozygous CPVT-mutant RyR2-R2474S/WT (RS/WT) or wild-type (WT) channels were isolated from the hearts of 1) resting, control mice or 2) mice which underwent exercise stress testing (‘stressed’). Using the lipid bilayer method (cytosolic [Ca2+]cis = 150 nM to approximate diastolic concentrations), RyR2-RS/WT from ‘stressed’ mice showed a significant gain-of-function defect as evidenced by increased open probability (Po = 0.134±0.008, n=7) versus WT (0.018±0.008, n=7; P<0.05). When isolated cardiac myocytes were pre-treated with isoproterenol (1 μM) to mimic catecholaminergic stress and following 1 Hz field pacing, fluo-4-AM loaded RS/WT cells showed a significantly increased spark rate (341.8 ±115.7% vs. 108.1±44.6%) when compared to WT cells. These findings suggest that changes in RyR2 Po, Ca2+ sparks and arrhythmogenesis are linked mechanistically. How the ensemble of findings are interrelated dynamically, however, is model-dependent and this modeling will be presented. These findings suggest that our approach to the investigation of Ca2+ dependent arrhythmogenesis broadens understanding of molecular cardiac defects in disease and lays the foundation for the development and testing of novel therapeutic agents.

A mutation in calsequestrin, CASQ2D307H, impairs Sarcoplasmic Reticulum Ca2+ handling and causes complex ventricular arrhythmias in mice

A naturally-occurring mutation in cardiac calsequestrin (CASQ2) at amino acid 307 was discovered in a highly inbred family and hypothesized to cause Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). The goal of this study was to establish a causal link between CASQ2(D307H) and the CPVT phenotype using an in vivo model. Cardiac-specific expression of the CASQ2(D307H) transgene was achieved using the alpha-MHC promoter. Multiple transgenic (TG) mouse lines expressing CASQ2(D307H) from 2- to 6-fold possess structurally normal hearts without any sign of hypertrophy. The hearts displayed normal ventricular function. Myocytes isolated from TG mice had diminished I(Ca)-induced Ca2+ transient amplitude and duration, as well as increased Ca2+ spark frequency. These myocytes, when exposed to isoproterenol and caffeine, displayed disturbances in their rhythmic Ca2+ oscillations and membrane potential, and delayed afterdepolarizations. ECG monitoring revealed that TG mice challenged with isoproterenol and caffeine developed complex ventricular arrhythmias, including non-sustained polymorphic ventricular tachycardia. The findings of the present study demonstrate that expression of mutant CASQ2(D307H) in the mouse heart results in abnormal myocyte Ca2+ handling and predisposes to complex ventricular arrhythmias similar to the CPVT phenotype observed in human patients.

A mutation in Calsequestrin can cause polymorphic ventricular Tachycardia

Calsequestrin (CASQ2) is the major calcium binding protein in the cardiac junctional sarcoplasmic reticulum (SR). CASQ2 has been shown to function both as a luminal Ca2+ sensor and a modulator of calcium release. Mutations in the human CASQ2 gene have been linked to ventricular tachycardia and sudden cardiac death. A naturally occurring point mutation (CASQ2D307H) in the human CASQ2 gene at amino acid 307 (Aspartate→Histidine) has been proposed to cause Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT).

Molecular genetics of exercise-induced polymorphic ventricular tachycardia: identification of three novel cardiac ryanodine receptor mutations and two common calsequestrin 2 amino-acid polymorphisms.

Mutations of two myocardial calcium signaling molecules, ryanodine receptor 2 (RYR2) and calsequestrin 2 (CASQ2), may cause catecholaminergic polymorphic ventricular tachycardia (CPVT), a severe inherited arrhythmic disease manifesting with salvoes of exercise-induced bidirectional and polymorphic tachycardias. We screened 12 Finnish CPVT probands for mutations in these genes and identified three novel RYR2 mutations (V2306I, P4902L, R4959Q), which were absent in unaffected and control individuals. Although no obvious disease-causing mutations were identified in the CASQ2 gene, the molecular screening revealed two novel amino-acid polymorphisms (T66A and V76M). The frequencies of these polymorphisms in 185 unrelated probands with long QT syndrome and in 280 healthy blood donors were not significantly different. These data, combined with our previous findings, show that RYR2 mutations are present in at least 6/16 (38%) of the catecholaminergic polymorphic ventricular tachycardia families, while CASQ2 mutations must be a rare cause of CPVT.