Catecholaminergic Polymorphic Ventricular Tachycardia Mutations Research Articles

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca(2+) release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca(2+) on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [(3)H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G(+/-)) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G(+/-) hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca(2+) release during systole, gradually overloading the sarcoplasmic reticulum with Ca(2+). The resultant Ca(2+) overload then randomly caused bursts of prolonged Ca(2+) release, activating electrogenic Na(+)-Ca(2+) exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.

Calmodulin mutations causing catecholaminergic polymorphic ventricular tachycardia confer opposing functional and biophysical molecular changes.

Calmodulin (CaM) is the central mediator of intracellular Ca(2+) signalling in cardiomyocytes, where it conveys the intricate Ca(2+) transients to the proteins controlling cardiac contraction. We recently linked two separate mutations in CaM (N53I and N97S) to dominantly inherited catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmic disorder in which exercise or acute emotion can lead to syncope and sudden cardiac death. Given the ubiquitous presence of CaM in all eukaryote cells, it is particular intriguing that carriers of either mutation show no additional symptoms. Here, we investigated the effects of the CaM CPVT mutations in a zebrafish animal model. Three-day-old embryos injected with either CaM mRNA showed no detectable pathologies or developmental abnormalities. However, embryos injected with CPVT CaM mRNA displayed increased heart rate compared to wild-type CaM mRNA under β-adrenergic stimulation, demonstrating a conserved dominant cardiac specific effect between zebrafish and human carriers of these mutations. Motivated by the highly similar physiological phenotypes, we compared the effects of the N53I and N97S mutations on the biophysical and functional properties of CaM. Surprisingly, the mutations have opposing effects on CaM C-lobe Ca(2+) binding affinity and kinetics, and changes to the CaM N-lobe Ca(2+) binding are minor and specific to the N53I mutation. Furthermore, both mutations induce differential perturbations to structure and stability towards unfolding. Our results suggest different molecular disease mechanisms for the CPVT (N53I and N97S mutations) and strongly support that cardiac contraction is the physiological process most sensitive to CaM integrity.

Effects of Arrhythmogenic Mutations on Ca2+-Induced Ca2+ Release Activities of Type 2 Ryanodine Receptors

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the sarcoplasmic reticulum and plays a pivotal role in excitation-contraction coupling in heart. RyR2 is the major target for arrhythmogenic diseases, e.g., catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy (ARVC). To date, over 150 mutations have been identified in the RyR2 gene of CPVT and ARVC patients. It is widely believed that CPVT and ARVC mutations cause hyperactivation of Ca2+ release and result in ventricular arrhythmia by abnormal Ca2+ homeostasis in cardiac muscle. The RyR2 channel is known to be regulated by both cytoplasmic Ca2+ (Ca2+ induced Ca2+ release: CICR) and luminal Ca2+ (store overload induced Ca2+ release: SOICR). It is well documented that the SOICR threshold and termination levels are affected in the disease-associated mutations, whereas it remains unclear how these mutations affect CICR. CICR shows biphasic Ca2+ dependence against cytoplasmic Ca2+, thus the activity can be determined by three parameters: sensitivity to activating Ca2+, sensitivity to inactivating Ca2+, and the gain (i.e., peak activity). In this study, we expressed RyR2 channels carrying CPVT and ARVC mutations in HEK cells and tested their Ca2+ homeostasis by live-cell Ca2+ imaging using gene-encoded Ca2+ indicators for optical imaging (GECO) and calcium-measuring organelle-entrapped protein indicators (CEPIA). In addition, three parameters of CICR activity was determined with [3H]ryanodine binding assay. Our results suggest that the disease-associated mutations divergently affects the parameters of CICR depending on the sites for mutation. The underlying molecular mechanism will be discussed.

Arrhythmogenic mechanisms in ryanodine receptor channelopathies.

Ryanodine receptors (RyRs) are the calcium release channels of sarcoplasmic reticulum (SR) that provide the majority of cal-cium ions (Ca2+) necessary to induce contraction of cardiac and skeletal muscle cells. In their intracellular environment, RyR channels are regulated by a variety of cytosolic and luminal factors so that their output signal (Ca2+) induces finely-graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity (ventricular arrhythmias) or cellular remodeling. The importance of RyR dysfunction has been recently highlighted with the demonstration that point mutations in RYR2, the gene encoding for the cardiac isoform of the RyR (RyR2), are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart. Here we summarize the state of the field in regards to the main arrhythmogenic mechanisms triggered by RyR2 channels harboring mutations linked to CPVT. Most CPVT mutations characterized to date endow RyR2 channels with a gain of function, resulting in hyperactive channels that release Ca2+ spontaneously, especially during diastole. The spontaneous Ca2+ release is extruded by the electrogenic Na+/Ca2+ exchanger, which depolarizes the external membrane (delayed afterdepolarization or DAD) and may trigger untimely action potentials. However, a rare set of CPVT mutations yield RyR2 channels that are intrinsically hypo-active and hypo-responsive to stimuli, and it is unclear whether these channels release Ca2+ spontaneously during diastole. We discuss novel cellular mechanisms that appear more suitable to explain ventricular arrhythmias due to RyR2 loss-of-function mutations.

Dynamics of Calcium Sparks and SR Calcium Leak During Excitation-Contraction Coupling in Mouse Heart Cells

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cell contraction during cardiac excitation-contraction (EC) coupling in heart cells. The fundamental element of CICR in heart is the calcium (Ca2+) spark which arises from the regenerative release of Ca2+ from a cluster of ryanodine receptors (RyR2) in the junctional sarcoplasmic reticulum membrane. We have shown how stochastic gating of RyR2s could produce an SR Ca2+ leak capable of balancing SR Ca2+-ATPase (SERCA2a) activity under quiescent conditions (Williams et al. BJ 2011). This investigation suggested the surprising finding that a single, or even multiple, RyR2 openings could fail probabilistically to trigger a Ca2+ spark. To further investigate this effect, we expanded upon that formulation to create a detailed, local control model of EC coupling in mouse heart. A number of features were added or modified, including the addition of a novel seven-state Markov chain model of the sarcolemmal L-type Ca2+ channel (LCC). This model features dynamic action-potential (AP) generation, robust Ca2+ spark initiation and termination, realistic [Ca2+]i transients, and true SR Ca2+ pump/leak balance. The model suggests that numerous LCC openings are often required to trigger a single Ca2+ spark. This is consistent with our prior work where multiple RyR2 openings were needed to trigger a spontaneous Ca2+ spark during quiescent conditions. We also investigated how RyR2 mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) influence the dynamics of Ca2+ sparks, “invisible” non-spark Ca2+ leak, [Ca2+]i transients, and SR Ca2+ content. We observe that CPVT mutations can lead to unstable Ca2+ spark dynamics, altering SR Ca2+ content and promoting [Ca2+]i signaling instability. Our new model provides significant insights into the dynamics of local control of CICR under physiological and pathological conditions.

Divergent Effects of Disease-Associated Mutations on Type 2 Ryanodine Receptor Channel

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel in the sarcoplasmic reticulum and plays a pivotal role in excitation-contraction coupling in heart. RyR2 is the major target for arrhythmogenic diseases, e.g., catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy (ARVC). To date, over 150 mutations have been identified in the RyR2 gene of CPVT and ARVC patients. It is widely believed that CPVT and ARVC mutations cause hyperactivation of the Ca2+-induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in cardiac muscle. CICR shows biphasic Ca2+ dependence against cytoplasmic Ca2+, thus the activity can be determined by three parameters: sensitivity to activating Ca2+, sensitivity to inactivating Ca2+, and the gain (i.e., peak activity). In addition, CICR is also regulated by luminal Ca2+; high luminal Ca2+ activates CICR, and vise versa. However, it remains unclear how the disease-associated mutations affect these parameters. In this study, we expressed RyR2 channels carrying several CPVT/ARVC mutations in HEK cells and tested their CICR by live-cell Ca2+ imaging and [3H]ryanodine binding. Our results suggest that the disease-associated mutations divergently affects the parameters of CICR depending on the sites for mutation. The underlying molecular mechanism will be discussed.

Type 2 Ryanodine Receptor Domain A Contains a Unique and Dynamic α-Helix That Transitions to a β-Strand in a Mutant Linked with a Heritable Cardiomyopathy

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca2+) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca2+ sensitivity, propagating sarcoplasmic reticulum luminal Ca2+ release in the process of excitation–contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1–559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the β-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense “columns” projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.

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Atrial arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia – is there a mechanistic link between sarcoplasmic reticulum Ca2+ leak and re‐entry?

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in the genes encoding the cardiac ryanodine receptor channel (RyR2) or the major sarcoplasmic reticulum (SR) Ca2+ buffer calsequestrin-2 (Casq2) (Leenhardt et al., 2012). Traditionally, CPVT has been described as a bi-directional or polymorphic ventricular arrhythmia occurring during conditions of increased sympathetic tone, which may originate from ectopic activity due to abnormal (sub)cellular Ca2+ handling (Leenhardt et al., 2012). Recent studies, however, have suggested that CPVT mutations in RyR2 are also associated with atrial arrhythmias. In this edition of Acta Physiologica, King et al. (King et al., 2012) show for the first time that atria from mice homozygous for the CPVT-associated mutation P2328S in RyR2 (RyR2S/S) have a reduced conduction velocity (CV) and lower maximum rates of action potential (AP) upstroke velocity compared to wild-type (WT) mice. Moreover, these parameters correlated strongly with arrhythmia susceptibility suggesting a potential novel arrhythmogenic mechanism due to RyR2 mutations.

ATP and Caffeine Binding to the Human RyR2 Central Domain Encompassing a CPVT Mutation Cluster

The sarcoplasmic reticulum (SR) of cardiomyocytes contains the cardiac ryanodine receptor (RyR2) - a calcium release channel that plays a pivotal role in mediating the SR calcium release essential for cardiac excitation-contraction coupling. Discrete clusters of point mutations, that may comprise important regulatory regions within RyR2, have been associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). In response to a physiological trigger, RyR2 bearing CPVT mutations are believed to cause diastolic calcium leak, which can result in a fatal arrhythmia. A RyR2 central domain, which encompasses both a CPVT-associated region and a predicted ATP binding motif, has been expressed as a soluble recombinant protein. The wild-type protein was compared with three constructs each containing a different CPVT mutation (P2328S, F2331S and N2368I). ATP is a physiological activator of RyR2 activity and caffeine has a similar effect. Fluorescence spectroscopy was used to record changes in the intrinsic fluorescence of the single tryptophan residue present within the expressed domain, and we found evidence for protein conformational changes upon ATP or caffeine binding. We observed a similar binding affinity of the expressed RyR2 central domain for both compounds, with an EC50 ∼100μM. The presence of the individual mutations made no apparent difference to the EC50. However, there was an increased initial fluorescence value observed for each of the mutant constructs compared to WT. Analysis of the central domain sequence revealed homology with conserved motifs typical of P-loop kinases. Interestingly, many of the CPVT mutations present in this region are coincident with sites corresponding to the Walker A, Walker B and LID motifs in ATP binding proteins.

Abstract 19541: Impaired Calcium Release, Prolonged Action Potentials and Early Afterdepolarizations in Ventricular Myocytes from Mice Harboring a Ryanodine Receptor 2 Loss-of-Function Mutation Associated with Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) patients harboring the cardiac ryanodine receptor (RyR2) mutation RyR2-A4860G exhibit sympathetically-driven idiopathic ventricular fibrillation without cardiac structural defects. In sharp contrast to other CPVT mutations characterized to date, RyR2-A4860G fails to activate in response to increasing luminal [Ca 2+ ] and, when expressed in HEK293 or HL-1 cells, it exhibits attenuated store overload-induced Ca 2+ release. Thus, RyR2-A4860G is the first loss-of-function mutation linked to CPVT, but the arrhythmogenic mechanisms are unknown. We used homologous recombination to generate a mouse harboring the RyR2-A4860G mutation and used isolated ventricular myocytes to investigate the cellular mechanisms by which this mutation generates ventricular arrhythmias. Mice heterozygous for the mutation (RyR2-A4860G +/- ) display no apparent cardiac structural defects. Whole cell current-clamp combined with confocal Ca 2+ imaging to simultaneously record actions potential (AP) and intracellular Ca 2+ transient ([Ca 2+ ] i ) revealed that AP duration at 90% repolarization (APD 90 ) in RyR2-A4860G +/- was longer than in WT cardiomyocytes at 1 Hz (209±51 vs. 118±37 ms, n=12 and 7, respectively). As a result of prolonged APs, RyR2-A4860G +/- cells exhibited early afterdepolarizations (EADs) at significantly greater frequency than WT (50% vs 14%, respectively). Ca 2+ imaging of RyR2-A4860G +/- cells showed that during the longer APs, Ca 2+ release events are often interspaced between two successive AP-triggered Ca 2+ transients. Since the [Ca 2+ ] i amplitude in RyR2-A4860G +/- cells is significantly lower than in WT (3.5±0.6 , 2.5±0.3, and 2.5±0.2 vs. 3.8±0.3, 4.3±0.5, and 4.4±0.7, at 1, 2 and 3 Hz, respectively), the presence of Ca 2+ release suggests decreased Ca 2+ -dependent inactivation, and subsequent re-activation, of L-type Ca 2+ current during the AP plateau. In conclusion, our results demonstrate that RyR2-A4860G mutation is accompanied by profound impairment of SR Ca 2+ release, which leads to AP prolongation and EADs. This is a novel mechanism of arrhythmias in CPVT, distinct from delayed afterdepolarizations that are commonly detected in all gain-of-function RyR2 mutations.

Heterogeneity of Ryanodine Receptor Dysfunction in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Most cardiac ryanodine receptor (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) are postulated to cause a distinctive form of Ca(2+) release dysfunction. Considering the spread distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity also was feasible. To determine the molecular and cellular mechanisms by which a novel RyR2-V2475F mutation associated with CPVT in humans triggers Ca(2+)-dependent arrhythmias in whole hearts and intact mice. Recombinant channels harboring CPVT-linked RyR2 mutations were functionally characterized using tritiated ryanodine binding and single-channel recordings. Homologous recombination was used to generate a knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice heterozygous for the mutation (RyR2-V2475F(+/-)) and their wild-type littermates were Ca(2+)-imaged by confocal microscopy under conditions that mimic stress. The propensity of wild-type and RyR2-V2475F(+/-) mice to have development of arrhythmias was tested at the whole heart level and in intact animals. Recombinant RyR2-V2475F channels displayed increased cytosolic Ca(2+) activation, abnormal protein kinase A phosphorylation, and increased activation by luminal Ca(2+). The RyR2-V2475F mutation appears embryonic-lethal in homozygous mice, but heterozygous mice have no alterations at baseline. Spontaneous Ca(2+) release events were more frequent and had shorter latency in isoproterenol-stimulated cardiomyocytes from RyR2-V2475F(+/-) hearts, but their threshold was unchanged with respect to wild-type. Adrenergically triggered tachyarrhythmias were more frequent in RyR2-V2475F(+/-) mice. The mutation RyR2-V2475F is phenotypically strong among other CPVT mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living mice, this mutation appears too severe to be harbored in all RyR2 channels but remains undetected under basal conditions if expressed at relatively low levels. β-adrenergic stimulation breaks the delicate Ca(2+) equilibrium of RyR2-V2475F(+/-) hearts and triggers life-threatening arrhythmias.

Modeling Catecholaminergic Polymorphic Ventricular Tachycardia using Induced Pluripotent Stem Cell-derived Cardiomyocytes

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias induced by physical or emotional stress, in the absence structural heart abnormalities. The arrhythmias may cause syncope or degenerate into cardiac arrest and sudden death which usually occurs during childhood. Recent studies have shown that CPVT is caused by mutations in the cardiac ryanodine receptor type 2 (RyR2) or calsequestrin 2 (CASQ2) genes. Both proteins are key contributors to the intracellular Ca2+ handling process and play a pivotal role in Ca2+ release from the sarcoplasmic reticulum to the cytosol during systole. Although the molecular pathogenesis of CPVT is not entirely clear, it was suggested that the CPVT mutations promote excessive sarcoplasmic reticulum Ca2+ leak, which initiates delayed afterdepolarizations (DADs) and triggered arrhythmias in cardiac myocytes. The recent breakthrough discovery of induced pluripotent stem cells (iPSC) generated from somatic cells (e.g. fibroblasts, keratinocytes) now enables researches to investigate mutated cardiomyocytes generated from the patient’s iPSC. To this end, in the present article we review recent studies on CPVT iPSC-derived cardiomyocytes, thus demonstrating in the mutated cells catecholamine-induced DADs and triggered arrhythmias.

Calcium Leak Through Ryanodine Receptors Leads to Atrial Fibrillation in 3 Mouse Models of Catecholaminergic Polymorphic Ventricular Tachycardia

Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca2+) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. In the present study, we sought to determine whether intracellular diastolic SR Ca2+ leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. We generated 3 knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca2+ leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the 3 CPVT mouse models (RyR2-R2474S+/-, 70%; RyR2-N2386I+/-, 60%; RyR2-L433P+/-, 35.71%) but not in wild-type (WT) mice (P<0.05). Consistent with these in vivo results, there was a significant diastolic SR Ca2+ leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca2+ leak through the channel. Atrial RyR2 from RyR2-R2474S+/- mice were oxidized, and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca2+ leak in atrial myocytes and decreased burst pacing-induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing-induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug. The present study demonstrates that RyR2-mediated diastolic SR Ca2+ leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca2+ leak through RyR2 and pacing-induced AF associated with CPVT mutations.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

In Vitro Characterization of a Novel N-Terminal CPVT RyR Mutation

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an arrhythmogenic disease characterized by stress-triggered syncope and sudden death. Most of the CPVT mutations are concentrated in RyR “hotspots”: the C terminus, the central and the N terminal domains. Although the phenotypic manifestations are common, it is likely that the intrinsic mechanism of RyR dysfunction varies with the location of the mutation within the RyR. Previously, we characterized RyR dysfunction in a CPVT mutation located at the C terminal domain, identifying an increased cytosolic Ca2+ sensitivity as the causative of a gain-of-funcion defect. Recently, we have identified a mutation in a family with CPVT syndrome, which is located in the N terminal portion: RyR2R420Q. To analyze the function of the mutated RyR channel, we have created an expression plasmids of RyR2R420Q and RyR2WT. Heterologous expression in HEK cells showed decreased caffeine sensitivity, suggesting loss-of-function defect. In order to be able to further characterize this mutation in a cardiac context, we expressed both RyR2R420Q and RyR2WT in cultured neonatal rat cardiac myocytes and analyzed the amplitude and frequency of spontaneous [Ca2+]i transients and Ca2+ sparks by confocal microscopy. We found that the spontaneous [Ca2+]i transients were more frequent but of lower amplitude in RyR2R420Q expressing cells. Further experiments are being conducted to fully understand the underlying mechanism.

A Novel Ryanodine Receptor Mutation Linked to Sudden Death Increases Sensitivity to Cytosolic Calcium

Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young individuals during β-adrenergic stimulation. This study sought to determine the effects of a novel RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca(2+)). Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2. RyR2 channels were measured to examine the regulation of the channels by cytosolic versus luminal sarcoplasmic reticulum Ca(2+). A 50-year-old white man with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. cAMP-dependent protein kinase-phosphorylated RyR2-G230C channels exhibited a significantly higher open probability at diastolic Ca(2+) concentrations, associated with a depletion of calstabin2. The luminal Ca(2+) sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like. The RyR2-G230C mutant exhibits similar biophysical defects compared with previously characterized CPVT mutations: decreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca(2+) dependence for activation under conditions that simulate exercise, consistent with a "leaky" channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca(2+) activation. Thus, diastolic sarcoplasmic reticulum Ca(2+) leak caused by reduced calstabin2 binding and a leftward shift in the Ca(2+) dependence for activation by diastolic levels of cytosolic Ca(2+) is a common mechanism underlying CPVT.

Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants

CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.

Effect of Human RyR2 CPVT Mutations on Interaction with Calmodulin

The ryanodine receptor (RyR) is a member of a family of intracellular calcium release channels that regulate calcium efflux from intracellular stores. The RyR2 isoform is most abundant in the heart and plays a key role in cardiac muscle excitation-contraction coupling. Clusters of mutations associated with the inherited arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), have been found in specific regions throughout RyR2, a large protein of ∼5000 amino acids. Many of these CPVT mutations (total >100) are thought to occur in significant functional domains and result in the dysregulation of RyR channel function.One such region of RyR2 is believed to comprise a calmodulin (CaM) interaction site and two EF hand motifs. The calcium-sensitive binding of CaM has been shown to regulate the opening of RyR. Hence, examining the RyR2 interaction with CaM and the potential effects of CPVT mutations on this binding may help reveal mutation-dependent mechanisms of channel dysfunction.We have prepared bacterial expression plasmid constructs containing the wild-type human RyR2 CaM-interacting domain and introduced a series of CPVT mutations that have previously been identified to occur within this region. Expression and purification of the corresponding recombinant fusion proteins has enabled calcium-dependent binding of CaM to be determined with all these constructs. Examining the distinct functional role of calcium concentration on CaM binding kinetics and further comparative structural analyses of the wild-type and mutant RyR2 domains may help reveal the specific effect(s) that CaM-mediated regulation may have in mediating CPVT-linked arrhythmogenesis.

Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT. Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca2+ imaging and [3H]ryanodine binding. A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca2+ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine. The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.