Arrhythmogenic Mutations Research Articles

PO-614-08 SODIUM CHANNEL NAV1.6 AND NA-CA EXCHANGER REMODELING CONTRIBUTES TO ARRHYTHMOGENIC LATE SODIUM CURRENT AND CA2+ SPARKS IN THE PRESENCE OF D96V MUTANT CALMODULIN

Calmodulin (CaM) facilitates sodium channel (NaV) inactivation, thereby preventing proarrhythmic late sodium current (INa). To date, a link between arrhythmogenic mutations in CaM and NaV dysfunction is not well established. Outside of NaV1.5, dysfunctional inactivation of Nav1.6 promotes late INa-mediated arrhythmias.

Stabilizer Cell Gene Therapy: A Less-Is-More Strategy to Prevent Cardiac Arrhythmias.

In cardiac gene therapy to improve contractile function, achieving gene expression in the majority of cardiac myocytes is essential. In preventing cardiac arrhythmias, however, this goal may not be as important since transduction efficiencies as low as 40% suppressed ventricular arrhythmias in genetically modified mice with catecholaminergic polymorphic ventricular tachycardia. Using computational modeling, we simulated 1-, 2-, and 3-dimensional tissue under a variety of conditions to test the ability of genetically engineered nonarrhythmogenic stabilizer cells to suppress triggered activity due to delayed or early afterdepolarizations. Due to source-sink relationships in cardiac tissue, a minority (20%-50%) of randomly distributed stabilizer cells engineered to be nonarrhythmogenic can suppress the ability of arrhythmogenic cells to generate delayed and early afterdepolarizations-related arrhythmias. Stabilizer cell gene therapy strategy can be designed to correct a specific arrhythmogenic mutation, as in the catecholaminergic polymorphic ventricular tachycardia mice studies, or more generally to suppress delayed or early afterdepolarizations from any cause by overexpressing the inward rectifier K channel Kir2.1 in stabilizer cells. This promising antiarrhythmic strategy warrants further testing in experimental models to evaluate its clinical potential.

The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia-causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84-2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.

RyR2 mutation-linked arrhythmogenic diseases and its therapeutic strategies

The type 2 ryanodine receptor (RyR2) is a sarcoplasmic reticulum Ca2+ release channel that plays a central role in cardiac excitation-contraction coupling. Abnormal activity of the RyR2 is linked to abnormal Ca2+ signaling in cardiac cells, which often results in cardiac arrhythmias. For example, amino acid mutations in RyR2 have been reported to cause various types of arrhythmias, including catecholaminergic polymorphic ventricular tachycardia (CPVT), idiopathic ventricular fibrillation, and left ventricular non-compaction. At present, the total number of disease-associated RyR2 mutations exceeds 300. In addition, in chronic heart failure, modification of RyR2 by phosphorylation, oxidation or S-nitrosylation may cause abnormal channel activity. Arrhythmogenic mechanisms of these various disorders are not yet fully understood. We have recently established a method to quantitatively evaluate the effects of various arrhythmogenic mutations and modifications on RyR2 channels by using HEK293 expression system. We found that arrhythmogenic mutations in RyR2 are classified into two groups: gain-of-function and loss-of-function of the channel. Since they are indistinguishable in clinical diagnosis, our analysis is very useful for diagnosis and choice of treatment strategies for RyR2-linked arrhythmogenic diseases. This review describes the current advances and issues of research on RyR2 mutation-related arrhythmogenic disorders.

Abnormal RyR2 in arrhythmogenic disorders and CICR

Ca2+ induced Ca2+ release (CICR) via ryanodine receptor 2 (RyR2) plays a central role in E-C coupling in cardiac cells, i.e., Ca2+ influx via L-type Ca2+ channels during action potential (AP) activates RyR2 to release Ca2+ from the ER and causes muscle contractions. Many arrhythmogenic mutations in RyR2 are reported to increase AP-independent spontaneous Ca2+ release from ER that often lead to arrhythmia. Two explanations have been proposed for the increased propensity of spontaneous Ca2+ release: (1) CPVT mutations increase the cytoplasmic Ca2+ sensitivity of RyR2 to enhance CICR, or (2) mutations decrease threshold for store-overload induced Ca2+ release (SOICR) by reducing luminal Ca2+ sensitivity of RyR2. To understand the underlying mechanism for the increased spontaneous Ca2+ release by the mutations, we performed quantitative evaluation of CICR activity and cytoplasmic and ER Ca2+ signals in HEK293 cells expressing mutant RyR2s. Furthermore, the effects of RyR2 inhibitors, which had been found by recently established high-throughput screening method, were examined on Ca2+ signals in RyR2-HEK cells and cardiomyocytes from adult mice. Our results indicate that CICR plays critical role in generation of spontaneous Ca2+ release and that regulation of CICR is important in suppression of arrhythmia.

GW28-e0175 In silico assessment of the effects of drug disopyramide on electrical activity in human ventricular myocardium associated with short QT syndrome

Short QT syndrome (SQTS) is an inheritable electric heart disease characterized by abnormally short QT intervals on the ECG and a high incidence of sudden cardiac death (SCD) in individuals with a structurally normal heart. To our knowledge, multiple functional effects of arrhythmogenic mutations in

Characterization of Ca2+-Induced Ca2+ Release via RyR2 Carrying Arrhythmogenic Mutations

The type 2 ryanodine receptor (RyR2) is the Ca2+ release channel on cardiac sarcoplasmic reticulum and plays a crucial role in cardiac E-C coupling. The mutations in RyR2 have been implicated in a number of arrhythmogenic disorders including catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular cardiomyopathy (ARVC), idiopathic ventricular fibrillation (I-VF) and atrial fibrillation (AF). In this study, we aimed to characterize Ca2+ release properties of RyR2s carrying the above 4 types of disease mutations. Wild type and mutant RyR2 channels were expressed in HEK293 cells and spontaneous Ca2+ oscillations were monitored by live-cell Ca2+ imaging using Ca2+ indicators for cytoplasm (fluo-4, GECOs) and ER (CEPIAs). In addition, the Ca2+-induced Ca2+ release (CICR) activity were determined with [3H]ryanodine binding assay. There were good correlations between CICR activity, Ca2+ oscillation frequencies and ER Ca2+ levels among all of mutants examined. While all CPVT mutants showed enhanced CICR activity, I-VF mutants showed divergent CICR profiles. Our results suggest that further analyses of experimental results and clinical data are needed to understand mechanisms of arrhythmogenic diseases due to RyR2 mutations.

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.

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias.

Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1-606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410-437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545-606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C(α) atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine-isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

FGF12 is a candidate Brugada syndrome locus

Less than 30% of the cases of Brugada syndrome (BrS) have an identified genetic cause. Of the known BrS-susceptibility genes, loss-of-function mutations in SCN5A or CACNA1C and their auxiliary subunits are most common. On the basis of the recent demonstration that fibroblast growth factor (FGF) homologous factors (FHFs; FGF11-FGF14) regulate cardiac Na(+) and Ca(2+) channel currents, we hypothesized that FHFs are candidate BrS loci. The goal of this study was to test whether FGF12 is a candidate BrS locus. We used quantitative polymerase chain reaction to identify the major FHF expressed in the human ventricle and then queried a phenotype-positive, genotype-negative BrS biorepository for FHF mutations associated with BrS. We queried the effects of an identified mutant with biochemical analyses combined with electrophysiological assessment. We designed a novel rat ventricular cardiomyocyte system in which we swapped the endogenous FHF with the identified mutant and defined its effects on multiple ionic currents in their native milieu and on the cardiac action potential. We identified FGF12 as the major FHF expressed in the human ventricle. In 102 individuals in the biorepository, we identified a single missense mutation in FGF12-B (Q7R-FGF12). The mutant reduced binding to the NaV1.5 C terminus, but not to junctophilin-2. In adult rat cardiac myocytes, Q7R-FGF12, but not wild-type FGF12, reduced Na(+) channel current density and availability without affecting Ca(2+) channel function. Furthermore, the mutant, but not wild-type FGF12, reduced action potential amplitude, which is consistent with a mutant-induced loss of Na(+) channel function. These multilevel investigations strongly suggest that Q7R-FGF12 is a disease-associated BrS mutation. Moreover, these data suggest for the first time that FHF effects on Na(+) and Ca(2+) channels are separable. Most significantly, this study establishes a new method to analyze effects of human arrhythmogenic mutations on cardiac ionic currents.

Direct Detection of Domain Peptide Binding to the Cardiac Ryanodine Receptor (RYR2) using FRET

The RyR2 Ca release channel is activated by the synthetic domain peptide DPc10, which corresponds to a 36-residue sequence within the channel's central mutation hot-spot region. DPc10 activation is hypothesized to result from the destabilization of a critical intramolecular interaction between the N-terminal and central hot-spot regions of the full-length RyR2, and to thereby mimic the effects of arrhythmogenic mutations on this putative intramolecular interaction controlling channel activation. However, the site of DPc10 binding within the RyR2 3D structure is uncertain, and factors that may modulate binding are undefined. To directly monitor and map DPc10 binding to the RyR2, we attached a FRET acceptor at DPc10's N-terminus. A FRET donor was targeted to the RyR2 cytoplasmic assembly via a fluorescent-labeled FKBP12.6. Addition of the acceptor-labeled DPc10 (30 µM) resulted in a marked decrease in fluorescence of the RyR2-bound FKBP12.6. Fluorescence was partially restored upon FKBP12.6 dissociation from the RyR2, indicating that a major fraction of the total fluorescence decrease was attributable to FRET between FKBP12.6 and DPc10 when bound to the channel. The DPc10 dependence of FRET was similar to the DPc10 dependence of RyR2 activation observed previously in bilayer and ryanodine binding studies (EC50 ∼25 µM), consistent with the likelihood that FRET reflected DPc10 binding at its regulatory site on the RyR2. FRET decreased as a function of increasing Ca (30 nM to 300 µM), suggesting that Ca activation of the RyR2 altered either the affinity of DPc10 binding or its proximity to the FKBP12.6 subunit. We conclude that DPc10 binds to a site on the RyR2 within 10 nm of FKBP12.6. Regulatory interactions and structural changes at this site can be monitored using FRET.

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations

BackgroundMutations in HERG and KCNQ1 potassium channels have been associated with Long QT syndrome and atrial fibrillation, and more recently with sudden infant death syndrome and sudden unexplained death. In other proteins, disease-associated amino acid mutations have been analyzed according to the chemical severity of the changes and the locations of the altered amino acids according to their conservation over metazoan evolution. Here, we present the first such analysis of arrhythmia-associated mutations (AAMs) in the HERG and KCNQ1 potassium channels.ResultsUsing evolutionary analyses, AAMs in HERG and KCNQ1 were preferentially found at evolutionarily conserved sites and unevenly distributed among functionally conserved domains. Non-synonymous single nucleotide polymorphisms (nsSNPs) are under-represented at evolutionarily conserved sites in HERG, but distribute randomly in KCNQ1. AAMs are chemically more severe, according to Grantham's Scale, than changes observed in evolution and their severity correlates with the expected chemical severity of the involved codon. Expected chemical severity of a given amino acid also correlates with its relative contribution to arrhythmias. At evolutionarily variable sites, the chemical severity of the changes is also correlated with the expected chemical severity of the involved codon.ConclusionUnlike nsSNPs, AAMs preferentially locate to evolutionarily conserved, and functionally important, sites and regions within HERG and KCNQ1, and are chemically more severe than changes which occur in evolution. Expected chemical severity may contribute to the overrepresentation of certain residues in AAMs, as well as to evolutionary change.

M-type KCNQ2–KCNQ3 potassium channels are modulated by the KCNE2 subunit

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K + channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.