Julianne Wojciak

Calcium Transients Closely Reflect Prolonged Action Potentials in iPSC Models of Inherited Cardiac Arrhythmia

Summary Long-QT syndrome mutations can cause syncope and sudden death by prolonging the cardiac action potential (AP). Ion channels affected by mutations are various, and the influences of cellular calcium cycling on LQTS cardiac events are unknown. To better understand LQTS arrhythmias, we performed current-clamp and intracellular calcium ([Ca2+]i) measurements on cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPS-CM). In myocytes carrying an LQT2 mutation (HERG-A422T), APs and [Ca2+]i transients were prolonged in parallel. APs were abbreviated by nifedipine exposure and further lengthened upon releasing intracellularly stored Ca2+. Validating this model, control iPS-CM treated with HERG-blocking drugs recapitulated the LQT2 phenotype. In LQT3 iPS-CM, expressing NaV1.5-N406K, APs and [Ca2+]i transients were markedly prolonged. AP prolongation was sensitive to tetrodotoxin and to inhibiting Na+-Ca2+ exchange. These results suggest that LQTS mutations act partly on cytosolic Ca2+ cycling, potentially providing a basis for functionally targeted interventions regardless of the specific mutation site.

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

Rationale: 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 &bgr;-adrenergic stimulation. Objective: 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 (Ca2+). Methods and Results: 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 Ca2+. 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 Ca2+ concentrations, associated with a depletion of calstabin2. The luminal Ca2+ sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like. Conclusions: 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 Ca2+ dependence for activation under conditions that simulate exercise, consistent with a “leaky” channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca2+ activation. Thus, diastolic sarcoplasmic reticulum Ca2+ leak caused by reduced calstabin2 binding and a leftward shift in the Ca2+ dependence for activation by diastolic levels of cytosolic Ca2+ is a common mechanism underlying CPVT.

Plasma BIN1 correlates with heart failure and predicts arrhythmia in patients with arrhythmogenic right ventricular cardiomyopathy

BACKGROUND Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder involving diseased cardiac muscle. Bridging integrator 1 (BIN1) is a membrane-associated protein important to cardiomyocyte homeostasis and is downregulated in cardiomyopathy. We hypothesized that BIN1 could be released into the circulation and that blood-available BIN1 can provide useful data on the cardiac status of patients whose hearts are failing secondary to ARVC. OBJECTIVE To determine whether plasma BIN1 levels can be used to measure disease severity in patients with ARVC. METHODS We performed a retrospective cohort study of 24 patients with ARVC. Plasma BIN1 levels were assessed for their ability to correlate with cardiac functional status and predict ventricular arrhythmias. RESULTS Mean plasma BIN1 levels were decreased in patients with ARVC with heart failure (15 ± 7 vs 60 ± 17 in patients without heart failure, P <.05; the plasma BIN1 level was 60 ± 10 in non-ARVC normal controls). BIN1 levels correlated inversely with number of previous ventricular arrhythmia (R = -.47; P <.05), and low BIN1 levels correctly classified patients with advanced heart failure or ventricular arrhythmia (receiver operator curve area under the curve of 0.88 ± 0.07). Low BIN1 levels also predicted future ventricular arrhythmias (receiver operator curve area under the curve of 0.89 ± 0.09). In a stratified analysis, BIN1 levels could predict future arrhythmias in patients without severe heart failure (n = 20) with an accuracy of 82%. In the 7 patients with ARVC with serial blood samples, all of whom had evidence of disease progression during follow-up, plasma BIN1 levels decreased significantly (a decrease of 63%; P <.05). CONCLUSIONS Plasma BIN1 level seems to correlate with cardiac functional status and the presence or absence of sustained ventricular arrhythmias in a small cohort of patients with ARVC and can predict future ventricular arrhythmias.

Characterization and Mechanisms of Action of Novel NaV1.5 Channel Mutations Associated With Brugada Syndrome

Background—Brugada syndrome is a heterogeneous heart rhythm disorder characterized by an atypical right bundle block pattern with ST-segment elevation and T-wave inversion in the right precordial leads. Loss-of-function mutations in SCN5A encoding the cardiac sodium channel NaV1.5 are associated with Brugada syndrome. We found novel mutations in SCN5A in 2 different families diagnosed with Brugada syndrome and investigated how those affected NaV1.5 channel function. Methods and Results—We performed genetic testing of the probands’ genomic DNA. After site-directed mutagenesis and transfection, whole-cell currents were recorded for NaV1.5 wild type and mutants heterologously expressed in Chinese hamster ovary-K1 cells. Proband 1 had two novel NaV1.5 mutations: NaV1.5-R811H and NaV1.5-R620H. The NaV1.5-R811H mutation showed a significant loss of function in peak Na+ current density and alteration of biophysical kinetic parameters (inactivation and recovery from inactivation), whereas NaV1.5-R620H had no significant effect on the current. Proband 2 had a novel NaV1.5-S1218I mutation. NaV1.5-S1218I had complete loss of function, and 1:1 expression of NaV1.5-wild type and NaV1.5-S1218I mimicking the heterozygous state revealed a 50% reduction in current compared with wild type, suggesting a functional haploinsufficiency in the patient. Conclusions—NaV1.5-S1218I and R811H are novel loss-of-function mutations in the SCN5A gene causing Brugada syndrome.

Loss-of-Function KCNE2 Variants: True Monogenic Culprits of Long-QT Syndrome or Proarrhythmic Variants Requiring Secondary Provocation?

Background— Insight into type 6 long-QT syndrome (LQT6), stemming from mutations in the KCNE2-encoded voltage-gated channel &bgr;-subunit, is limited. We sought to further characterize its clinical phenotype. Methods and Results— Individuals with reported pathogenic KCNE2 mutations identified during arrhythmia evaluation were collected from inherited arrhythmia clinics and the Rochester long-QT syndrome (LQTS) registry. Previously reported LQT6 cases were identified through a search of the MEDLINE database. Clinical features were assessed, while reported KCNE2 mutations were evaluated for genotype–phenotype segregation and classified according to the contemporary American College of Medical Genetics guidelines. Twenty-seven probands possessed reported pathogenic KCNE2 mutations, while a MEDLINE search identified 17 additional LQT6 cases providing clinical and genetic data. Sixteen probands had normal resting QTc values and only developed QT prolongation and malignant arrhythmias after exposure to QT-prolonging stressors, 10 had other LQTS pathogenic mutations, and 10 did not have an LQTS phenotype. Although the remaining 8 subjects had an LQTS phenotype, evidence suggested that the KCNE2 variant was not the underlying culprit. The collective frequency of KCNE2 variants implicated in LQT6 in the Exome Aggregation Consortium database was 1.4%, in comparison with a 0.0005% estimated clinical prevalence for LQT6. Conclusions— On the basis of clinical phenotype, the high allelic frequencies of LQT6 mutations in the Exome Aggregation Consortium database, and absence of previous documentation of genotype–phenotype segregation, our findings suggest that many KCNE2 variants, and perhaps all, have been erroneously designated as LQTS-causative mutations. Instead, KCNE2 variants may confer proarrhythmic susceptibility when provoked by additional environmental/acquired or genetic factors, or both.

High incidence of functional ion-channel abnormalities in a consecutive Long QT cohort with novel missense genetic variants of unknown significance

The Long QT syndrome (LQTS) is a disorder characterized by a prolongation of the QT interval and a propensity to ventricular tachyarrhythmias, which may lead to syncope, cardiac arrest, or sudden death. Our objective was to (1) determine the incidence of variants with unknown significance (VUS) in a cohort of consecutive LQTS patients and (2) to determine the percentage of those with novel missense VUS that have demonstrable functional channel abnormalities from a single referral center. We performed genetic screening of candidate genes in 39 probands with a diagnosis of LQTS to identify mutations and variants. Seven variants of unknown significance were identified, six were missense variants and one was a splice site variant. We investigated the six novel missense VUS in five patients; three missense variants in KCNQ1 (L236R, W379R, Y522S) and three missense variants in KCNH2 (R35W, S620G, V491I). We employed two-electrode voltage-clamp experiments in Xenopus laevis oocytes and confocal imaging to characterize the novel missense mutations functionally. We revealed electrophysiological and trafficking loss-of-function phenotypes. This report emphasizes the frequency of adverse channel function in patients with LQTS and the importance of heterologous studies to define channel function.

Multiple genetic variations in sodium channel subunits in a case of sudden infant death syndrome

Dysfunction of NaV1.5 encoded by SCN5A accounts for approximately half of the channelopathic SIDS cases. We investigated the functional effect of two gene variants identified in the same patient, one in SCN5A and one in SCN1Bb. The aim of the study was to risk stratify the probands family.

Arrhythmogenic Right Ventricular Cardiomyopathy Caused by a Novel Frameshift Mutation

Arrhythmogenic right ventricular cardiomyopathy is a rare cardiomyopathy that might be asymptomatic or symptomatic, causing palpations or syncope, and might lead to sudden cardiac death. It is recommended that physical exertion be reduced. It is also recommended that those with syncope and ventricular tachycardia/ventricular fibrillation have an implantable cardioverter-defibrillator placed. β-Blockers, antiarrhythmic drugs, and radiofrequency ablation should be used to control the ventricular arrhythmia burden in arrhythmogenic right ventricular cardiomyopathy.