Yanfei Ruan

Sodium channel mutations and arrhythmias.

Since the identification of the first SCN5A mutation associated with long QT syndrome in 1995, several mutations in this gene for the α subunit of the cardiac sodium channel have been identified in a heterogeneous subset of cardiac rhythm syndromes, including Brugada syndrome, progressive cardiac conduction defect, sick sinus node syndrome, atrial fibrillation and dilated cardiomyopathy. Robust clinical evidence has been accompanied by bench studies performed in different models spanning from in vitro expression systems to transgenic mice. Together, these studies have helped establish genotype–phenotype correlations and have shaped our understanding of the role of the cardiac sodium channel in health and in disease. Remarkably, these advances in understanding have impacted on clinical management by allowing us to start developing gene-specific risk stratification schemes and mutation-specific management strategies. In this Review, we summarize the current understanding of the molecular mechanism of SCN5A-associated inherited arrhythmias, focusing on the most recent development of mutation-specific management in SCN5A-associated long QT syndrome type 3. We also briefly discuss arrhythmia-causing mutations in the genes encoding the β subunit of the cardiac sodium channel and in those encoding proteins in the associated macromolecular complex.

Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a highly malignant form of arrhythmogenic disorder characterized by exercise- or emotional-induced polymorphic ventricular tachycardia in the absence of detectable structural heart disease. Because of the typical pattern of arrhythmias (bidirectional ventricular tachycardia and the occurrence and severity of arrhythmia correlated well with exercise workload) during exercise stress test, CPVT can be identified promptly. Molecular genetic screening of the genes encoding the cardiac ryanodine receptor and calsequestrin is critical to confirm uncertain diagnosis of CPVT. With the exception of beta-blockers, no pharmacologic therapy of proven effectiveness is available: although beta-blockers reduce the occurrence of ventricular tachycardia, 30% of patients treated with beta-blockers still experience cardiac arrhythmias and eventually require implantable cardioverter defibrillator implantation to prevent cardiac arrest.

Gating properties of SCN5A mutations and the response to mexiletine in long-QT syndrome type 3 patients

Background— Mexiletine (Mex) has been proposed as a gene-specific therapy for patients with long-QT syndrome type 3 (LQT3) caused by mutations in the cardiac sodium channel gene (SCN5A). The degree of QT shortening and the protection from arrhythmias vary among patients harboring different mutations. We tested whether the clinical response to Mex in LQT3 could be predicted by the biophysical properties of the different mutations. Methods and Results— We identified 4 SCN5A mutations in 5 symptomatic LQT3 patients with different responses to Mex (6 to 8 mg · kg−1 · d−1). We classified the mutations as sensitive to Mex (P1332L, R1626P; ≥10% of QTc shortening and QTc <500 ms or no arrhythmias) or insensitive to Mex (S941N, M1652R; negligible or no QTc shortening and sudden death). We measured Na+ current from HEK 293 cells transfected with wild-type (WT) or mutant Nav1.5. All mutations showed impaired inactivation of Na+ current, but the mutations identified in patient responders to Mex (P1332L, R1626P) showed a hyperpolarizing shift of V1/2 of steady-state inactivation. Furthermore, Mex produced use-dependent block with the order R1626P=P1332L>S941N=WT>M1652R, suggesting that Mex-sensitive mutants present prolonged recovery from Mex block. Conclusions— We propose that voltage dependence of channel availability and shifts of V1/2 of steady-state inactivation correlate with the clinical response observed in LQT3 patients. This supports the view that the response to Mex is mutation specific and that in vitro testing may help to predict the response to therapy in LQT3.

Short Communication: Flecainide Exerts an Antiarrhythmic Effect in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia by Increasing the Threshold for Triggered Activity

Rationale: Flecainide prevents arrhythmias in catecholaminergic polymorphic ventricular tachycardia, but the antiarrhythmic mechanism remains unresolved. It is possible for flecainide to directly affect the cardiac ryanodine receptor (RyR2); however, an extracellular site of action is suggested because of the hydrophilic nature of flecainide. Objective: To investigate the mechanism for the antiarrhythmic action of flecainide in a RyR2R4496C+/− knock-in mouse model of catecholaminergic polymorphic ventricular tachycardia. Methods and Results: Flecainide prevented catecholamine-induced sustained ventricular tachycardia in RyR2R4496C+/− mice. Cellular studies were performed with isolated RyR2R4496C+/− myocytes. Isoproterenol caused the appearance of spontaneous Ca2+ transients, which were unaffected by flecainide (6 &mgr;mol/L). Flecainide did not affect Ca2+ transient amplitude, decay, or sarcoplasmic reticulum Ca2+ content. Moreover, it did not affect the frequency of spontaneous Ca2+ sparks in permeabilized myocytes. In contrast, flecainide effectively prevented triggered activity induced by isoproterenol. The threshold for action potential induction was increased significantly (P<0.01), which suggests a primary extracellular antiarrhythmic effect mediated by Na+ channel blockade. Conclusions: Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in RyR2R4496C+/− mice; however, at variance with previous reports, we observed minimal effects on intracellular Ca2+ homeostasis. Our data suggest that the antiarrhythmic activity of the drug is caused by reduction of Na+ channel availability and by an increase in the threshold for triggered activity.

Calmodulin kinase II inhibition prevents arrhythmias in RyR2R4496C+/− mice with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by life-threatening arrhythmias elicited by adrenergic activation. CPVT is caused by mutations in the cardiac ryanodine receptor gene (RyR2). In vitro studies demonstrated that RyR2 mutations respond to sympathetic activation with an abnormal diastolic Ca(2+) leak from the sarcoplasmic reticulum; however the pathways that mediate the response to adrenergic stimulation have not been defined. In our RyR2(R4496C+/-) knock-in mouse model of CPVT we tested the hypothesis that inhibition of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) counteracts the effects of adrenergic stimulation resulting in an antiarrhythmic activity. CaMKII inhibition with KN-93 completely prevented catecholamine-induced sustained ventricular tachyarrhythmia in RyR2(R4496C+/-) mice, while the inactive congener KN-92 had no effect. In ventricular myocytes isolated from the hearts of RyR2(R4496C+/-) mice, CaMKII inhibition with an autocamtide-2 related inhibitory peptide or with KN-93 blunted triggered activity and transient inward currents induced by isoproterenol. Isoproterenol also enhanced the activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), increased spontaneous Ca(2+) release and spark frequency. CaMKII inhibition blunted each of these parameters without having an effect on the SR Ca(2+) content. Our data therefore indicate that CaMKII inhibition is an effective intervention to prevent arrhythmogenesis (both in vivo and in vitro) in the RyR2(R4496C+/-) knock-in mouse model of CPVT. Mechanistically, CAMKII inhibition acts on several elements of the EC coupling cascade, including an attenuation of SR Ca(2+) leak and blunting catecholamine-mediated SERCA activation. CaMKII inhibition may therefore represent a novel therapeutic target for patients with CPVT.

KCNJ2 mutation in short QT syndrome 3 results in atrial fibrillation and ventricular proarrhythmia

We describe a mutation (E299V) in KCNJ2, the gene that encodes the strong inward rectifier K+ channel protein (Kir2.1), in an 11-y-old boy. The unique short QT syndrome type-3 phenotype is associated with an extremely abbreviated QT interval (200 ms) on ECG and paroxysmal atrial fibrillation. Genetic screening identified an A896T substitution in a highly conserved region of KCNJ2 that resulted in a de novo mutation E299V. Whole-cell patch-clamp experiments showed that E299V presents an abnormally large outward IK1 at potentials above −55 mV (P < 0.001 versus wild type) due to a lack of inward rectification. Coexpression of wild-type and mutant channels to mimic the heterozygous condition still resulted in a large outward current. Coimmunoprecipitation and kinetic analysis showed that E299V and wild-type isoforms may heteromerize and that their interaction impairs function. The homomeric assembly of E299V mutant proteins actually results in gain of function. Computer simulations of ventricular excitation and propagation using both the homozygous and heterozygous conditions at three different levels of integration (single cell, 2D, and 3D) accurately reproduced the electrocardiographic phenotype of the proband, including an exceedingly short QT interval with merging of the QRS and the T wave, absence of ST segment, and peaked T waves. Numerical experiments predict that, in addition to the short QT interval, absence of inward rectification in the E299V mutation should result in atrial fibrillation. In addition, as predicted by simulations using a geometrically accurate three-dimensional ventricular model that included the His–Purkinje network, a slight reduction in ventricular excitability via 20% reduction of the sodium current should increase vulnerability to life-threatening ventricular tachyarrhythmia.

Trafficking Defects and Gating Abnormalities of a Novel SCN5A Mutation Question Gene-Specific Therapy in Long QT Syndrome Type 3

Rationale: Sodium channel blockers are used as gene-specific treatments in long-QT syndrome type 3, which is caused by mutations in the sodium channel gene (SCN5A). Response to treatment is influenced by biophysical properties of mutations. Objective: We sought to investigate the unexpected deleterious effect of mexiletine in a mutation combining gain-of- function and trafficking abnormalities. Methods and Results: A long-QT syndrome type 3 child experienced paradoxical QT prolongation and worsening of arrhythmias after mexiletine treatment. The SCN5A mutation F1473S expressed in HEK293 cells presented a right-ward shift of steady-state inactivation, enlarged window current, and huge sustained sodium current. Unexpectedly, it also reduced the peak sodium current by 80%. Immunostaining showed that mutant Nav1.5 is retained in the cytoplasm. Incubation with 10 &mgr;mol/L mexiletine rescued the trafficking defect of F1473S, causing a significant increase in peak current, whereas sustained current was unchanged. Using a Markovian model of the Na channel and a model of human ventricular action potential, we showed that simulated exposure of F1473S to mexiletine paradoxically increased action potential duration, mimicking QT prolongation seen in the index patient on mexiletine treatment. Conclusions: Sodium channel blockers are largely used to shorten QT intervals in carriers of SCN5A mutations. We provided evidence that these agents may facilitate trafficking of mutant proteins, thus exacerbating QT prolongation. These data suggest that caution should be used when recommending this class of drugs to carriers of mutations with undefined electrophysiological properties.

Therapeutic Strategies for Long-QT Syndrome: Does the Molecular Substrate Matter?

The discovery of genetic defects underlying long-QT syndrome (LQTS) has allowed to identify important genotype-phenotype correlations that are now being used for risk stratification. The next challenge is to exploit the new information on the pathophysiology of the disease derived from molecular genetics to devise more effective therapies. The successful response of LQT1 patients to β-blockers, the QT-shortening action of sodium channel blockers in at least some LQT3 patients, and the importance of maintaining adequate plasma potassium levels in LQT2 patients clearly demonstrate the importance of selecting therapy in the context of the molecular substrate. The hurdles on the road toward the development of novel therapeutic strategies are represented by the variable expressivity of the disease, the high prevalence of “private” mutations, and the role of genetic and nongenetic factors that act as modifiers of the phenotype. Given the large number of mutations identified and their phenotypic complexity, it is clearly impossible to anticipate a scenario in which each mutation will be managed through a specific therapy. However, the evidence that mutations in the LQTS gene may be clustered based on their electrophysiological profile and on their response to specific drugs may provide the rationale for the development of mutation-specific therapies. In this article, we will review the most relevant genotype-phenotype features of LQTS and the strategies explored to develop novel therapeutic approaches. ### Definition and Prevalence The congenital LQTS is an inherited arrhythmogenic disease characterized by abnormally prolonged QT interval leading to life-threatening arrhythmias in the presence of a structurally normal heart.1 Different clinical variants of LQTS have been identified. Romano Ward syndrome, transmitted as an autosomal dominant trait, is the most common form of LQTS and presents only a cardiac phenotype. Less prevalent variants of LQTS, such as Andersen and Timothy syndromes or the recessive Jervell and Lange-Nielsen syndromes, also present …

Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

Short QT3 syndrome (SQT3S) is a cardiac disorder characterized by a high risk of mortality and associated with mutations in Kir2.1 (KCNJ2) channels. The molecular mechanisms leading to channel dysfunction, cardiac rhythm disturbances and neurodevelopmental disorders, potentially associated with SQT3S, remain incompletely understood. Here, we report on monozygotic twins displaying a short QT interval on electrocardiogram recordings and autism–epilepsy phenotype. Genetic screening identified a novel KCNJ2 variant in Kir2.1 that (i) enhanced the channels surface expression and stability at the plasma membrane, (ii) reduced protein ubiquitylation and degradation, (iii) altered protein compartmentalization in lipid rafts by targeting more channels to cholesterol-poor domains and (iv) reduced interactions with caveolin 2. Importantly, our study reveals novel physiological mechanisms concerning wild-type Kir2.1 channel processing by the cell, such as binding to both caveolin 1 and 2, protein degradation through the ubiquitin–proteasome pathway; in addition, it uncovers a potential multifunctional site that controls Kir2.1 surface expression, protein half-life and partitioning to lipid rafts. The reported mechanisms emerge as crucial also for proper astrocyte function, suggesting the need for a neuropsychiatric evaluation in patients with SQT3S and offering new opportunities for disease management.

L-Type Calcium Channel Disease

In recent years, the progress of molecular genetics of inherited arrhythmogenic diseases portrays an unexpected complexity of clinical phenotypes associated with mutations in several genes that control cardiac excitability. Among the most recent findings, the voltage-gated L-type cardiac calcium channel (Cav1.2) has been involved in the pathogenesis of Timothy syndrome (TS). TS is a variant of the long QT syndrome (also LQT8) and it is a rare and severe genetic disorder characterized by a spectrum of complex phenotypes including QT interval prolongation, congenital heart defects, syndactyly, and distinctive dysmorphic features. So far TS is the only inherited arrhythmogenic disorder linked to cardiac calcium channel mutations. In this chapter, we will briefly review the structure, physiology, and pathophysiology of the cardiac Cav1.2 encoded by the CACNA1c gene.