Rianne Wolswinkel

Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10−68; rs9388451, P = 5.1 × 10−17) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10−14). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10−81). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Intercalated disc abnormalities, reduced Na+ current density, and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes

AIMS Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC). However, the consequences of these mutations in early disease stages are unknown. We investigated whether mutation-induced intercalated disc remodelling impacts on electrophysiological properties before the onset of cell death and replacement fibrosis. METHODS AND RESULTS Transgenic mice with cardiac overexpression of mutant Desmoglein2 (Dsg2) Dsg2-N271S (Tg-NS/L) were studied before and after the onset of cell death and replacement fibrosis. Mice with cardiac overexpression of wild-type Dsg2 and wild-type mice served as controls. Assessment by electron microscopy established that intercellular space widening at the desmosomes/adherens junctions occurred in Tg-NS/L mice before the onset of necrosis and fibrosis. At this stage, epicardial mapping in Langendorff-perfused hearts demonstrated prolonged ventricular activation time, reduced longitudinal and transversal conduction velocities, and increased arrhythmia inducibility. A reduced action potential (AP) upstroke velocity due to a lower Na(+) current density was also observed at this stage of the disease. Furthermore, co-immunoprecipitation demonstrated an in vivo interaction between Dsg2 and the Na(+) channel protein Na(V)1.5. CONCLUSION Intercellular space widening at the level of the intercalated disc (desmosomes/adherens junctions) and a concomitant reduction in AP upstroke velocity as a consequence of lower Na(+) current density lead to slowed conduction and increased arrhythmia susceptibility at disease stages preceding the onset of necrosis and replacement fibrosis. The demonstration of an in vivo interaction between Dsg2 and Na(V)1.5 provides a molecular pathway for the observed electrical disturbances during the early ARVC stages.

Functional NaV1.8 Channels in Intracardiac Neurons The Link Between SCN10A and Cardiac Electrophysiology

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A/Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

PDZ Domain–Binding Motif Regulates Cardiomyocyte Compartment-Specific NaV1.5 Channel Expression and Function

Background— Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain–binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results— To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (&Dgr;SIV). &Dgr;SIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of &Dgr;SIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and &Dgr;SIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued &Dgr;SIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. Conclusions— Our results demonstrate the in vivo significance of the PDZ domain–binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Functional NaV1.8 Channels in Intracardiac NeuronsNovelty and Significance: The Link Between SCN10A and Cardiac Electrophysiology

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A/Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

A common co-morbidity modulates disease expression and treatment efficacy in inherited cardiac sodium channelopathy

Aims Management of patients with inherited cardiac ion channelopathy is hindered by variability in disease severity and sudden cardiac death (SCD) risk. Here, we investigated the modulatory role of hypertrophy on arrhythmia and SCD risk in sodium channelopathy. Methods and results Follow-up data was collected from 164 individuals positive for the SCN5A-1795insD founder mutation and 247 mutation-negative relatives. A total of 38 (obligate) mutation-positive patients died suddenly or suffered life-threatening ventricular arrhythmia. Of these, 18 were aged >40 years, a high proportion of which had a clinical diagnosis of hypertension and/or cardiac hypertrophy. While pacemaker implantation was highly protective in preventing bradycardia-related SCD in young mutation-positive patients, seven of them aged >40 experienced life-threatening arrhythmic events despite pacemaker treatment. Of these, six had a diagnosis of hypertension/hypertrophy, pointing to a modulatory role of this co-morbidity. Induction of hypertrophy in adult mice carrying the homologous mutation (Scn5a1798insD/+) caused SCD and excessive conduction disturbances, confirming a modulatory effect of hypertrophy in the setting of the mutation. The deleterious effects of the interaction between hypertrophy and the mutation were prevented by genetically impairing the pro-hypertrophic response and by pharmacological inhibition of the enhanced late sodium current associated with the mutation. Conclusion This study provides the first evidence for a modulatory effect of co-existing cardiac hypertrophy on arrhythmia risk and treatment efficacy in inherited sodium channelopathy. Our findings emphasize the need for continued assessment and rigorous treatment of this co-morbidity in SCN5A mutation-positive individuals.

Functional NaV1.8 Channels in Intracardiac Neurons

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A/Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

Delayed AV-conduction in pressure-overloaded Scn5a-1798insD+/- mice rescued by genetic deletion of the calcium-dependent transcription factor Nfatc2

Introduction: Transgenic Scn5a-1798insD+/- mice display atrio-ventricular conduction disturbances and develop susceptibility to arrhythmias with age. Isolated cardiomyocytes from Scn5a-1798insD+/- mouse hearts exhibit increased sustained inward sodium current which is associated with elevated intracellular sodium (Na+), intracellular diastolic calcium ([Ca2+]i) levels and Ca2+ transient amplitudes. We investigated whether the Scn5a-1798insD+/- mutation leads to activation of Ca2+-dependent calcineurin/Nfat signaling pathway, and assessed its impact on atrio-ventricular conduction. Results: Despite increased [Ca2+]i, activation of the calcineurin/Nfat pathway was not observed in Scn5a-1798insD+/- hearts at baseline (as evidenced by unaltered nuclear levels of Nfat protein as compared to wild type hearts). Wild-type and Scn5a-1798insD+/- mice displayed a similar hypertrophic response to 2 weeks of TAC (as evidenced by similar increases in heart weight and Anf mRNA levels), indicating equal activation of the calcineurin/Nfat pathway. However, Scn5a-1798insD+/–TAC mice displayed increased incidence of sudden death preceded by progressive bradycardia and AV-block. Furthermore, isolated Scn5a-1798insD+/–TAC hearts showed AV-block and significantly prolonged AV-delay as compared to wild-type-TAC hearts. Since Nfatc2 is a well-described downstream effector of the calcineurin pathway, we next investigated whether the observed electrical abnormalities after TAC could be rescued by crossing Scn5a-1798insD+/- with Nfatc2-KO mice. Scn5a-1798insD+/-/Nfatc2-KO mice showed a lower response to TAC as Scn5a-1798insD+/–TAC mice (as evidenced by lower increase in heart weight and Anf mRNA levels). The ex vivo AV-delay after TAC was attenuated in Scn5a-1798insD+/-/Nfatc2-KO TAC hearts, indicating rescue of the AV-conduction disturbances in Scn5a-1798insD+/–TAC mice by Nfatc2 deficiency. Moreover, His-ventricular conduction was unaffected by TAC, indicating that the increase in AV-delay in Scn5a-1798insD+/–TAC mice occurs proximal to the His bundle. Conclusions: Scn5a-1798insD+/- mice show intracellular Na+/Ca2+-dysregulation at baseline and severe AV-conduction abnormalities and sudden death upon increased activation of the calcineurin/Nfat-pathway by TAC challenge. Nfatc2 deficiency prevents these electrical disturbances secondary to TAC in Scn5a-1798insD+/-/Nfatc2-KO mice, indicating a role of the calcineurin/Nfat pathway on atrio-ventricular conduction.

Functional NaV1.8 Channels in Intracardiac NeuronsNovelty and Significance

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A/Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

The Selective Nav1.8 Sodium Channel Blocker A-803467 Affects Electrical Activity in Intracardiac Neurons, but not in Cardiomyocytes

Background. Recently we observed differential myocardial expression of the brain-type sodium channel isoform Scn10a between two inbred mouse strains, both harboring the Scn5a-1798insD+/- mutation and displaying different severity of conduction disease. The functional role of Scn10a in the heart is as yet unknown, and we therefore investigated expression and channel activity of NaV1.8 (encoded by Scn10a) in intracardiac neurons and myocardium of the murine heart.Methods. Immunocytochemistry was performed using anti-NaV1.8 antibody on mouse embryos and adult murine cardiac tissue sections. The effect of the NaV1.8 blocker A-803467 (500 nM) on action potentials (APs) and sodium current (INa) properties was assessed in isolated intracardiac neurons and ventricular myocytes.Results. In embryonic and adult heart tissue sections, NaV1.8 staining was observed at the epicardial surface, and within the myocardium in between cardiomyocytes. The NaV1.8 blocker A-803467 had no effect on either mean INa density or INa kinetic properties in isolated myocytes, but clearly reduced INa density in intracardiac neurons (−344±51 pA/pF versus control −448±61; mean±SEM, n=11). In addition, the slow component of the current decay (τslow) at −20 mV was accelerated in the presence of A-803467 (2.8±0.3 ms versus control 3.4±0.4 ms; mean±SEM, n=5) and V1/2 of voltage-dependent inactivation was shifted by −9.6 mV (−73.6±2.0 mV versus control −64.0±1.6 mV; mean±SEM, n=5). This is consistent with a reduction in slowly inactivating brain-type sodium current with depolarized voltage-dependent inactivation. In AP measurements A-803467 did not affect cardiomyocyte upstroke velocity, but reduced AP firing frequency in intracardiac neurons by 50%.Conclusion. The sodium channel NaV1.8 is expressed in murine heart, and is functionally present in intracardiac neurons, but absent in cardiomyocytes. Thus, NaV1.8 may influence myocardial electrophysiological properties through its contribution to cardiac neuronal activity.