Screening for acute IKr block is insufficient to detect torsades de pointes liability: role of late sodium current.

BACKGROUND: New drugs are routinely screened for IKr blocking properties thought to predict QT prolonging and arrhythmogenic liability. However, recent data suggest that chronic (hours) drug exposure to phosphoinositide 3-kinase inhibitors used in cancer can prolong QT by inhibiting potassium currents and increasing late sodium current (INa-L) in cardiomyocytes. We tested the extent to which IKr blockers with known QT liability generate arrhythmias through this pathway. METHODS AND RESULTS: Acute exposure to dofetilide, an IKr blocker without other recognized electropharmacologic actions, produced no change in ion currents or action potentials in adult mouse cardiomyocytes, which lack IKr. By contrast, 2 to 48 hours of exposure to the drug generated arrhythmogenic afterdepolarizations and ≥15-fold increases in INa-L. Including phosphatidylinositol 3,4,5-trisphosphate, a downstream effector for the phosphoinositide 3-kinase pathway, in the pipette inhibited these effects. INa-L was also increased, and inhibitable by phosphatidylinositol 3,4,5-trisphosphate, with hours of dofetilide exposure in human-induced pluripotent stem cell-derived cardiomyocytes and in Chinese hamster ovary cells transfected with SCN5A, encoding sodium current. Cardiomyocytes from dofetilide-treated mice similarly demonstrated increased INa-L and afterdepolarizations. Other agents with variable IKr-blocking potencies and arrhythmia liability produced a range of effects on INa-L, from marked increases (E-4031, d-sotalol, thioridazine, and erythromycin) to little or no effect (haloperidol, moxifloxacin, and verapamil). CONCLUSIONS: Some but not all drugs designated as arrhythmogenic IKr blockers can generate arrhythmias by augmenting INa-L through the phosphoinositide 3-kinase pathway. These data identify a potential mechanism for individual susceptibility to proarrhythmia and highlight the need for a new paradigm to screen drugs for QT prolonging and arrhythmogenic liability.

Blocking Scn10a channels in heart reduces late sodium current and is antiarrhythmic.

RATIONALE: Although the sodium channel locus SCN10A has been implicated by genome-wide association studies as a modulator of cardiac electrophysiology, the role of its gene product Nav1.8 as a modulator of cardiac ion currents is unknown. OBJECTIVE: We determined the electrophysiological and pharmacological properties of Nav1.8 in heterologous cell systems and assessed the antiarrhythmic effect of Nav1.8 block on isolated mouse and rabbit ventricular cardiomyocytes. METHODS AND RESULTS: We first demonstrated that Scn10a transcripts are identified in mouse heart and that the blocker A-803467 is highly specific for Nav1.8 current over that of Nav1.5, the canonical cardiac sodium channel encoded by SCN5A. We then showed that low concentrations of A-803467 selectively block "late" sodium current and shorten action potentials in mouse and rabbit cardiomyocytes. Exaggerated late sodium current is known to mediate arrhythmogenic early afterdepolarizations in heart, and these were similarly suppressed by low concentrations of A-803467. CONCLUSIONS: Scn10a expression contributes to late sodium current in heart and represents a new target for antiarrhythmic intervention.

Informatic and functional approaches to identifying a regulatory region for the cardiac sodium channel.

RATIONALE: Although multiple lines of evidence suggest that variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation. OBJECTIVE: We used in silico and in vitro experiments to identify possible noncoding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted. METHODS AND RESULTS: We identified 92 noncoding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved noncoding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as a potential regulatory region. Using recombinase-mediated cassette exchange (RMCE), we generated mice in which a 435-base pair region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, Na(V)1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2 ms in controls versus 9.7±0.2 ms in knockouts), indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3' segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6%±0.3% of baseline in CHO cells and 16%±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62%±4% and 57%±2% of baseline in CHO cells and myocytes, respectively (both P<0.05). CONCLUSIONS: These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a noncoding region underlying this regulation.

Striking In vivo phenotype of a disease-associated human SCN5A mutation producing minimal changes in vitro.

BACKGROUND: The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. METHODS AND RESULTS: We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of ß1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a dilated cardiomyopathy phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose-dependent increases in SCN5A mRNA abundance but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, 41.0±2.9 pA/pF at -30 mV; DN/H, 19.2±3.1 pA/pF, P<0.001 vs. H/H; DN/DN, 9.3±1.1 pA/pF, P<0.001 versus H/H). CONCLUSIONS: Although D1275N produces near-normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a dilated cardiomyopathy phenotype by reducing cardiac sodium current.

Voltage-gated sodium channels are required for heart development in zebrafish.

RATIONALE: Voltage-gated sodium channels initiate action potentials in excitable tissues. Mice in which Scn5A (the predominant sodium channel gene in heart) has been knocked out die early in development with cardiac malformations by mechanisms which have yet to be determined. OBJECTIVE: Here we addressed this question by investigating the role of cardiac sodium channels in zebrafish heart development. METHODS AND RESULTS: Transcripts of the functionally-conserved Scn5a homologs scn5Laa and scn5Lab were detected in the gastrulating zebrafish embryo and subsequently in the embryonic myocardium. Antisense knockdown of either channel resulted in marked cardiac chamber dysmorphogenesis and perturbed looping. These abnormalities were associated with decreased expression of the myocardial precursor genes nkx2.5, gata4, and hand2 in anterior lateral mesoderm and significant deficits in the production of cardiomyocyte progenitors. These early defects did not appear to result from altered membrane electrophysiology, as prolonged pharmacological blockade of sodium current failed to phenocopy channel knockdown. Moreover, embryos grown in calcium channel blocker-containing medium had hearts that did not beat but developed normally. CONCLUSIONS: These findings identify a novel and possibly nonelectrogenic role for cardiac sodium channels in heart development.

SCN5A variant R222Q generated abnormal changes in cardiac sodium current and action potentials in murine myocytes and Purkinje cells.

BACKGROUND: The cardiac sodium channel (SCN5A) mutation R222Q neutralizes a positive charge in the domain I voltage sensor. Mutation carriers display very frequent ectopy and dilated cardiomyopathy. OBJECTIVES: To describe the effect of SCN5A R222Q on murine myocyte and Purkinje fiber electrophysiology, and identify underlying mechanisms. METHODS: We generated mice carrying humanized wild-type (H) and mutant (RQ) SCN5A channels. We characterized whole-heart and isolated ventricular and Purkinje myocyte properties. RESULTS: RQ/RQ mice were not viable. INa from RQ/H ventricular myocytes displayed increased "window current" and hyperpolarizing shifts in both inactivation and activation compared to H/H, as previously reported in heterologous expression systems. Surprisingly, action potentials were markedly abbreviated in RQ/H myocytes (action potential durations at 90% repolarization: 12.6 ± 1.3 ms vs 29.1 ± 1.0 ms in H/H, P < .01, n = 10 each). We identified a large [K+]o-dependent outward gating pore current in RQ/H but not H/H myocytes, and decreasing [K+]o elicited early afterdepolarizations (EADs) and triggered activity in isolated myocytes and ectopic beats in whole hearts. Further, RQ/H Purkinje cells displayed striking, consistent low-voltage EADs. In vivo, however, RQ/H mice displayed little ectopy and contractile function was normal. CONCLUSION: While SCN5A R222Q increases plateau inward sodium current, action potentials were unexpectedly shortened, likely reflecting an outward gating-pore current. Low extracellular potassium increased this pore current, and was arrhythmogenic in vitro and ex vivo.

Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal targeted deletion of Alk3/Bmpr1a receptor.

BACKGROUND: The atrioventricular (AV) node is essential for the sequential excitation and optimized contraction of the adult multichambered heart; however, relatively little is known about its formation from the embryonic AV canal. A recent study demonstrated that signaling by Alk3, the type 1a receptor for bone morphogenetic proteins, in the myocardium of the AV canal was required for the development of both the AV valves and annulus fibrosus. To test the hypothesis that bone morphogenetic protein signaling also plays a role in AV node formation, we investigated conduction system function and AV node morphology in adult mice with conditional deletion of Alk3 in the AV canal. METHODS AND RESULTS: High-resolution optical mapping with correlative histological analysis of 28 mutant hearts revealed 4 basic phenotypic classes based on electrical activation patterns and volume-conducted ECGs. The frequency of AV node conduction and morphological abnormalities increased from no detectable anomalies (class I) to severe defects (class IV), which included the presence of bypass tracts, abnormal ventricular activation patterns, fibrosis of the AV node, and twin AV nodes. CONCLUSIONS: The present findings demonstrate that bone morphogenetic protein signaling is required in the myocardium of the AV canal for proper AV junction development, including the AV node.

Contrasting Nav1.8 Activity in Scn10a-/- Ventricular Myocytes and the Intact Heart.

BACKGROUND: Genome-wide association studies have implicated variants in SCN10A, which encodes Nav1.8, as modulators of cardiac conduction. Follow-up work has indicated the SCN10A sequence includes an intronic enhancer for SCN5A. Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this, we use homozygous knockout mice (Scn10a-/-) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact. METHODS AND RESULTS: We previously reported that pharmacologic blockade of Nav1.8 in wild-type animals blunts action potential prolongation by ATX-II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a-/- compared to wild-type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low-dose ATX-II exposure in lightly anesthetized animals and Langendorff-perfused hearts prolonged QTc and generated arrhythmias to the same extent in wild-type and Scn10a-/-. RNA sequencing failed to identify full-length Scn10a transcripts in wild-type or knockout isolated ventricular myocytes. However, loss of late current in Scn10a-/- myocytes was replicated independently in a blinded set of experiments. CONCLUSIONS: While Scn10a transcripts are not detectible in ventricular cardiomyocytes, gene deletion results in reproducible loss of late sodium current under extreme experimental conditions. However, there are no identifiable consequences of this Scn10a deletion in the intact mouse heart at usual rates. These findings argue that common variants in SCN10A that affect ventricular conduction do so by modulating SCN5A.

Increased late sodium current contributes to long QT-related arrhythmia susceptibility in female mice.

AIMS: Female gender is a risk factor for long QT-related arrhythmias, but the underlying mechanisms remain uncertain. Here, we tested the hypothesis that gender-dependent function of the post-depolarization 'late' sodium current (I(Na-L)) contributes. METHODS AND RESULTS: Studies were conducted in mice in which the canonical cardiac sodium channel Scn5a locus was disrupted, and expression of human wild-type SCN5A cDNA substituted. Baseline QT intervals were similar in male and female mice, but exposure to the sodium channel opener anemone toxin ATX-II elicited polymorphic ventricular tachycardia in 0/9 males vs. 6/9 females. Ventricular I(Na-L) and action potential durations were increased in myocytes isolated from female mice compared with those from males before and especially after treatment with ATX-II. Further, ATX-II elicited potentially arrhythmogenic early afterdepolarizations in myocytes from 0/5 male mice and 3/5 female mice. CONCLUSION: These data identify variable late I(Na) as a modulator of gender-dependent arrhythmia susceptibility.

Complex genomic rearrangement in CCS-LacZ transgenic mice.

The cardiac conduction system (CCS)-lacZ insertional mouse mutant strain genetically labels the developing and mature CCS. This pattern of expression is presumed to reflect the site of transgene integration rather than regulatory elements within the transgene proper. We sought to characterize the genomic structure of the integration locus and identify nearby gene(s) that might potentially confer the observed CCS-specific transcription. We found rearrangement of chromosome 7 between regions D1 and E1 with altered transcription of multiple genes in the D1 region. Several lines of evidence suggested that regulatory elements from at least one gene, Slco3A1, influenced CCS-restricted reporter gene expression. In embryonic hearts, Slco3A1 was expressed in a spatial pattern similar to the CCS-lacZ transgene and was similarly neuregulin-responsive. At later stages, however, expression patterns of the transgene and Slco3A1 diverged, suggesting that the Slco3A1 locus may be necessary, but not sufficient to confer CCS-specific transgene expression in the CCS-lacZ line.