Dina Myers Stroud

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.

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 &bgr;1 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.

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.

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. Conclusion— 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.

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.

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, NaV1.5 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.

Proliferation of embryonic cardiomyocytes in zebrafish requires the sodium channel scn5Lab

In mice, homozygous deletion of the cardiac sodium channel Scn5a results in defects in cardiac morphology and embryonic death before robust sodium current can be detected. In zebrafish, morpholino knockdown of cardiac sodium channel orthologs scn5Laa and scn5Lab perturbs specification of precardiac mesoderm and inhibits growth of the embryonic heart. It is not known which developmental processes are perturbed by sodium channel knockdown and whether reduced cell number is from impaired migration of cardiac progenitors into the heart, impaired myocyte proliferation, or both. We found that embryos deficient in scn5Lab displayed defects in primary cardiogenesis specific to loss of nkx2.5, but not nkx2.7. We generated kaede reporter fish and demonstrated that embryos treated with anti‐scn5Lab morpholino showed normal secondary differentiation of cardiomyocytes at the arterial pole between 30 and 48 h post‐fertilization. However, while proliferating myocytes were readily detected at 48 hpf in wild type embryos, there were no BrdU‐positive cardiomyocytes in embryos subjected to anti‐scn5Lab treatment. Proliferating myocytes were present in embryos injected with anti‐tnnt2 morpholino to phenocopy the silent heart mutation, and absent in embryos injected with anti‐tnnt2 and anti‐scn5Lab morpholinos, indicating cardiac contraction is not required for the loss of proliferation. These data demonstrate that the role of scn5Lab in later heart growth does not involve contribution of the secondary heart field, but rather proliferation of cardiomyocytes, and appears unrelated to the role of the channel in cardiac electrogenesis. genesis 51:562–574.

Response to Letters Regarding Article, “Abnormal Conduction and Morphology in the Atrioventricular Node of Mice with Atrioventricular Canal Targeted Deletion of Alk3/Bmpr1a Receptor”

We would like to thank Drs Gourdie and Sedmera for their comments about our recent study on the role of bone morphogenic protein (BMP) signaling in atrioventricular (AV) node function.1 We agree that the mechanism responsible for the “split” or “twin” AV nodes we observed in Class IV hearts is as yet unknown. Our data suggest that disruption of BMP signaling affects normal fibrotic deposition, but it was not our contention that this was the primary means of abnormal AV node morphology. In the Discussion section we presented 2 alternative mechanisms (outlined on pages 2541 to 2542), “… BMP signaling is required to maintain proper continuity between the AV node and …

Development of the Conduction System: Picking up the Pace

Abstract The cardiac conduction system initiates and maintains electrical activity that must pass through every cell of the heart to trigger contraction. How are these “specialized” cells differentiated from working cardiomyocytes? The question is simple, the story complex, and the answers remain elusive. Discrepancies between morphological studies and functional data leave genetics and molecular biology to fill the gaps in our understanding. In this chapter, we travel the path from the early gross anatomy discoveries to high‐resolution optical mapping studies, focusing on the latest work from both murine and avian systems. We speculate on the future direction of investigation into conduction system development and highlight the clinical significance of this work.