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Background: Mutations in RBM20 (RNA-binding motif protein 20) cause a clinically aggressive form of dilated cardiomyopathy, with an increased risk of malignant ventricular arrhythmias. RBM20 is a splicing factor that targets multiple pivotal cardiac genes, such as Titin (TTN) and CAMK2D (calcium/calmodulin-dependent kinase II delta). Aberrant TTN splicing is thought to be the main determinant of RBM20-induced dilated cardiomyopathy, but is not likely to explain the increased risk of arrhythmias. Here, we investigated the extent to which RBM20 mutation carriers have an increased risk of arrhythmias and explore the underlying molecular mechanism. Methods: We compared clinical characteristics of RBM20 and TTN mutation carriers and used our previously generated Rbm20 knockout (KO) mice to investigate downstream effects of Rbm20-dependent splicing. Cellular electrophysiology and Ca 2+ measurements were performed on isolated cardiomyocytes from Rbm20 KO mice to determine the intracellular consequences of reduced Rbm20 levels. Results: Sustained ventricular arrhythmias were more frequent in human RBM20 mutation carriers than in TTN mutation carriers (44% versus 5%, respectively, P =0.006). Splicing events that affected Ca 2+ - and ion-handling genes were enriched in Rbm20 KO mice, most notably in the genes CamkIIδ and RyR2. Aberrant splicing of CamkIIδ in Rbm20 KO mice resulted in a remarkable shift of CamkIIδ toward the δ-A isoform that is known to activate the L-type Ca 2+ current ( I Ca,L ). In line with this, we found an increased I Ca,L , intracellular Ca 2+ overload and increased sarcoplasmic reticulum Ca 2+ content in Rbm20 KO myocytes. In addition, not only complete loss of Rbm20, but also heterozygous loss of Rbm20 increased spontaneous sarcoplasmic reticulum Ca 2+ releases, which could be attenuated by treatment with the I Ca,L antagonist verapamil. Conclusions: We show that loss of Rbm20 disturbs Ca 2+ handling and leads to more proarrhythmic Ca 2+ releases from the sarcoplasmic reticulum. Patients that carry a pathogenic RBM20 mutation have more ventricular arrhythmias despite a similar left ventricular function, in comparison with patients with a TTN mutation. Our experimental data suggest that RBM20 mutation carriers may benefit from treatment with an I Ca,L blocker to reduce their arrhythmia burden.

Inheritable cardiac disorders, which may be associated with cardiomyopathic changes, are often associated with increased risk of sudden death in the young. Early linkage analysis studies in Mendelian forms of these diseases, such as hypertrophic cardiomyopathy and long-QT syndrome, uncovered large-effect genetic variants that contribute to the phenotype. In more recent years, through genotype-phenotype studies and methodological advances in genetics, it has become evident that most inheritable cardiac disorders are not monogenic but, rather, have a complex genetic basis wherein multiple genetic variants contribute (oligogenic or polygenic inheritance). Conversely, studies on genes underlying these disorders uncovered pleiotropic effects, with a single gene affecting multiple and apparently unrelated phenotypes. In this review, we explore these 2 phenomena: on the one hand, the evidence that variants in multiple genes converge to generate one clinical phenotype, and, on the other, the evidence that variants in one gene can lead to apparently unrelated phenotypes. Although multiple conditions are addressed to illustrate these concepts, the experience obtained in the study of long-QT syndrome, Brugada syndrome, and arrhythmogenic cardiomyopathy, and in the study of functions related to SCN5A (the gene coding for the α-subunit of the most abundant sodium channel in the heart) and PKP2 (the gene coding for the desmosomal protein plakophilin-2), as well, is discussed in more detail.

Background— Sodium channel Na V 1.5 underlies cardiac excitability and conduction. The last 3 residues of Na V 1.5 (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 Na V 1.5 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 (ΔSIV). ΔSIV mice displayed reduced Na V 1.5 expression and sodium current ( I Na ), specifically at the lateral myocyte membrane, whereas Na V 1.5 expression and I Na at the intercalated disks were unaffected. Optical mapping of ΔSIV 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 ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV I Na , suggesting that the SIV motif is important for regulation of Na V 1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased Na V 1.5 cell surface expression and I Na when expressed in HEK293 cells. Conclusions— Our results demonstrate the in vivo significance of the PDZ domain–binding motif in the correct expression of Na V 1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral Na V 1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Background— Patients carrying the cardiac sodium channel ( SCN5A ) mutation 1795insD show sudden nocturnal death and signs of multiple arrhythmia syndromes including bradycardia, conduction delay, QT prolongation, and right precordial ST-elevation. We investigated the electrophysiological characteristics of a transgenic model of the murine equivalent mutation 1798insD. Methods and Results— On 24-hour continuous telemetry and surface ECG recordings, Scn5a 1798insD/+ heterozygous mice showed significantly lower heart rates, more bradycardic episodes (pauses ≥500 ms), and increased PQ interval, QRS duration, and QTc interval compared with wild-type mice. The sodium channel blocker flecainide induced marked sinus bradycardia and/or sinus arrest in the majority of Scn5a 1798insD/+ mice, but not in wild-type mice. Epicardial mapping using a multielectrode grid on excised, Langendorff-perfused hearts showed preferential conduction slowing in the right ventricle of Scn5a 1798insD/+ hearts. On whole-cell patch-clamp analysis, ventricular myocytes isolated from Scn5a 1798insD/+ hearts displayed action potential prolongation, a 39% reduction in peak sodium current density and a similar reduction in action potential upstroke velocity. Scn5a 1798insD/+ myocytes displayed a slower time course of sodium current decay without significant differences in voltage-dependence of activation and steady-state inactivation, slow inactivation, or recovery from inactivation. Furthermore, Scn5a 1798insD/+ myocytes showed a larger tetrodotoxin-sensitive persistent inward current compared with wild-type myocytes. Conclusions— Mice carrying the murine equivalent of the SCN5A -1795insD mutation display bradycardia, right ventricular conduction slowing, and QT prolongation, similar to the human phenotype. These results demonstrate that the presence of a single SCN5A mutation is indeed sufficient to cause an overlap syndrome of cardiac sodium channel disease.

Background and Aim: Selective inhibition of cardiac late sodium current (I Na,L ) is an emerging target in the treatment of ventricular arrhythmias. The electrophysiological effects of GS967, a potent I Na,L inhibitor, were investigated in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from human SCN5A -1795insD induced pluripotent stem cells (iPSC-CMs) and mice carrying the homologous mutation Scn5a -1798insD. Methods and Results: On patch-clamp analysis, isolated mouse Scn5a -1798insD cardiomyocytes and human SCN5A -1795insD iPSC-CMs showed decreased peak I Na and action potential (AP) upstroke velocity (Vmax) and increased I Na,L and AP duration at 90% repolarization (APD 90 ) as compared to wild-type. GS967 (50-300 nM) significantly decreased APD 90 in mouse Scn5a -1798insD cardiomyocytes by 8±2% (mean±SEM) at 50 nM (n=7), 13±3% at 100 nM (n=11) and 20±5% at 300 nM (n=6) (all p NaL in mouse Scn5a -1798insD cardiomyocytes (GS967-sensitive current of 0.7±0.1 pA/pF, n=6), but had no effect on peak I Na . Furthermore, GS967 (100 nM) suppressed fast (5 Hz) pacing-induced afterpotentials and triggered activity. In human SCN5A -1795insD iPSC-CMs (n=6), GS967 (300 nM) significantly reduced APD 90 without affecting the resting membrane potential or Vmax. In Langendorff-perfused, isolated mouse Scn5a -1798insD hearts (n=5), GS967 (300 nM) had no effect on ventricular activation time or conduction velocity (as assessed by epicardial mapping). Conclusion: Selective inhibition of I NaL by GS967 attenuated AP prolongation and prevented pro-arrhythmic activity in mouse Scn5a -1798insD cardiomyocytes and human SCN5A-1795insD iPSC-CMs, thus suppressing the gain-of-function features of this overlap syndrome mutation. Importantly, these beneficial actions of GS967 occurred in the absence of deleterious effects on sodium channel availability or cardiac conduction, despite a pre-existing decrease in peak I Na . Thus, selective inhibition of I Na,L constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced I NaL , even in overlap syndromes whereby peak I Na is decreased.

Introduction: The mechanism of enhanced atrioventricular nodal conduction (EAVNC), including Lown-Ganong-Levine (LGL) syndrome, is not well described. Transgenic mice with the over-expression SCN5A results in a shorter P-R interval; hence, we tested the hypothesis that overexpression of SCN5A may provide an animal model mimicking EAVNC. Methods: Electrocardiogram (ECG), atrial, His-bundle and ventricular electrograms were recorded from wild type (WT) and transgenic mice over-expressing SCN5A (TG). Peak Na + current (I Na ) and Nav1.5 expression were measured using patch-clamp technique and immunohistochemistry method, respectively. Results: P-R interval in TG was much shorter than in WT mice (13.6±1.20 vs. 40.2±0.59 ms, n=20 and 35). In TG isolated hearts the A-V conduction during right atrial pacing remains shorter than that of WT isolated hearts (14.4±0.81 vs. 39.5±0.62 ms, n=28 each). His-bundle electrogram (HBE) revealed a short A-H and H-V intervals in TG, compared to WT mice (Figure 1A-D). In addition, TG mice had a short Wenckebach cycle length (CL) and atrioventricular effective refractory period (AVERP). Peak I Na was 2-fold greater in TG compared to WT ventricular myocytes (198.2±16.22 vs. 89.6±5.35 pA/pF, n=12). Flecainide (6 μM, n=6) led to prolongation of A-V conduction time in TG to a value close to that of WT hearts. Nifedipine (1 μM, n=) prolonged A-H interval in WT but not in TG hearts. Immunohistochemistry studies revealed increased Nav1.5 labeling in TG atrial and ventricular tissue, and Nav1.5 expression in A-V junction and A-V ring regions of TG compared to WT hearts. Conclusion: SCN5A over expression in TG mice enhanced the A-V conduction mimicking EAVNC. Thus, higher level of sodium channel expression in the A-V nodal region may be an electrophysiological substrate responsible for the syndrome of EAVNC. Figure 1