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Atrial Fibrillation (AF) is the most common cardiac arrhythmia. Its pathogenesis is complex and poorly understood. Whole exome sequencing of Danish families with AF revealed a novel four nucleotide deletion c.1041_1044del in CLCN2 shared by affected individuals. We aimed to investigate the role of genetic variation of CLCN2 encoding the inwardly rectifying chloride channel ClC-2 as a risk factor for the development of familiar AF. The effect of the CLCN2 variant was evaluated by electrophysiological recordings on transiently transfected cells. We used quantitative PCR to assess CLCN2 mRNA expression levels in human atrial and ventricular tissue samples. The nucleotide deletion CLCN2 c.1041_1044del results in a frame-shift and premature stop codon. The truncated ClC-2 p.V347fs channel does not conduct current. Co-expression with wild-type ClC-2, imitating the heterozygote state of the patients, resulted in a 50% reduction in macroscopic current, suggesting an inability of truncated ClC-2 protein to form channel complexes with wild type channel subunits. Quantitative PCR experiments using human heart tissue from healthy donors demonstrated that CLCN2 is expressed across all four heart chambers. Our genetic and functional data points to a possible link between loss of ClC-2 function and an increased risk of developing AF.

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K+ current ( Ito,f) and with CaV1.2 to mediate the inward L-type Ca2+ current ( ICa,L). Anesthetized KChIP2−/− mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ2 protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca2+ entry into the cell, thereby augmenting Ca2+-induced Ca2+ release. Ca2+ transients in disaggregated KChIP2−/− cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca2+-induced Ca2+ release event. We next functionally probed dyad structure, ryanodine receptor Ca2+ sensitivity, and sarcoplasmic reticulum Ca2+ load and found that increased temporal synchronicity of the Ca2+ release in KChIP2−/− cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2−/− mice.

Background Dysfunction of NaV 1.5 encoded by SCN5A accounts for approximately half of the channelopathic SIDS cases. We investigated the functional effect of two gene variants identified in the same patient, one in SCN5A and one in SCN1Bb. The aim of the study was to risk stratify the proband's family. Methods The family was referred for cardiovascular genetic evaluation to assess familial risk of cardiac disease. Functional analysis of the identified variants was performed with patch-clamp electrophysiology in HEK293 cells. Results A 16-month-old healthy boy died suddenly in the context of nonspecific illness and possible fever. Postmortem genetic testing revealed variants in the SCN5A and SCN1Bb genes. The proband's father carries the same variants but is asymptomatic. Electrophysiological analysis of the NaV 1.5_1281X truncation revealed complete loss-of-function of the channel. Coexpression of NaV 1.5 with NaV β1b significantly increased INa density when compared to NaV 1.5 alone. The NaV β1b _V268I variant abolished this INa density increase. Moreover, it shifted the activation curve toward more depolarized potentials. Conclusions Genetic variation of both sodium channel and its modifiers may contribute to sudden unexplained death in childhood. However, the asymptomatic father suggests that genetic variation of these genes is not sufficient to cause sudden death or clinically detectable SCN5A phenotypes.

Stem cell based replacement therapy is envisioned as a method to repair failing hearts suffering from cardiomyocyte loss. To prevent potentially lethal arrhythmias, the donor cellular electrophysiological make-up should match with the acceptor tissue. To engineer the desired electrophysiological phenotype, the underlying molecular regulation of ion channels and gap-junction proteins should be clarified first. We established the expression of seven main cardiac ion channel a-subunits and four b-subunits using semiquantitive RT-PCR and two major cardiac gap-junction proteins by immunohistochemistry during the differentiation process of murine ES cells into cardiomyocytes. Ion channel mRNA expression profiles display sequential upregulation. Connexin-40 and -43 expression is low in early cardiomyocytes, increased expression rates were found in subsequent differentiation phases. Cardiac differentiation of mouse embryonic stem cells is characterized by a sequential upregulation of the main cardiac ion channels and connexins

Aim The voltage-gated potassium channel KV11.1 is the molecular basis for the IKr current which plays an important role in cardiac physiology. Its malfunction is associated with both inherited and acquired cardiac arrhythmias. Native currents differ from those in experimental models, suggesting additional regulatory mechanisms. We hypothesised that the post-translational modification sumoylation finetunes channel activity. Methods The functional effects of sumoylation on KV11.1 were addressed by employing two-electrode voltage-clamp (TEVC) experiments in Xenopus laevis oocytes. Site-directed mutagenesis enabled a further analyse of the SUMO-target amino acids. We assessed protein expression levels and used confocal imaging for localization studies. Results Co-expression with Ubc9 and SUMO alters the electrophysiological properties of KV11.1 leading to a decrease in steady-state current amplitude largely due to faster inactivation and alteration of deactivation kinetics. We identified three lysines (K21, K93, and K116) in the PAS-domain as the putative SUMO-targets. Conclusion This study indicates KV11.1 as a sumoylation target, and offers the three main targets; K21, K93, and K116. Furthermore, it proposes an underlying mechanism for the observed kinetic impact of the PAS domain. This article is protected by copyright. All rights reserved.

Background— Brugada syndrome is a heterogeneous heart rhythm disorder characterized by an atypical right bundle block pattern with ST-segment elevation and T-wave inversion in the right precordial leads. Loss-of-function mutations in SCN5A encoding the cardiac sodium channel Na V 1.5 are associated with Brugada syndrome. We found novel mutations in SCN5A in 2 different families diagnosed with Brugada syndrome and investigated how those affected Na V 1.5 channel function. Methods and Results— We performed genetic testing of the probands’ genomic DNA. After site-directed mutagenesis and transfection, whole-cell currents were recorded for Na V 1.5 wild type and mutants heterologously expressed in Chinese hamster ovary-K1 cells. Proband 1 had two novel Na V 1.5 mutations: Na V 1.5-R811H and Na V 1.5-R620H. The Na V 1.5-R811H mutation showed a significant loss of function in peak Na + current density and alteration of biophysical kinetic parameters (inactivation and recovery from inactivation), whereas Na V 1.5-R620H had no significant effect on the current. Proband 2 had a novel Na V 1.5-S1218I mutation. Na V 1.5-S1218I had complete loss of function, and 1:1 expression of Na V 1.5-wild type and Na V 1.5-S1218I mimicking the heterozygous state revealed a 50% reduction in current compared with wild type, suggesting a functional haploinsufficiency in the patient. Conclusions— Na V 1.5-S1218I and R811H are novel loss-of-function mutations in the SCN5A gene causing Brugada syndrome.

Lateralization of the ventricular gap junction protein connexin 43 (Cx43) occurs in epicardial border zone myocytes following myocardial infarction (MI) and is arrhythmogenic. Alterations in Cx43 protein partners have been hypothesized to play a role in lateralization although mechanisms by which this occurs are unknown. To examine potential mechanisms we did nuclear magnetic resonance, yeast 2-hybrid, and surface plasmon resonance studies and found that the SH3 domain of the tyrosine kinase c-Src binds to the Cx43 scaffolding protein zonula occludens-1 (ZO-1) with a higher affinity than does Cx43. This suggests c-Src outcompetes Cx43 for binding to ZO-1, thus acting as a chaperone for ZO-1 and causing unhooking from Cx43. To determine whether c-Src/ZO-1 interactions affect Cx43 lateralization within the epicardial border zone, we performed Western blot, immunoprecipitation, and immunolocalization for active c-Src (p-cSrc) post-MI using a canine model of coronary occlusion. We found that post-MI p-cSrc interacts with ZO-1 as Cx43 begins to decrease its interaction with ZO-1 and undergo initial loss of intercalated disk localization. This indicates that the molecular mechanisms by which Cx43 is lost from the intercalated disk following MI includes an interaction of p-cSrc with ZO-1 and subsequent loss of scaffolding of Cx43 leaving Cx43 free to diffuse in myocyte membranes from areas of high Cx43, as at the intercalated disk, to regions of lower Cx43 content, the lateral myocyte membrane. Therefore shifts in Cx43 protein partners may underlie, in part, arrhythmogenesis in the post-MI heart.

INTRODUCTION: The Brugada syndrome (BrS) is characterized by an atypical right bundle block pattern with ST segment elevation and T wave inversion in the right precordial leads. The disease transmission is autosomal dominant with incomplete penetrance. We found 3 novel mutations in SCN5A in two independent families diagnosed with BrS.METHODS: We performed genetic testing of the probands’ genomic DNA. After site-directed mutagenesis and transfection, whole-cell currents were recorded by voltage clamp for NaV1.5 WT and mutants heterologously expressed in CHO-K1 cells.RESULTS: Proband 1, a 44 male with history of recurrent episodes of syncope was found to have two novel NaV1.5 mutations; NaV1.5-R811H and NaV1.5-R620H. The NaV1.5-R811H mutation showed a significant loss-of-function in peak Na+ current density. In addition, a negative shift in steady-state inactivation as well as a slowing of Ina recovery was observed for R811H. NaV1.5-R620H had no significant effect on the current. Proband 2, 33 year-old male with history of recurrent syncope which occurred both at rest and with exercise, had a novel NaV1.5-S1218I mutation. NaV1.5-S1218I had complete loss-of-function, and 1:1 expression of NaV1.5-WT and NaV1.5-S1218I mimicking the heterozygous state, revealed a 50% reduction of current compared to WT suggesting a functional haploinsufficiency in the patient.CONCLUSION: NaV1.5-S1218I and R811H are novel loss-of-function mutations in the SCN5A gene causing Brugada syndrome, whereas R620H did not appear to alter NaV1.5 function.