Roles and regulation of the cardiac sodium channel Na v 1.5: recent insights from experimental studies.

During the past decade, Na(v)1.5, the main voltage-gated Na(+) channel in the heart, has been shown to be involved in many cardiac diseases. Genetic variants in the gene SCN5A, encoding Na(v)1.5, have been linked to various cardiac phenotypes, such as the congenital and acquired long QT syndromes, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and even cases of dilated cardiomyopathy. This unexpected phenotypic diversity may reflect that Na(v)1.5 is not only restricted to the initiation of the action potential and rapid cardiac conduction, but may also be involved in other, not-yet elucidated, functions. Despite the fact that our understanding of the regulation of expression, localization, and function of Na(v)1.5 is deepening, we are still far from a comprehensive view. Much of our current knowledge has been obtained by carrying out experiments using "cellular expression systems", e.g. host cells expressing exogenous Na(v)1.5. Although very informative, these techniques are limited, in that Na(v)1.5 is not expressed in the physiological cellular environment of a cardiac cell. Recently, however, there have been several studies published which used approaches closer to "normal" or pathological physiology. In an attempt to summarize recently published data, this article will review the phenotypes of genetically-modified mouse strains where Na(v)1.5 expression and activity are directly or indirectly modified, as well as the regulation of Na(v)1.5 function using native cardiac myocytes. Despite obvious limitations, the reviewed studies provide an overview of the complex multi-factorial and multi-protein regulation of Na(v)1.5

Molecular autopsy in sudden cardiac death and its implication for families: discussion of the practical, legal and ethical aspects of the multidisciplinary collaboration.

Sudden cardiac death (SCD) is a major cause of premature death in young adults and children in developed countries. Standard forensic autopsy procedures are often unsuccessful in determining the cause of SCD. Post-mortem genetic testing, also called molecular autopsy, has revealed that a non-negligible number of these deaths are a result of inherited cardiac diseases, including arrhythmic disorders such as congenital long QT syndrome and Brugada syndrome. Due to the heritability of these diseases, the potential implications for living relatives must be taken into consideration. Advanced diagnostic analyses, genetic counselling, and interdisciplinary collaboration should be integral parts of clinical and forensic practice. In this article we present a multidisciplinary collaboration established in Lausanne, with the goal of properly informing families of these pathologies and their implications for surviving family members. In Switzerland, as in many other countries, legal guidelines for genetic testing do not address the use of molecular tools for post-mortem genetic analyses in forensic practice. In this article we present the standard practice guidelines established by our multidisciplinary team

Molecular and clinical determinants of drug-induced long QT syndrome: an iatrogenic channelopathy.

More than 70 drugs present on the Swiss market can cause drug-induced long QT syndrome (LQTS), which is associated with torsades de pointes (TdP) arrhythmias, potentially leading to sudden cardiac death. Basic and clinical investigations performed during the last decade have helped a better understanding of the mechanisms and risk factors of this serious public health problem. In their vast majority, QT interval prolonging drugs block the human ERG (hERG) channel involved in the repolarisation phase of the cardiac action potential, and thus lengthen the QT interval. Beside the well-known QT interval prolonging action of class IA, IC and III anti-arrhythmic drugs, many antibiotics, neurotropic, antifungal, and antimalarial drugs are also able to cause drug-induced LQTS. Reviewing the literature indicates that the risk of QT interval prolongation and TdP is increased in females, in patients with organic heart diseases and hypokalaemia. Furthermore in a few cases, genetic factors have also been reported. However thus far, no genetic test is available to detect at-risk patients, and in consequence, drug prescribers are still relying only on the clinical history and findings to perform an evaluation of the risk. Treatment of drug-induced LQTS and TdP includes identifying and withdrawing the culprit drug(s), infusing magnesium and, in resistant cases acceleration of the heart rate. In this review article we provide a list of QT interval prolonging drugs adapted to the pharmaceuticals found on the Swiss market that can be used as a check-list for drug prescribers and at-risk patients

A Distinct Pool of Nav1.5 Channels at the Lateral Membrane of Murine Ventricular Cardiomyocytes.

Background: In cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Na <sub>v</sub> 1.5 at the lateral membrane depends in part on the interaction between the dystrophin-syntrophin complex and the Na <sub>v</sub> 1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Na <sub>v</sub> 1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Na <sub>v</sub> 1.5 in which the SIV motif is deleted (ΔSIV), sodium current (I <sub>Na</sub> ) recordings from the lateral membrane revealed a SIV-motif-independent I <sub>Na</sub> . Since immunostaining has suggested that Na <sub>v</sub> 1.5 is expressed in transverse (T-) tubules, this remaining I <sub>Na</sub> might be carried by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Na <sub>v</sub> 1.5 N-terminus, which may explain the SIV-motif independent I <sub>Na</sub> at the lateral membrane of cardiomyocytes. Aim: To address the role of α1-syntrophin in regulating the I <sub>Na</sub> at the lateral membrane of cardiac cells. Methods and Results: Patch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knockdown, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral I <sub>Na</sub> was observed in myocytes from α1-syntrophin knockdown hearts. Similar to ΔSIV myocytes, a remaining I <sub>Na</sub> was still recorded. In addition, cell-attached I <sub>Na</sub> recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents carried by channels in T-tubules such as calcium channels. Conclusion: Altogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Na <sub>v</sub> 1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Na <sub>v</sub> 1.5 channels, however, remains open

The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family.

OBJECTIVE: The voltage-gated KCNQ1 potassium channel regulates key physiological functions in a number of tissues. In the heart, KCNQ1 alpha-subunits assemble with KCNE1 beta-subunits forming a channel complex constituting the delayed rectifier current I(Ks). In epithelia, KCNQ1 channels participate in controlling body electrolyte homeostasis. Several regulatory mechanisms of the KCNQ1 channel complexes have been reported, including protein kinase A (PKA)-phosphorylation and beta-subunit interactions. However, the mechanisms controlling the membrane density of KCNQ1 channels have attracted less attention. METHODS AND RESULTS: Here we demonstrate that KCNQ1 proteins expressed in HEK293 cells are down-regulated by Nedd4/Nedd4-like ubiquitin-protein ligases. KCNQ1 and KCNQ1/KCNE1 currents were reduced upon co-expression of Nedd4-2, the isoform among the nine members of the Nedd4/Nedd4-like family displaying the highest expression level in human heart. In vivo expression of a catalytically inactive form of Nedd4-2, able to antagonize endogenous Nedd4-2 in guinea-pig cardiomyocytes, increased I(Ks) significantly, but did not modify I(K1). Concomitant with the reduction in current induced by Nedd4-2, an increased ubiquitylation as well as a decreased total level of KCNQ1 proteins were observed in HEK293 cells. Pull-down and co-immunoprecipitation experiments showed that Nedd4-2 interacts with the C-terminal part of KCNQ1. The Nedd4/Nedd4-like-mediated regulation of the KCNQ1 channel complexes is strictly dependent on a PY motif located in the distal part of the C-terminal domain. When this motif was mutated, the current and ubiquitylation levels were unaffected by Nedd4-2, and Nedd4-2 proteins were neither pulled-down nor co-immunoprecipitated. CONCLUSIONS: These results suggest that KCNQ1 internalization and stability is physiologically regulated by its Nedd4/Nedd4-like-dependent ubiquitylation. This mechanism may thereby be important in regulating the surface density of the KCNQ1 channels in cardiomyocytes and other cell types

Brugada syndrome and fever: genetic and molecular characterization of patients carrying SCN5A mutations.

OBJECTIVE: Brugada syndrome (BrS) is characterized by ventricular tachyarrhythmias leading to sudden cardiac death and is caused, in part, by mutations in the SCN5A gene encoding the sodium channel Na(v)1.5. Fever can trigger or exacerbate the clinical manifestations of BrS. The aim of this work was to characterize the genetic and molecular determinants of fever-dependent BrS. METHODS: Four male patients with typical BrS ST-segment elevation in V1-V3 or ventricular arrhythmias during fever were screened for mutations in the SCN5A gene. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. The sodium currents (I(Na)) were analysed using the whole-cell patch clamp technique at various temperatures. Protein expression of WT and mutant channels was studied by Western blot experiments. RESULTS: Two mutations in SCN5A, L325R and R535X, were identified. Expression of the two mutant Na(v)1.5 channels in HEK293 cells revealed in each case a severe loss-of-function. Upon the increase of temperature up to 42 degrees C, we observed a pronounced acceleration of Na(v)1.5 activation and fast inactivation kinetics. Cardiac action potential modelling experiments suggest that in patients with reduced I(Na), fever could prematurely shorten the action potential by virtue of its effect on WT channels. Further experiments revealed that L325R channels are likely misfolded, since their function could be partially rescued by mexiletine or curcumin. In co-expression experiments, L325R channels interfered with the proper function of WT channels, suggesting that a dominant negative phenomenon may underlie BrS triggered by fever. CONCLUSIONS: The genetic background of BrS patients sensitive to fever is heterogeneous. Our experimental data suggest that the clinical manifestations of fever-exacerbated BrS may not be mutation specific

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a+/− Mouse Model

BACKGROUND: Loss-of-function mutations in SCN5A, the gene encoding Na(v)1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a(+/-) mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. METHODOLOGY/PRINCIPAL FINDINGS: Based on ECG, 10-week-old Scn5a(+/-) mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a(+/-) mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A-mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a(+/-) mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a(+/-) mice had similar Na(v)1.5 mRNA but higher Na(v)1.5 protein expression, and moderately larger I(Na) current than severely affected Scn5a(+/-) mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a(+/-) mice than in mildly affected ones. CONCLUSIONS: Scn5a(+/-) mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a(+/-) mice, phenotype severity correlates with wild-type Na(v)1.5 protein expression