@article {2881, title = {A leaky voltage sensor domain of cardiac sodium channels causes arrhythmias associated with dilated cardiomyopathy.}, journal = {Sci Rep}, volume = {8}, year = {2018}, month = {2018 Sep 14}, pages = {13804}, abstract = {

Dilated cardiomyopathy (DCM) is a structural heart disease that causes dilatation of cardiac chambers and impairs cardiac contractility. The SCN5A gene encodes Na1.5, the predominant cardiac sodium channel alpha subunit. SCN5A mutations have been identified in patients with arrhythmic disorders associated with DCM. The characterization of Na1.5 mutations located in the voltage sensor domain (VSD) and associated with DCM revealed divergent biophysical defects that do not fully explain the pathologies observed in these patients. The purpose of this study was to characterize the pathological consequences of a gating pore in the heart arising from the Na1.5/R219H mutation in a patient with complex cardiac arrhythmias and DCM. We report its properties using cardiomyocytes derived from patient-specific human induced pluripotent stem cells. We showed that this mutation generates a proton leak (called gating pore current). We also described disrupted ionic homeostasis, altered cellular morphology, electrical properties, and contractile function, most probably linked to the proton leak. We thus propose a novel link between SCN5A mutation and the complex pathogenesis of cardiac arrhythmias and DCM. Furthermore, we suggest that leaky channels would constitute a common pathological mechanism underlying several neuronal, neuromuscular, and cardiac pathologies.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-31772-0}, author = {Moreau, Adrien and Gosselin-Badaroudine, Pascal and Mercier, Aur{\'e}lie and Burger, Bettina and Keller, Dagmar I and Mohamed Chahine} } @article {2457, title = {Biophysical, Molecular, and Pharmacological Characterization of Voltage-Dependent Sodium Channels From Induced Pluripotent Stem Cell-Derived Cardiomyocytes.}, journal = {Can J Cardiol}, volume = {33}, year = {2017}, month = {2017 Feb}, pages = {269-278}, abstract = {

BACKGROUND: The ability to differentiate patient-specific human induced pluripotent stem cells in cardiac myocytes (hiPSC-CM) offers novel perspectives for cardiovascular research. A number of studies, that reported mainly on current-voltage curves used hiPSC-CM to model voltage-gated Na(+) channel (Nav) dysfunction. However, the expression patterns and precise biophysical and pharmacological properties of Nav channels from hiPSC-CM remain unknown. Our objective was to study the characteristics of Nav channels from hiPSC-CM and assess the appropriateness of this novel cell model.

METHODS: We generated hiPSC-CM using the recently described monolayer-based differentiation protocol.

RESULTS: hiPSC-CM expressed cardiac-specific markers, exhibited spontaneous electrical and contractile activities, and expressed distinct Nav channels subtypes. Electrophysiological, pharmacological, and molecular characterizations revealed that, in addition to the main Nav1.5 channel, the neuronal tetrodotoxin (TTX)-sensitive Nav1.7 channel was also significantly expressed in hiPSC-CM. Most of the Na(+) currents were resistant to TTX block. Therapeutic concentrations of lidocaine, a class I antiarrhythmic drug, also inhibited Na(+) currents in a use-dependent manner. Nav1.5 and Nav1.7 expression and maturation patterns of hiPSC-CM and native human cardiac tissues appeared to be similar. The 4 Navβ regulatory subunits were expressed in hiPSC-CM, with β3 being the preponderant subtype.

CONCLUSIONS: The findings indicated that hiPSC-CM robustly express Nav1.5 channels, which exhibited molecular and pharmacological properties similar to those in native cardiac tissues. Interestingly, neuronal Nav1.7 channels were also expressed in hiPSC-CM and are likely to be responsible for the TTX-sensitive Nav current.

}, keywords = {Biochemical Phenomena, Biophysical Phenomena, Blotting, Western, Humans, Immunohistochemistry, Induced Pluripotent Stem Cells, Myocytes, Cardiac, Patch-Clamp Techniques, Sodium Channels, Voltage-Gated Sodium Channels}, issn = {1916-7075}, doi = {10.1016/j.cjca.2016.10.001}, author = {Moreau, Adrien and Mercier, Aur{\'e}lie and Th{\'e}riault, Olivier and Boutjdir, Mohamed and Burger, Bettina and Keller, Dagmar I and Mohamed Chahine} } @article {1716, title = {Mexiletine differentially restores the trafficking defects caused by two brugada syndrome mutations.}, journal = {Front Pharmacol}, volume = {3}, year = {2012}, month = {2012}, pages = {62}, abstract = {

The human cardiac sodium channel Na(v)1.5 encoded by the SCN5A gene plays a critical role in cardiac excitability and the propagation of action potentials. Na(v)1.5 dysfunctions due to mutations cause cardiac diseases such as the LQT3 form of long QT syndrome, conduction disorders, and Brugada syndrome (BrS). They have also recently been associated with dilated cardiomyopathy. BrS is characterized by coved ST-segment elevation on surface ECGs and lethal ventricular arrhythmias in an apparently structurally normal heart. Na(v)1.5 mutations that cause BrS result in a loss of channel function. Our aim was to functionally characterize two novel Na(v)1.5 mutations (A124D and V1378M) in BrS patients. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in tsA201 cells in the presence of the β(1)-auxiliary subunit. The patch-clamp technique and immunocytochemistry approaches were used to study the mutant channels and their cellular localization. The two mutant channels displayed a dramatic reduction in current density but had normal gating properties. The reduction in current density was caused by the retention of channel proteins in the endoplasmic reticulum (ER). Mutant channel retention could be partially reversed by incubating transfected cells at 25{\textdegree}C and by treating them with mexiletine (for V1378M but not A124D), or with curcumin or thapsigargin, two drugs that target ER resident proteins. It is likely that the clinical phenotypes observed in these two BrS patients were related to a surface expression defect caused by ER retention.

}, issn = {1663-9812}, doi = {10.3389/fphar.2012.00062}, author = {Moreau, Adrien and Keller, Dagmar I and Huang, Hai and Fressart, V{\'e}ronique and Schmied, Christian and Timour, Quadiri and Mohamed Chahine} } @article {1680, title = {A proton leak current through the cardiac sodium channel is linked to mixed arrhythmia and the dilated cardiomyopathy phenotype.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e38331}, abstract = {

Cardiac Na(+) channels encoded by the SCN5A gene are essential for initiating heart beats and maintaining a regular heart rhythm. Mutations in these channels have recently been associated with atrial fibrillation, ventricular arrhythmias, conduction disorders, and dilated cardiomyopathy (DCM).We investigated a young male patient with a mixed phenotype composed of documented conduction disorder, atrial flutter, and ventricular tachycardia associated with DCM. Further family screening revealed DCM in the patient{\textquoteright}s mother and sister and in three of the mother{\textquoteright}s sisters. Because of the complex clinical phenotypes, we screened SCN5A and identified a novel mutation, R219H, which is located on a highly conserved region on the fourth helix of the voltage sensor domain of Na(v)1.5. Three family members with DCM carried the R219H mutation.The wild-type (WT) and mutant Na(+) channels were expressed in a heterologous expression system, and intracellular pH (pHi) was measured using a pH-sensitive electrode. The biophysical characterization of the mutant channel revealed an unexpected selective proton leak with no effect on its biophysical properties. The H(+) leak through the mutated Na(v)1.5 channel was not related to the Na(+) permeation pathway but occurred through an alternative pore, most probably a proton wire on the voltage sensor domain.We propose that acidification of cardiac myocytes and/or downstream events may cause the DCM phenotype and other electrical problems in affected family members. The identification of this clinically significant H(+) leak may lead to the development of more targeted treatments.

}, keywords = {Adult, Amino Acid Sequence, Amino Acid Substitution, Animals, Arrhythmias, Cardiac, Base Sequence, Cardiomyopathy, Dilated, Cell Line, Humans, Hydrogen-Ion Concentration, Male, Molecular Sequence Data, Mutation, Myocytes, Cardiac, NAV1.5 Voltage-Gated Sodium Channel, Oocytes, Pedigree, Phenotype, Protons, Sodium Channels, Xenopus}, issn = {1932-6203}, doi = {10.1371/journal.pone.0038331}, author = {Gosselin-Badaroudine, Pascal and Keller, Dagmar I and Huang, Hai and Pouliot, Val{\'e}rie and Chatelier, Aur{\'e}lien and Osswald, Stefan and Brink, Marijke and Mohamed Chahine} } @article {1648, title = {Characterization of novel KCNH2 mutations in type 2 long QT syndrome manifesting as seizures.}, journal = {Can J Cardiol}, volume = {25}, year = {2009}, month = {2009 Aug}, pages = {455-62}, abstract = {

BACKGROUND: Long QT syndrome (LQTS) is characterized by corrected QT interval prolongation leading to torsades de pointes and sudden cardiac death. LQTS type 2 (LQTS2) is caused by mutations in the KCNH2 gene, leading to a reduction of the rapidly activating delayed rectifier K+ current and loss of human ether-{\`a}-go-go-related gene (hERG) channel function by different mechanisms. Triggers for life-threatening arrhythmias in LQTS2 are often auditory stimuli.

OBJECTIVES: To screen KCNH2 for mutations in patients with LQTS2 on an electrocardiogram and auditory-induced syncope interpreted as seizures and sudden cardiac death, and to analyze their impact on the channel function in vitro.

METHODS: The KCNH2 gene was screened for mutations in the index patients of three families. The novel mutations were reproduced in vitro using site-directed mutagenesis and characterized using the Xenopus oocyte expression system in voltage clamp mode.

RESULTS: Novel KCNH2 mutations (Y493F, A429P and del234-241) were identified in the index patients with mostly typical LQTS2 features on their electrocardiograms. The biochemical data revealed a trafficking defect. The biophysical data revealed a loss of function when mutated hERG channels were coexpressed with the wild type.

CONCLUSIONS: In all families, at least one patient carrying the mutation had a history of seizures after auditory stimuli, which is a major trigger for arrhythmic events in LQTS2. Seizures are likely due to cardiac syncope as a consequence of mutation-induced loss of function of the rapidly activating delayed rectifier K+ current.

}, issn = {1916-7075}, author = {Keller, Dagmar I and Grenier, Julie and Christ{\'e}, Georges and Dubouloz, Fr{\'e}d{\'e}rique and Osswald, Stefan and Brink, Marijke and Ficker, Eckhard and Mohamed Chahine} } @article {1720, title = {A novel SCN5A mutation, F1344S, identified in a patient with Brugada syndrome and fever-induced ventricular fibrillation.}, journal = {Cardiovasc Res}, volume = {70}, year = {2006}, month = {2006 Jun 1}, pages = {521-9}, abstract = {

OBJECTIVE: Brugada syndrome (BS) is an inherited electrical cardiac disorder characterized by right bundle branch block pattern and ST segment elevation in leads V1 to V3 on surface electrocardiogram that can potentially lead to malignant ventricular tachycardia and sudden cardiac death. About 20\% of patients have mutations in the only so far identified gene, SCN5A, which encodes the alpha-subunit of the human cardiac voltage-dependent sodium channel (hNa(v)1.5). Fever has been shown to unmask or trigger the BS phenotype, but the associated molecular and the biophysical mechanisms are still poorly understood. We report on the identification and biophysical characterization of a novel heterozygous missense mutation in SCN5A, F1344S, in a 42-year-old male patient showing the BS phenotype leading to ventricular fibrillation during fever.

METHODS: The mutation was reproduced in vitro using site-directed mutagenesis and characterized using the patch clamp technique in the whole-cell configuration.

RESULTS: The biophysical characterization of the channels carrying the F1344S mutation revealed a 10 mV mid-point shift of the G/V curve toward more positive voltages during activation. Raising the temperature to 40.5 degrees C further shifted the mid-point activation by 18 mV and significantly changed the slope factor in Na(v)1.5/F1344S mutant channels from -6.49 to -10.27 mV.

CONCLUSIONS: Our findings indicate for the first time that the shift in activation and change in the slope factor at a higher temperature mimicking fever could reduce sodium currents{\textquoteright} amplitude and trigger the manifestation of the BS phenotype.

}, issn = {0008-6363}, doi = {10.1016/j.cardiores.2006.02.030}, author = {Keller, Dagmar I and Huang, Hai and Zhao, Juan and Frank, Rudolf and Suarez, Vivian and Delacr{\'e}taz, Etienne and Brink, Marijke and Osswald, Stefan and Schwick, Nicola and Mohamed Chahine} } @article {1673, title = {A novel nonsense mutation in the SCN5A gene leads to Brugada syndrome and a silent gene mutation carrier state.}, journal = {Can J Cardiol}, volume = {21}, year = {2005}, month = {2005 Sep}, pages = {925-31}, abstract = {

BACKGROUND: Brugada syndrome (BS) is an electrical cardiac disorder with a right bundle branch block and ST segment elevation in leads V1 to V3 on surface electrocardiograms (ECGs), and is a syndrome that may lead to sudden cardiac death.

PURPOSE: The aim of the present study was to screen for mutations in the SCN5A gene in a family with BS, and to characterize the consequences of the mutation on channel function.

RESULTS: A heterozygous nonsense SCN5A mutation (W822X) was identified in the index patient. The mutation was confirmed in the patient{\textquoteright}s asymptomatic 16-year-old brother and 48-year-old father. The mutation was absent in the index patient{\textquoteright}s sister and mother. The ECG of the index patient showed a BS type 2 ECG phenotype, which converted into a type 1 ECG phenotype in the presence of flecainide. The ECG of the patient{\textquoteright}s brother was not typical for BS, but ajmaline treatment unmasked a type 1 ECG phenotype. The ECG of the asymptomatic father was normal at baseline and in the presence of ajmaline. No Na+ currents could be measured in tsA201 cells transfected with W822X mutant channels. Heterozygote expression showed a nearly 50\% reduction in Na+ current amplitude with no significant alterations of biophysical properties, indicating a loss of functional Na+ channels, obviously without any dominant-negative activity on wild type channels.

CONCLUSIONS: The haploinsufficiency of the Nav1.5 protein is the plausible explanation for the clinical BS phenotype in this family. Because the heterozygous W822X mutation theoretically leads to channel expression at one-half of the normal level, the authors suggest that a modifier gene may influence or rescue the phenotype in the asymptomatic family members.

}, issn = {0828-282X}, author = {Keller, Dagmar I and Barrane, Fatima-Zahara and Gouas, Laetitia and Martin, Johannie and Pilote, Sylvie and Suarez, Vivian and Osswald, Stefan and Brink, Marijke and Guicheney, Pascale and Schwick, Nicola and Mohamed Chahine} } @article {1657, title = {A novel mutation in SCN5A, delQKP 1507-1509, causing long QT syndrome: role of Q1507 residue in sodium channel inactivation.}, journal = {J Mol Cell Cardiol}, volume = {35}, year = {2003}, month = {2003 Dec}, pages = {1513-21}, abstract = {

Inherited long QT syndrome (LQTS) is caused by mutations in six genes including SCN5A, encoding the alpha-subunit of the human cardiac voltage-dependent sodium channel hNa(v)1.5. In LQT3, various mutations in SCN5A were identified, which produce a gain of channel function. The aim of this study was to screen SCN5A for mutations in a family with the LQT3 phenotype and to analyze the consequences of the mutation on the channel function. By polymerase chain reaction-denaturating high performance liquid chromatography-sequencing, we identified a novel deletion in SCN5A, delQKP 1507-1509, in the DIII-DIV linker of the sodium channel. The hNa(v)1.5/delQKP1507-1509, hNa(v)1.5/delQ1507 and hNa(v)1.5/Q1507A mutants were constructed in vitro, mutant channels were expressed in the tsA201 human cell line and studied using the whole-cell configuration of the patch clamp technique. A persistent inward sodium current of 1-1.5\% of maximum currents measured at -30 mV in all mutant sodium channels was recorded, which was nearly completely blocked by the sodium-channel blockers tetrodotoxin and lidocaine. The deletion mutants resulted in a significant shift of steady-state activation to more depolarized voltages. The delQ1507 showed a small shift of steady-state inactivation towards more negative potentials, whereas no significant shifts were observed in both steady-state activation and inactivation in Q1507A compared to the wild-type Na(v)1.5 sodium channels. The novel SCN5A mutation, delQKP, induces a residual current as previously shown for other SCN5A mutations causing LQTS. DelQKP shares the deletion of Q1507 with the formerly known delKPQ 1505-1507. Our data suggest that Q1507 plays an important role in fast sodium channel inactivation.

}, issn = {0022-2828}, author = {Keller, Dagmar I and Acharfi, Said and Delacr{\'e}taz, Etienne and Benammar, Nawal and Rotter, Martin and Pfammatter, Jean Pierre and Fressart, V{\'e}ronique and Guicheney, Pascale and Mohamed Chahine} }