MD Azza Ziyadeh-Isleem

PhD Jérôme Clatot

PhD Sabine Duchatelet

MD, PhD Estelle Gandjbakhch

MD Isabelle Denjoy

MD Françoise Hidden-Lucet

MD, PhD Stéphane Hatem

PhD, FHRS Isabelle Deschênes

PhD Alain Coulombe

PhD ‡ Nathalie Neyroud

PharmD Pascale Guicheney

Both

A truncating SCN5A mutation combined with genetic variability causes sick sinus syndrome and early atrial fibrillation

Keywords: Ankyrin-B syndrome AF, Atrial fibrillation AFL, Atrial flutter AP, Action potential ARVC, Arrhythmogenic right ventricular cardiomyopathy ATS, Andersen-Tawil syndrome AV, Atrio-ventricular AVN, Atrio-ventricular node AVNRT, Atrio-ventricular nodal re-entrant tachycardia AVRT, Atrio-ventricular reciprocating tachycardia bpm, Beats per minute BrS, Brugada syndrome BSA, Bovine serum albumin Ca 2+, Calcium ion CaM, Calmodulin CaMKII, Ca2 + /calmodulin-dependent protein kinase II cAMP, Cyclic adenosine monophosphate CASQ2, Calsequestrin 2 CCD, Cardiac conduction diseases CCW, Counterclockwise CFTR, Cystic fibrosis transmembrane conductance regulator CHO, Chinese hamster ovary CPVT, Catecholaminergic polymorphic ventricular tachycardia Sarcomere Actin Myosin Actin Relaxed Contracted Sarcomere Actin Myosin Actin Actin Myosin Actin Actin Myosin Actin Actin Myosin Actin

Résumé des travaux de thèse

Titre : Nouveaux mécanismes contribuant à la variabilité phénotypiques de mutations N-et C-terminales du canal sodique cardiaque L'activité électrique cardiaque est étroitement contrôlée par l'activité coordonnée de multiples canaux ioniques voltage-dépendants. L'un des plus importants est le canal sodique cardiaque formé d'une sous-unité α, Na v 1.5, constituant le canal lui-même et de nombreuses protéines partenaires régulatrices. Ce canal permet la génération du courant sodique entrant I Na , qui est un déterminant essentiel de la vitesse de conduction de l'influx électrique cardiaque, qui se transmet du noeud sinusal aux oreillettes, puis aux ventricules le long du tissu conducteur, puis à travers le myocarde ventriculaire. Des mutations dans le gène SCN5A, codant Na v 1.5, ont été impliquées dans de nombreuses arythmies cardiaques héréditaires, telles que le syndrome du QT long (SQTL), le syndrome de Brugada (SBr), les dysfonctions du noeud sinusal (DNS), et la fibrillation auriculaire (FA). Les arythmies liées à des mutations de SCN5A ont d'abord été considérées comme des entités cliniques distinctes. Plus récemment, un large spectre de phénotypes mixtes a été rapporté dans les arythmies liées aux mutations de SCN5A regroupés sous le terme d'«overlap syndromes». En plus de leur expression variable, les arythmies liées à SCN5A sont caractérisées par une pénétrance incomplète. L'origine des phénotypes mixtes causés par une même mutation de SCN5A, et la pénétrance incomplète de ces canalopathies, restent encore mal comprises. Cela a conduit à souligner la complexité des maladies liées aux mutations de SCN5A, et à suggérer que d'autres facteurs génétiques ou environnementaux pouvaient moduler le phénotype clinique. Dans notre travail, nous avons étudié trois facteurs qui pourraient expliquer les phénotypes variables induits par des mutations de SCN5A : l'interaction entre les sous-unités α de Na v 1.5, les propriétés électriques différentes entre les cardiomyocytes auriculaires et ventriculaires, et le terrain génétique des patients.

Dans la première partie de ce travail de thèse, nous avons caractérisé la mutation R104W située dans la partie N-terminale du canal sodique cardiaque Na v 1.5, identifiée chez un patient de 33 ans atteint de SBr. Ce syndrome est une canalopathie cardiaque héréditaire à transmission autosomique dominante et à pénétrance incomplète, où des mutations perte-defonction de Na v 1.5 sont trouvées chez environ 25% des cas. Par des études biochimiques et électrophysiologiques, nous avons montré que le mutant R104W exprimé seul dans les cellules HEK293 n'était associé à aucun courant sodique I Na , et est dégradé par le système ubiquitine-protéasome. Lorsqu'il est co-exprimé avec le canal sauvage, imitant l'état hétérozygote du patient, le mutant exerce un effet dominant négatif sur les canaux sauvages, et conduit à un décalage de la courbe d'activation vers des potentiels positifs. L'immunocytochimie de cardiomyocytes de rats nouveaux-nés transfectés avec R104W et/ou Na v 1.5 sauvage a montré que le mutant seul est principalement retenu dans le réticulum endoplasmique. De plus, sa co-expression avec le sauvage conduit à la rétention de ce dernier.

Par ailleurs, la co-transfection de R104W avec R878C, un canal mutant capable d'atteindre la membrane mais non fonctionnel, restaure une très petite activité de R104W. Ces résultats nous ont amené à penser qu'une coopération entre les sous-unités α Na v 1.5 pouvait exister.

Par des études de co-immunoprécipitation, nous avons démontré pour la première fois que les sous-unités α sauvages interagissaient entre elles, ainsi qu'avec les sous-unités α mutées R104W. L''interaction entre les sous-unités α Na v 1.5 est un nouveau mécanisme qui pourrait moduler l'effet fonctionnel de mutations de SCN5A ainsi que le phénotype clinique ; ceci démontre l'importance de l'expression hétérozygote lorsque l'on étudie les effets fonctionnels de mutations dans SCN5A.

Dans la deuxième partie de ce travail de thèse, nous avons caractérisé la mutation R1860Gfs*12, mutation tronquante dans la région C-terminale de Na v 1.5, identifiée dans une famille présentant un tableau clinique mixte de DNS, FA, flutter atrial, et de troubles de la conduction sans SBr. La mutation a été identifiée chez le cas index, son père et son oncle.

Les analyses biochimiques et les études de patch-clamp ont objectivé que dans les cellules HEK293 les canaux mutants R1860Gfs*12 sont partiellement dégradés par le système ubiquitine-protéasome, et associés à une réduction de I Na , un décalage positif de la courbe d'activation, un décalage négatif de la courbe d'inactivation et une augmentation du courant persistant I NaL par rapport au canal sauvage. À l'état hétérozygote, le mutant n'exerce pas d'effet dominant négatif sur le sauvage, mais induit un décalage négatif de la courbe d'inactivation, ainsi qu'un courant sodique persistant. Le décalage négatif de la courbe d'inactivation conduit à une réduction marquée de la disponibilité de Na v 1.5, ce qui indique une perte-de-fonction du mutant R1860Gfs*12. Par ailleurs, en mesurant I Na dans des cellules HEK293 exprimant le mutant, maintenues à des potentiels imitant le potentiel de repos des myocytes auriculaires ou celui des myocytes ventriculaires, nous avons montré que le mutant induit une perte-de-fonction nettement plus prononcée dans les cellules auriculaires que dans les cellules ventriculaires. Ces résultats ont été confirmés par modélisation informatique (des potentiels d'action membranaires de cellules uniques auriculaires ou ventriculaires) et pourraient expliquer la survenue d'arythmies auriculaires dans cette famille sans SBr, ni arythmies ventriculaires. En outre, en recherchant la présence de polymorphismes associés au risque de FA dans cette famille, nous avons mis en évidence que le cas index, qui a développé la maladie à un jeune âge avec des symptômes plus sévères que son père et son oncle, est porteur d'allèles à risque en amont de PITX2, un gène largement associé au développement de la FA. De plus, dans cette étude, nous avons mis en évidence que les sous-unités α tronquées R1860Gfs*12 étaient encore capables d'interagir entre elles ainsi qu'avec des sous-unités α sauvages, indiquant que les derniers 156 acides aminés de Na v 1.5 ne sont pas essentiels pour l'interaction entre les sous-unités α. Ces résultats suggèrent que les différences électrophysiologiques entre les oreillettes et les ventricules, combinées au terrain génétique des patients, sont des facteurs modulant l'expression phénotypique des mutations SCN5A. Na v 1.5 interagit avec de nombreuses protéines partenaires, y compris le facteur homologue au facteur de croissance des fibroblastes (FHF1B), qui se lie à la partie Cterminale de Na v 1.5 et module l'inactivation du canal. Cependant, le site exact de l'interaction de FHF1B sur le C-terminus de Na v 1.5 fait l'objet d'un débat et serait situé dans sa région proximale ou ses régions proximale et distale. Dans la troisième partie de ce travail de thèse, nous avons confirmé par des études de co-immunoprécipitation de FHF1B et de deux mutants tronqués R1860Gfs*12*10 et L1821fs*10, que les deux régions proximale et distale de Na v 1.5 sont essentielles à l'interaction entre FHF1B et Na v 1.5. En outre, nous avons démontré par PCR quantitative que FHF1B est l'isoforme FHF la plus exprimée dans les ventricules humains.

Puisque 75% des cas de SBr ne sont pas liés aux gènes connus à l'origine de SBr, nous avons criblé le gène candidat FGF12B codant une protéine partenaire, FHF1B, chez 182 patients atteints de SBr avec un génotype négatif pour la plupart des gènes connus et nous avons identifié le variant V127M chez un patient. La valine en position 127 est conservée dans plusieurs espèces, ainsi que dans les autres FHFs humains (FHF2, FHF3, FHF4), ce qui est en faveur d'un possible rôle dans la fonction de cette protéine. Néanmoins, l'analyse électrophysiologique de cellules HEK293 transfectées avec Na v 1.5 et FHF1B sauvage ou muté n'a pas montré de différence fonctionnelle.

Conclusion: l'interaction entre les sous-unités α Na v 1.5 est un nouveau mécanisme qui pourrait moduler les effets fonctionnels de certaines mutations du SCN5A ainsi que les phénotypes cliniques. Cela met en évidence l'importance de l'expression hétérozygote lorsque l'on étudie les effets fonctionnels des mutations SCN5A. En outre, les propriétés électriques différentes entre les cardiomyocytes auriculaires et ventriculaires, et la présence de polymorphismes chez les patients porteurs des mutations pourraient modifier le phénotype clinique et aider à expliquer la grande variabilité phénotypique des canalopathies sodiques. The R104W mutant identified in a patient with Brugada syndrome is retained in the endoplasmic reticulum (ER), degraded by the proteasome and abolishes the sodium current.

Mots

Co-expressed with wild type (WT) channels, R104W leads to WT channels ER retention, causing a dominant-negative effect. We demonstrated that interaction between Na v 1.5 αsubunits is responsible for the retention and the dominant-negative effect. The R1860Gfs*12 mutation was identified in a family with atrial arrhythmias. In a heterologous system, this mutant induces both loss-and gain-of-function effects on Na v 1.5.

Computer-model simulation showed that the loss-of-function was more pronounced in atrial than in ventricular cells. In addition, we showed that the presence of polymorphisms upstream of the PITX2 gene could explain the observed phenotypic variability in this family.

In conclusion, the interaction between the α-subunits of Na v 1.5, the different electrical properties between atria and ventricles and the presence of polymorphisms in patients with SCN5A mutations, are important factors in the interpretation of the functional effects of these mutations, which could explain the phenotypic variability of sodium channelopathies.

Keywords: Arrhythmia, Brugada syndrome, Atrial fibrillation, Na v 1.5, SCN5A, PITX2,

Polymorphism

Acknowledgement

I would like to thank all the people who have contributed, in one way or another, to the work described in this thesis. My first and sincere gratitude goes to Dr. Pascal Guicheney, my thesis supervisor, whose expertise, understanding, direction and invaluable ideas and suggestions added considerably to make this work a success. I appreciate her vast knowledge in her domain, and her assistance in writing the non-stop reports concerning my scholarship and stay in France. Besides, I am deeply grateful to my co-supervisor, Dr. Nathalie Neyroud, for her continuous support during the last four years, her patience, presence and kindness. I owe her most, if not all, the techniques I learnt in the laboratory since my arrival. The guidance and support of both of my supervisors have facilitated remarkably my research experience and the writing of the articles and this thesis, it would have been so difficult to accomplish this whole work without your insights.

My special thanks go to Dr. Alain Coulombe and my colleague Dr. Jérôme Clatot, for their valuable inputs in the work of this thesis and its articles. I am also grateful to every member in our team; Myriam Berthet, Dr. Martin Grauso, Dr. Eric Villard, Agathe Korniat, Dr. Alexia Vite, Dr. Darouna Kattygnarath, Gilles Dilanian and Dr. Sabine Duchatelet. I must also acknowledge Valérie Cornillault for her kindness and help with the administrative paper work, Pr. Stéphane Hatem, director of the whole unit. I would also like to thank Dr. Rachel Peat, for the time she spent to help correct my written English. You are adorable as usual.

Furthermore, I have been privileged to know and collaborate with many other great people. Dr. Estelle Gandjbakhch, cardiologist at the Pitié-Salpêtrière University Hospital, and her team, for providing patients' medical data as well as ECG interpretations. Dr. Isabelle Denjoy, cardiologist at the Bichat University Hospital, for her nice help in revising and correcting the section of cardiac arrhythmias in the introduction. Dr. Isabelle Deschenes, from Cleveland Heart and Vascular Research Centre in USA, thank you for your suggestions and help.

In addition, I would like to express my gratitude to Pr. Eric Raddatz from Lausanne University, and Pr. Vincent Probst from Nantes University Hospital, for their acceptance to be members in my thesis jury, their valuable time spent to critically read and comment on my thesis work. My special thanks are owed to Pr. Rose Katz, president of the jury and representative of Paris VI University in the cooperation program with the Palestinian Medical School for more than ten years. Thank you for your patience and availability to solve our problems in difficult moments. You are unforgettable! I am greatly indebted to Pr. Anwar Dudin, ex-Dean of Annajah Medical School, who encouraged and helped me come here to do my first research experience. To Annajah University in Nablus, Palestine, for the grant I have been awarded to do this PhD degree.

Finally, I would like to thank from the depth of my heart my husband, Jawad, for his unconditional support, encouragement, love and assistance. I am deeply and forever indebted to my father, the soul of my mother, and my brothers and sister who have accompanied me from far away during my stay in France. My parents-in-law for their continuous payers for our success. Love you all, and forever!

I dedicate this thesis to my husband Jawad and my family

Love you all

Table of contents

List of figures

List of tables

Preface

Sudden cardiac death (SCD) is defined as an unexpected death due to a cardiac cause, which occurs within one hour following the onset of symptoms. Ventricular tachycardia (VT) degenerating into ventricular fibrillation (VF) and to asystole is the most common mechanism underlying SCD. The disorganized and ineffective ventricular contraction during VF will deprive the vital organs from the oxygenated blood and result in a loss of consciousness. If not treated quickly by an electric shock using an automated external defibrillator, death occurs within few minutes. It is a major health problem accounting for 20% of total mortality, and 50% of cardiovascular mortality 1 . Consequently, identifying the patients at high risk of SCD and treating them, became one of the medical challenges of the beginning of the 21 st century.

Eighty percent of SCDs occur in individuals older than 40 years with coronary heart disease 2 . However, SCDs also occur in young people where cardiomyopathies and primary electrical diseases (channelopathies) are the major causes of VF 2 . Brugada syndrome (BrS), long QT syndrome (LQTS), and catecholaminergic polymorphic ventricular tachycardia (CPVT) constitute the main channelopathies leading to SCD 3 . Our knowledge of the cellular and molecular mechanisms underlying these channelopathies is still insufficient, leading to inadequate risk stratification of patients and families, and treatment options restricted mainly to cardioverter defibrillator implantation. Nevertheless, basic research, using heterologous expressing systems or animal models, has proved its efficacy in improving our understanding of the pathophysiology of these channelopathies.

In this thesis work, we studied the cardiac sodium channelopathies associated with SCN5A mutations, which are involved in a wide spectrum of disease phenotypes: LQTS, BrS, progressive cardiac conduction disease (PCCD), atrial fibrillation (AF), sick sinus syndrome (SSS), and dilated cardiomyopathies. Despite important progresses in understanding the mechanisms underlying the sodium channel dysfunction, risk stratification and management of patients with this channelopathy are still limited, mainly because of its reduced penetrance and variable expressivity. Moreover, some SCN5A mutations were linked to a mixed clinical picture known as cardiac sodium channel "overlap syndromes". The variable expressivity of the SCN5A mutations suggests an involvement of additional unknown mechanistic, genetic, and environmental factors in the determination of the phenotypic expression of a given SCN5A mutation. Better understanding of these modifiers is essential to ameliorate the diagnosis, risk stratification, and treatment options in patients with sodium channelopathy.

In this thesis we aimed to address the mechanisms underlying the variable phenotypes of SCN5A mutations, by the characterization of two SCN5A mutations, R104W and R1860Gfs*12 identified in patients with hereditary cardiac arrhythmias. Moreover, by candidate gene approach in genotype-negative BrS patients, which accounts approximately for 75% of BrS cases, we aimed to identify a new gene involved in this hereditary arrhythmia. ejected into the systemic circulation through the largest artery, the aorta (Figure 1B).

Each cardiac cycle consists of two basic phases: diastole and systole. During the diastolic phase, the ventricles are relaxed, and blood passes passively from right and left atria into the right and left ventricles. At the end of diastole, the atria contract to eject an additional amount of blood into the ventricles. In the systolic phase, the left and right ventricles contract and blood moves into the aorta and the pulmonary artery, respectively. This succession of diastolic and systolic phases is produced through the spontaneous activity of pacemaker cells.

The trigger activity of these cells is not under the control of the nervous system. However, cardiac frequency can be modulated by the autonomic nervous system.

I.2 Cardiac cells

The heart is composed of different cell types: the cardiomyocytes, which account for approximately 75% of the total volume of normal myocardial tissue but represent 30-40% of total cell numbers, and the non-myocyte cells which make up 70% of cardiac cells 4 . The nonmyocyte cells include (1) the fibroblasts which represent 90% of non-myocyte cells, having many critical functions such as extracellular matrix synthesis and deposition, and cardiac remodeling 5 , (2) endothelial cells that line the entire circulatory system from the heart to the smallest capillaries, (3) smooth muscle cells, (4) neurons and ( 5) inflammatory cells, mainly macrophages 6 .

The cardiomyocytes are divided into two main cell types: working (contracting) myocytes and conduction cells.

I.2.1 Working or contracting cardiomyocytes

Adult cardiomyocytes have a cylindrical shape, are approximately 100 µm long by 10-25 µm in diameter, and are organized as a network of branched fibers. They have a specialized structure called a sarcolemma, which is a fusion of plasma and basement membrane surrounding the cells. The sarcolemma forms two specialized structures: the intercalated discs at the junction between adjacent cells, and the transverse tubules at the lateral membrane.

A B

The

I.2.2 Cardiac conduction cells

Specialized cardiomyocytes are responsible for the initiation, conduction and propagation of electrical signals, due to their ability to autonomously fire. These cells are present in the sinoatrial node (SAN), the atrio-ventricular node (AVN), the bundle of His and Purkinje fibers (Figure 3). In the normal heart, the electrical signal begins by the spontaneous excitation in the SAN, which is an area of specialized cells located at the junction between the superior vena cava and the right atrium. It is the natural and primary pacemaker of the heart since it is responsible for the initiation of electrical signals at faster rate than other areas of the heart with pacemaker potential. This rate of electrical discharge, called sinus rhythm, will determine the number of heartbeats per minute (bpm), which is normally between 60-100 bpm. From the SAN, the signal is rapidly conducted through the atria to the AVN, which also contains specialized nodal cells, located between the atria and the ventricles, in the interatrial septum near the opening of the coronary sinus. The AVN delays the propagation of impulse to the ventricles, this delay is extremely important to ensure that the atria have contracted and

I.3

The electrical activity of the heart

I.3.1 Membrane potential

The sarcolemma of the cardiomyocyte defines two different aqueous ionic compositions of intracellular and extracellular, by maintaining large gradients of ions across these two compartments. This uneven distribution of ionic species, mainly potassium (K + ) and sodium (Na + ), across the membrane, leads to an electro-diffusion membrane potential, which is equal to the differential electrical potential across the sarcolemma. Cations diffuse passively (following their concentration gradients) across the sarcolemma through embedded selective ion channels. The opening and closing of these channels depend on the change in membrane potential. Thus these ion channels are called voltage-dependent. The intracellular medium is rich in K + (150 mM) and the extracellular medium is rich in both Na + (135-145 mM) and Ca 2+ (1-2 mM). These ion concentration gradients are maintained by the presence of ion pumps such as the Na + /K + ATPase, PMCA (Plasma Membrane Ca 2+ ATPase) and the Na + /Ca 2+ exchanger (NCX), which move ions against their electrochemical gradients.

I.3.2 Resting membrane potential

Like most excitable cells, atrial and ventricular cardiomyocytes have a resting state in which the electrical potential is stable across the sarcolemma. This state is called the resting membrane potential. It results from the selective permeability of the sarcolemma to K + ions.

At resting membrane potential, most ion channels are closed except the potassium channel Kir2.1 (responsible for I K1 ). The efflux of K + ions through this channel leads to a potential difference across the membrane of about -80 and -85 mV for atrial and ventricular cardiomyocytes, respectively, a value that is closest to the Nernst equilibrium potential of the K + ion. The resting membrane potential is less polarised in the atrial cells (Figure 5B) since they have lower expression of Kir2.1 transcripts compared to ventricular cells (Figure 4) 7 . In addition, atrial I K1 has 5-to 10-fold smaller current density, smaller elementary conductance,

and shorter channel open times compared to ventricular I K1 8,9 . As a consequence, less depolarizing current is required to reach the firing action potential threshold in atrial cells compared to ventricular cells, explaining the higher excitability of atrial cells.

In contrast to atrial and ventricular myocytes, SAN myocytes do not polarize enough to reach the level of the resting membrane potential and show slow spontaneous depolarization during diastole (Figure 5B). These diastolic depolarizations contribute to the ability of SAN to generate spontaneous and repetitive action potentials in the absence of external electrical stimulation. This depolarization results from low intensity of I K1, and the high expression of hyperpolarization-activated channel that conducts the inward-rectifying mixed Na + -K + 'funny' current (I f ) when the membrane potentials descend below -40 to -45 mV during the repolarization phase 10 .

I.3.3 Cardiac action potential and ionic currents

The cardiac action potential (AP) represents a transient depolarization wave that starts when the membrane depolarizes. It is the consequence of a cascade of ion movements, depending mostly on changes in ion channel permeability. Each cell type exhibits a different shape and duration of cardiac AP (Figure 4). This diversity is due to the variable expression of ion channels 7,11 .

The cardiac AP consists of five phases (Figure 5):

• Phase 0: The rapid depolarization phase (initial upstroke)

Excitation by electrical signals from adjacent cells activates the voltage-dependent sodium channels. Opening of these channels gives rise to the inward sodium current I Na , which has a short duration but high intensity, and which triggers the very fast change in the membrane potential (upstroke of the AP) toward the equilibrium (Nernst) potential of Na + .

Indeed, entrance of sodium ions further depolarizes the membrane, opening more sodium channels and leading to phase 0 depolarization (initial upstroke). Closing of these channels is rapid, but they need time to be reactivated. This time is called the refractory period, it is essential for cardiac cells as it prevents the genesis of any new AP until these channels are able to open a second time.

In nodal cells, the slow depolarization during the resting membrane potential leads to the inactivation of most sodium channels, inhibiting them from participating in the depolarization phase. As a consequence, the depolarization phase in these cells is mainly due to the I Ca,L and the I Ca,T calcium currents (Figure 5B).

• Phase 1: The early repolarization phase Inactivation of sodium channels, and opening of certain potassium channels (K v 4.2, K v 4.3, K v 1.4) responsible for the transient potassium current I to, lead to a rapid outflow of K + and early membrane repolarization. In human ventricles, differences in density and ratedependent properties of I to in endocardial and epicardial layers lead to transmural electrical gradients and variation in the AP duration 12 .

• Phase 2: The plateau phase

The plateau phase of the cardiac AP represents the balance between the inward depolarizing and the outward repolarizing currents. The inward L-type Ca 2+ current, generated by Ca v 1.2 calcium channels, is the main current maintaining the plateau phase. The Ca v 1.2 channels activate slowly at membrane potentials higher than -40mV, and their inactivation depends on membrane potential and intracellular Ca 2+ concentration. They also have an important role in triggering the entry of Ca + into the cell, which, in turn, leads to SR Ca 2+ release that is responsible for excitation-contraction coupling. At the end of the plateau phase, the rectifier potassium channels start to be activated accounting for the replarization phase.

• Phase 3: The repolarization phase

The rectifying potassium channels are in charge of the predominant outward repolarizing currents. They consist of two channels with different kinetics and conductance: the rapid activating (K v 11.1, I Kr current) and the slow activating (K v 7.1, I Ks current) channels.

In the atrium, the ultra rapid activating potassium channel (K v 1.5, I Kur current) also exists.

Calcium channel closure and the increase in the outward potassium currents I Ks , I Kr , I Kur and I K1 lead to membrane repolarization and phase 3 of the cardiac AP. In the ventricles, the beginning of Phase 3 is mainly due to the I Ks and I Kr currents, then I K1 current participates at the end of the membrane repolarization and in the maintenance of the resting membrane potential. The atrial AP (Figure 5B) has a less pronounced plateau phase compared to the ventricular AP. This is due to differences in the repolarizing channels. The K v 1.5 channel is highly expressed in the atrium compared to the ventricles (Figure 4) as it is specific to atrial cardiomyocytes 7,9 , and I kur activates more rapidely than I Kr.

• Phase 4: The resting membrane potential When the repolarization is complete, the cell membranes return back to their resting membrane potential, which is maintained by the Kir2.1 potassium channel (I K1 current).

I.3.4 Electrical communication between cardiac cells

The propagation of electrical activity in the heart needs special intercellular junctions, called gap junctions. These structures directly connect the cytoplasm of two adjacent cardiomyocytes and form low resistance pathways allowing the electrical impulse to pass rapidly between cells, ensuring synchronous contraction of the myocardium. Gap junctions of Ca 2+ from the SR into the cytoplasm. This process is known as "calcium-induced calcium release". Ca 2+ released from the SR binds to troponin-C, which causes the conformational change of the tropomyosin and allows actin-myosin interaction. ATP hydrolysis releases energy that allows the myosin to pull the actin filaments leading to muscle contraction.

Binding of ATP to the myosin head leads to its detachment from actin, while ATP hydrolysis leads to rebinding of the myosin head to actin. Then, relaxation takes place when the intracellular Ca 2+ concentration returns to normal. Most of the intracellular Ca 2+ is reuptaken by the SR through the SR Ca 2+ -ATPase (SERCA pump) or it moves out of cells through the Na + /Ca 2+ exchanger or sarcolemmal Ca 2+ -ATPase 16 .

I.3.6 Electrocardiogram

The electrical activity of the heart can be recorded through electrodes placed on the patient's body. This recording is called an electrocardiogram (ECG). ECG is a non-invasive test that has a key role in the diagnosis of cardiac diseases, like ischemic heart disease and cardiac arrhythmias. ECG provides valuable informations, such as heart rate and regularity, conduction, and chamber sizes and position. These data are obtained through the standard interpretation of the different waves and intervals of the ECG (Figure 7) 17 . These waves represent the sequence of depolarization and repolarization of both the atria and ventricles: • P wave: rounded, positive wave that represents atrial depolarization. Usually it is between 80 to 100 ms in duration. We can calculate atrial rate by measuring the time interval between P waves. • QRS complex: represents ventricular depolarization, it is normally between 60 and 100 ms. Ventricular rate can be calculated by measuring the interval between QRS complexes.

• T wave: represents ventricular repolarization.

• PR interval: is the interval between the beginning of the P wave and the beginning of QRS complex. Normally it is between 120 and 200 ms. It represents the time the electrical signal needs to propagate from the SAN to the AVN. A PR interval longer than 200 ms means there is an AV block.

• ST segment: represents the time during which the whole ventricles are depolarized and is normally isoelectric. An abnormality of ionic currents during this period is most often translated to a ST segment elevation or depression relative to the isoelectric line. To evaluate these shifts, we use the J point, which is the point at which the QRS complex meets the ST segment.

J point J point

Physiologically, it is located in the extension of the PQ segment, which is used as the baseline in order to look for a shift of J point.

• QT interval: represents the time needed for ventricular depolarization and repolarization, it is approximately equal to the duration of the action potential of ventricular myocytes. The duration of the QT interval varies according to heart rate; it decreases when heart rate increases and vice versa. For this reason, it is better to use the corrected QT interval, which can be calculated according to Bazett's formula, QTc = QT/√RR. The normal values are 350 to 450 ms for males, and 360 to 460 ms for females 18 . Short and long QT intervals can be indicative of inherited cardiac arrhythmias called short and long QT syndromes (SQTS and LQTS), respectively.

To record an ECG, an electrode is attached to each of the limbs, and six other electrodes are placed on the chest. With these ten electrodes, twelve leads can be recorded (Figure 8). Each lead represents a view of the electrical activity of the heart from a particular angle across the body. Six limb leads (I, II, III, aVR, aVL, and aVF) view the heart in a vertical plane, and six precordial leads (V1-V6) view the heart in the horizontal plane.

Regarding the anatomical relationships, leads II, III, and aVF view the inferior surface of the heart, leads V1 to V4 view the anterior surface, leads I, aVL, V5, and V6 view the lateral surface.

II. Cardiac arrhythmias II.1 Mechanisms of cardiac arrhythmias

Any abnormality in the heart rate or in the regularity of heart rhythm is called cardiac arrhythmia. Cardiac arrhythmias can occur in a structurally normal heart as well as in heart diseases. They have a highly variable clinical presentation, but they share common electrophysiological pathways. Three well-known cellular mechanisms are responsible for most of cardiac arrhythmias: automaticity, triggered activity and reentry 19 .

II.1.1 Automaticity

Automaticity mechanisms of cardiac arrhythmia can be either due to altered normal automaticity or abnormal automaticity.

In altered normal automaticity, the electrical signal starts in one conductive cell region like the SA or AV nodes with abnormal suppression or enhancement. We can understand the different mechanisms underlying the altered normal automaticity by looking at the AP of nodal cells (Figure 5B). Any factor that changes the maximum diastolic potential, the threshold potential for AP initiation and the rate or slope of phase 4 depolarization, can be responsible for altered impulse initiation. For example, the parasympathetic nervous system decreases heart rate by hyperpolarizing the cells through increasing conductance of K + through the G-protein-gated K + channel I KACh and decreasing I f activity 19,20 . In contrast, the sympathetic nervous system increases heart rate by increasing I f current, enhancing the spontaneous depolarization of phase 4 19 .

In abnormal automaticity, atrial and ventricular cardiomyocytes, which normally do not have spontaneous activity, demonstrate automaticity properties. This occurs when the maximum diastolic potential is shifted toward the threshold potential. Many ion currents are involved in this process, resulting in a net inward depolarizing current with decreased K + conductance. Premature beats, as well as atrial and ventricular tachycardia are examples of abnormal automaticity related arrhythmias. Increased extracellular K + levels and low intracellular pH can cause cardiac arrhythmia through this mechanism 21 .

II.1.2 Triggered activity

Triggered activity is defined as impulse initiation caused by an after-depolarization, which is a membrane potential oscillation occuring during or immediately after an AP [START_REF] Zipes | Mechanisms of Clinical Arrhythmias[END_REF] .

Based on the time elapsed from the preceding AP, two types of after-depolarization are present: early after-depolarization (EAD) and delayed after-depolarization (DAD).

EADs occur during phase 2 or 3 of the AP (Figure 9A andB). AP prolongation, favored by lower outward potassium currents or an increase in calcium or sodium inward currents, as well as low heart rate, can all enhance the development of EAD. EAD is the underlying mechanism of arrhythmias seen in patients with LQTS, such as torsade de pointes due to polymorphic ventricular tachycardia (VT) [START_REF] Yan | Phase 2 Early Afterdepolarization as a Trigger of Polymorphic Ventricular Tachycardia in Acquired Long-QT Syndrome Direct Evidence From Intracellular Recordings in the Intact Left Ventricular Wall[END_REF] .

DADs (Figure 9C) occur during phase 4, due to an increased intracellular Ca + concentration, which mediates the oscillations of membrane potential leading to new AP initiation if they reached the stimulation threshold. Enhanced heart rate is a predisposing factor for DAD development. Digitalis toxicity-induced tachycardia [START_REF] Antman | Digitalis Toxicity[END_REF] and catecholaminergic polymorphic VT [START_REF] Leenhardt | Catecholaminergic Polymorphic Ventricular Tachycardia[END_REF] are examples of DAD induced arrhythmias.

II.1.3 Re-entry

The electrical signals in the heart normally propagate in a single direction, from the SAN down to the ventricles. Normally, depolarized cells enter a refractory period, during which they are unable to be re-excited, since most ion channels are inactivated. In re-entry excitation (Figure 10), an area that was not activated during the initial wave of depolarization due to unidirectional block (gray area), can be depolarized by the retrograde signal (blue line).

Action potentials exiting the block, could re-excite areas that have already recovered from the refractory period and AP can continue. However, if the AP finds non-excitable tissue, it will end. So, several elements are needed to lead to a re-entry: (1) substrate; myocardial areas that differ in conductivity and refractoriness, (2) unidirectional block to allow conduction propagation in one direction and conduction block in the other (3) slow conduction time to allow recovery from the refractory state of tissue present proximal to the block and (4) an initiating trigger.

II.2 Atrial arrhythmias

Abnormalities in heart rate or rhythm arising from the atrium are called atrial arrhythmias. There are several different types of atrial arrhythmias, which are discussed below.

II.2.1 Supraventricular tachycardia (SVT)

Supraventricular tachycardia is a rapid heart rhythm originating at or above the atrio-ventricular node. There are different types of SVT; here we will discuss those that are the most common.

• Atrial fibrillation (AF)

Discussed in chapter II.2.3.

• Atrial flutter (AFL)

Atrial flutter is the second most common pathological SVT. It is a macro-reentrant arrhythmia characterized by a regular atrial rhythm of 250-350 bpm, with AVN conduction block, usually 2:1, resulting in a ventricular rate of 140-150 bpm. In the presence of variable AV conduction, the heart rate is irregular. Patients with paroxysmal AFL and rapid ventricular response present with palpitation, dyspnea, chest discomfort, pre-syncope and weakness. There are different types of AFL according to the mechanisms causing the arrhythmia [START_REF] Lee | Atrial flutter: a review of its history, mechanisms, clinical features, and current therapy[END_REF] . The most common mechanism is counterclockwise (CCW) right AFL, characterized by circular activation around the tricuspid annulus. The activation wave propagates up over the septal right atrium, and then moves down over the anterolateral right atrium, passing through the tricuspid annulus and the inferior vena cava to again reach the septal right atrium 27 (Figure 11). On the ECG, there are flutter waves with a 'saw-tooth pattern that are negative in inferior leads (II, III, aVF) and positive in V1 (Figure 12). Atrial flutter is more common in men. Advanced age and other medical conditions, like heart failure, chronic pulmonary disease, previous stroke, and myocardial infarction can increase the risk of AFL. The main targets for the treatment of AFL are rate and rhythm control, prevention of stroke, ablation and treatment of the underlying disease [START_REF] Lee | Atrial flutter: a review of its history, mechanisms, clinical features, and current therapy[END_REF] .

• Atrial tachycardia

Atrial tachycardia is a focal tachycardia resulting from micro re-entrant circuits or an automatic focus. It is characterized by its occurence in short repetitive bursts as well as warmup phenomena in which the atrial rate increases slightly over the first 5 to 10 seconds before stabilizing. On ECG, there is a P-wave preceding each QRS complex (Figure13). However, if the rate is very rapid, P-waves may be hidden by QRS complexes. AVNRT is due to a re-entrant loop that involves the AVN and the atrium. It usually affects adult people. In this arrhythmia, AVN has two conduction pathways; both slow and rapid, and the electrical signal leaving the AV node can propagate into anterograde or retrograde directions. In sinus rhythm, the electrical impulse propagates down the fast pathway, enters the distal end of the slow pathway and both impulses cancel each other.

However, if premature atrial contraction reaches the AV node during the refractory period of the fast pathway, the impulse will propagate down the slow pathway and when it reaches the end of the slow pathway, the fast pathway will have recovered from the refractory state and impulse may then move retro-gradely through the fast pathway, creating a circuitous movement. In typical AVNRT (slow-fast) the retrograde conduction occurs via the fast pathway and the anterograde via the slow pathway. Consequently atrial activation appears before, at, or just after the onset of the QRS complex (Figure 14). PAC occurs when a region of the atria other than the SAN depolarizes before the SAN, leading to premature beats. It is a common and benign arrhythmia seen in stressful conditions, and may occur after caffeine or alcohol intake. On ECG, as the signal in PAC does not start in the SAN, the P-wave has an abnormal shape with longer RR interval after the PAC. QRS is normal as the signal propagates to the ventricles through the AVN (Figure 16).

Sometimes the atrial beat is so premature that it reaches the AVN during its refractory time, and as a consequence, the signal does not propagate to the ventricles, leading to the absence of QRS complex after the P-wave.

II.2.3 Atrial fibrillation (AF)

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting approximately 1-2% of the population [START_REF] Lemmens | Genetics of atrial fibrillation and possible implications for ischemic stroke[END_REF] . Its prevalence increases with age from 0.5% in those aged 50-59 years to 9.0% in those aged 80-90 years [START_REF]Atrial Fibrillation: National clinical guideline for management in primary and secondary care[END_REF] . AF is a tachy-arrhythmia characterized by uncoordinated activity of the atrial muscle leading to irregular heartbeat. This uncoordinated activity results in reduced ventricular filling and blood stasis in the atria, which can lead to heart failure and thrombembolic stroke. On the ECG, it is characterised by the absence of P wave, fibrillation (f) waves resulting in an oscillating irregular baseline, variable RR intervals, and normal QRS complexes with ventricular rate between 60 to 220 bpm, depending mostly on patient's age and the AVN state (if there is an underlying AV disease or if the patient has received medications that affect AVN conduction) (Figure 17). ECG is the gold standard test for the diagnosis of AF (Figure 17). However, a Holter monitor (a portable device for continuously recording ECG for at least 24 hours) may be needed, if the ECG does not demonstrate AF despite a strong suspicion.

II.2.3.3 Management of AF

The first step in the management of AF is the clinical evaluation of the patient's cardiac stability. If the patient is hemodynamically unstable, emergency electrical cardioversion is needed. However, if the patient has a stable clinical condition, then history, physical examination and diagnostic tests should be done in order to determine the potential causes, triggers, and co-morbid conditions.

AF treatment aims to improve symptoms and prevent future complications. Prevention of AF complications through anti-thrombotic therapy, ventricular rate control and treatment of underlying disease may improve the symptoms. However, in some cases rhythm control is also needed. The main approaches for the treatment of AF consist of anticoagulation, rate and rhythm control, treatment of any underlying diseases, and ablation [START_REF] Heart | Guidelines for the management of atrial fibrillation: the Task Force for the Management of Atrial Fibrillation of the European Society of Cardiology (ESC)[END_REF][START_REF] Camm | 2012 focused update of the ESC Guidelines for the management of atrial fibrillation: an update of the 2010 ESC Guidelines for the management of atrial fibrillation. Developed with the special contribution of the European Heart Rhythm Association[END_REF] (Figure 18).

Antithrombotic therapy

Treatment with antithrombotic drugs depends mainly on the presence or absence of risk factors for developing stroke and thrombo-embolism. There are many score systems that can be used to assess stroke risk [START_REF] Heart | Guidelines for the management of atrial fibrillation: the Task Force for the Management of Atrial Fibrillation of the European Society of Cardiology (ESC)[END_REF] . According to this score system, the choice of anticoagulant will be decided [START_REF] Camm | 2012 focused update of the ESC Guidelines for the management of atrial fibrillation: an update of the 2010 ESC Guidelines for the management of atrial fibrillation. Developed with the special contribution of the European Heart Rhythm Association[END_REF] .

Rate control

This After cardioversion, maintenance of sinus rhythm can be achieved with antiarrhythmic drugs, like flecainide and others, according to the cardiac status. It is recommended to start with the safer drug, even though it may be less efficacious, before using a more effective one with more side effects. Moreover, the type of medication used depends on the patient's medical background.

Catheter ablation

Catheter ablation is an invasive procedure used to ablate (destroy) the abnormal cardiac tissue causing the arrhythmia. In AF, it is mainly used to ablate the firing spots responsible for triggering activity, or to create lines of electrical block in the atria that interrupt the abnormal electrical circuits, allowing the pacemaker to regain its control of heart beats.

As ablation is an invasive procedure, it is usually reserved for patients who have remained symptomatic despite the use of optimal rate and rhythm control therapy. Before deciding to perform catheter ablation, benefit-risk ratios of ablation must be evaluated. For example, it is usually used in patients with symptomatic paroxysmal AF that is resistant to at least one anti-arrhythmic drug. However, in persistent or long standing persistent AF, or in patients with heart disease, medical therapy is recommended before ablation since the benefitrisk ratio in these cases is not well established.

II.2.3.4 Mechanisms of AF

AF results from abnormalities in the signaling pathway; where the electrical signal is generated from both the sinus node and multiple random regions in the atrium (Figure 19).

Three classical hypotheses underlie the development of AF: focal activity, singlecircuit re-entry and multiple-circuit re-entry [START_REF] Fatkin | Genes and Atrial Fibrillation A New Look at an Old Problem[END_REF] .

Focal activity results from spontaneous rapid firing of a single or multiple ectopic atrial foci, usually in the left atrium, due to an increase in depolarization, or the presence of after-depolarization. Pulmonary veins account for trigger activity in 85-95% of patients with paroxysmal AF, and other sites, other than the pulmonary veins, appear to be responsible for the rest [START_REF] Lee | Catheter ablation of atrial arrhythmias: state of the art[END_REF] .

Re-entrant arrhythmia circuits arise from differential impulse propagations within atrial tissue, and necessitate the existence of a focal region of conduction block. This block can be either fixed or dynamic. Fixed block can be structural or electrophysiological, like myocardial fibrosis and heterogeneity of ion channels. Dynamic or "functional" factors include membrane-voltage and intracellular Ca 2+ cycling properties that determine AP duration and conduction velocity.

In addition, according to Coumel's triangle of arrhythmogenesis [START_REF] Coumel | Arrhythmias as predictors of sudden death[END_REF] , the mechanism of AF can be explained in terms of substrate, triggers, and modulating factors [START_REF] Xiao | The genetics of atrial fibrillation: from the bench to the bedside[END_REF] .

Electrical and structural remodeling of the heart is the most prevalent substrate.

Electrical remodeling include mechanisms involved in intracellular Ca 2+ handling, leading to shortened refractory period and enhancement of re-entry. Restoration of sinus rhythm can reverse the electrical remodeling. In structural remodeling, there is a loss of cardiomyocytes, diffuse and patchy fibrosis, and scarring. These structural changes slow the conduction velocity and enhance electrical uncoupling, allowing the genesis of AF. Structural remodeling is irreversible even with the restoration of normal sinus rhythm.

Regarding the triggers, the exact mechanism that underlies the initiation of these firings is unclear. However, enhanced automaticity, after-depolarization and re-entry could be the possible underlying mechanisms. A trigger can then interact with the substrate to generate AF; and modulating factors, such as atrial and pulmonary vein stretch, or hormonal and autonomic nervous factors, may facilitate the initiation or the further development of AF.

II.2.3.5 Genetics of AF

The majority of AF is sporadic and non-familial, as most cases of AF occur with structural heart disease. Nevertheless, familial forms of AF have been identified. In 1943, the first familial form of AF was reported in three brothers diagnosed with autosomal dominant AF [START_REF] Wolff | Familial Auricular Fibrillation[END_REF] . After that, several studies have confirmed the heritability of AF [START_REF] Darbar | Familial atrial fibrillation is a genetically heterogeneous disorder[END_REF][START_REF] Fox | PArental atrial fibrillation as a risk factor for atrial fibrillation in offspring[END_REF][START_REF] Ellinor | Familial aggregation in lone atrial fibrillation[END_REF][START_REF] Arnar | Familial aggregation of atrial fibrillation in Iceland[END_REF][START_REF] Marcus | A first-degree family history in lone atrial fibrillation patients[END_REF][START_REF] Christophersen | Familial Aggregation of Atrial Fibrillation A Study in Danish Twins[END_REF][START_REF] Lubitz | Association between Familial Atrial Fibrillation and Risk of New-onset Atrial Fibrillation[END_REF] . Familial AF is particularly present in patients with lone AF, where approximately 30% of probands have first degree relatives with AF [START_REF] Darbar | Familial atrial fibrillation is a genetically heterogeneous disorder[END_REF][START_REF] Ellinor | Familial aggregation in lone atrial fibrillation[END_REF][START_REF] Marcus | A first-degree family history in lone atrial fibrillation patients[END_REF] . It has been shown that 5% of patients with AF, and 15% of patients with lone AF had a positive family history (one to nine additional relatives affected) [START_REF] Darbar | Familial atrial fibrillation is a genetically heterogeneous disorder[END_REF] . In addition, in the same study four multi-generation families were identified in which AF was transmitted in an autosomal dominant manner [START_REF] Darbar | Familial atrial fibrillation is a genetically heterogeneous disorder[END_REF] . Furthermore, other studies have showed that 38% [START_REF] Ellinor | Familial aggregation in lone atrial fibrillation[END_REF] , or 41% [START_REF] Marcus | A first-degree family history in lone atrial fibrillation patients[END_REF] of lone AF patients had a first or second degree family member with AF. Moreover, in a prospective cohort study within the Framingham Heart Study, it has been shown that having one parent with AF doubled the risk of predicted AF in the offspring compared to no parental AF. In addition, the risk increased when the parents developed AF before the age of 75 years [START_REF] Fox | PArental atrial fibrillation as a risk factor for atrial fibrillation in offspring[END_REF] . In agreement with this study, it has recently been shown that the risk of developing lone AF at a young age, increases with greater numbers of affected relatives and decreasing age at onset of AF in these relatives [START_REF] Oyen | Familial aggregation of lone atrial fibrillation in young persons[END_REF] . All this evidence supports the theory that genetic factors may have a role in AF development.

Rare genetic variants have been identified through either linkage analysis or candidate gene approaches in AF families. Even though these mutations have been identified in two or more family members with lone AF, the penetrance of AF in theses families is highly variable. In addition, in the more common secondary AF, not all patients with secondary risk factors, like hypertension or heart failure, develop AF, suggesting the presence of other modifiers such as single nucleotide polymorphisms (SNPs), which could alter AF susceptibility. Genome-wide association studies (GWAS) have identified a number of SNPs that can increase AF susceptibility.

• AF loci identified by linkage analysis (Table 1) Brugada et al. in 1997 discovered the first AF loci on chromosome 10 (10q22-q24) in three different families with autosomal dominant AF [START_REF] Brugada | Identification of a Genetic Locus for Familial Atrial Fibrillation[END_REF] . After that, several loci have been identified in familial AF (Table 1). Screening for candidate genes in these loci led to the identification of the first gene (KCNQ1) implicated in AF [START_REF] Chen | KCNQ1 gain-of-function mutation in familial atrial fibrillation[END_REF] , which encodes the α-subuint of the delayed-rectifier cardiac potassium channel (I Ks ). This identification led researchers to think that other genes encoding ion channels or subunits and factors that regulate ion channels, could be involved in AF development. Consequently, other genes have been identified by linkage studies and candidate-gene approaches. However, for more than half of the loci, the genes implicated in AF development have not yet been identified (Table 1). In most of these studies, AF has an autosomal dominant inheritance pattern [START_REF] Brugada | Identification of a Genetic Locus for Familial Atrial Fibrillation[END_REF][START_REF] Chen | KCNQ1 gain-of-function mutation in familial atrial fibrillation[END_REF][START_REF] Ellinor | Locus for Atrial Fibrillation Maps to Chromosome 6q14-16[END_REF][START_REF] Volders | Mapping a novel locus for familial atrial fibrillation on chromosome 10p11-q21[END_REF][START_REF] Darbar | Prolonged Signal-Averaged P-Wave Duration as an Intermediate Phenotype for Familial Atrial Fibrillation[END_REF][START_REF] Hodgson-Zingman | Atrial natriuretic peptide frameshift mutation in familial atrial fibrillation[END_REF] , except in one study where AF was inherited in an autosomal recessive manner, occurring during the fetal stage, and associated with neonatal sudden death [START_REF] Oberti | Genome-Wide Linkage Scan Identifies a Novel Genetic Locus on Chromosome 5p13 for Neonatal Atrial Fibrillation Associated With Sudden Death and Variable Cardiomyopathy[END_REF] .

Table 1: Summary of AF-susceptibility loci identified by linkage analysis

• Monogenic mutations in AF Several studies have identified rare variants associated with AF in genes encoding ion channels and accessory subunits, cardiac gap junctions, and signalling molecules. Up to date, AF has been associated with mutations in 25 different genes [START_REF] Olesen | Atrial fibrillation: the role of common and rare genetic variants[END_REF] . Most of these genes encode ion channel subunits involved in cardiac depolarization or repolarization. Functional studies of these mutations showed both gain-and loss-of-function effects, increasing the complexity of AF. Moreover, these rare mutations occur in less than 1% of AF patients [START_REF] Xiao | The genetics of atrial fibrillation: from the bench to the bedside[END_REF] , suggesting that other rare or common variants, in addition to the environmental factors, could contribute to AF genesis.

o Ion channel mutations (Table 2)

Mutations in potassium channel genes

The voltage-gated potassium currents (I Ks , I Kr , I Kur ) are involved in the repolarization phase of the atrial AP. Other potassium channels (I K1 ) are of importance for the control of resting potential. Gain-of-function as well as loss-of-function mutations within potassium channel genes have been identified in AF.

KCNQ1

The KCNQ1 gene encodes the pore-forming α subunit, K v LQT1, of the cardiac I Ks mutations have been identified in sporadic or familial cases of AF [START_REF] Hong | De novo KCNQ1 mutation responsible for atrial fibrillation and short QT syndrome in utero[END_REF][START_REF] Otway | Stretch-sensitive KCNQ1 mutation A link between genetic and environmental factors in the pathogenesis of atrial fibrillation?[END_REF][START_REF] Das | Mutation in the S3 segment of KCNQ1 results in familial lone atrial fibrillation[END_REF][START_REF] Abraham | Augmented potassium current is a shared phenotype for two genetic defects associated with familial atrial fibrillation[END_REF][START_REF] Bartos | A KCNQ1 Mutation Causes a High Penetrance for Familial Atrial Fibrillation[END_REF] .

Loss-of-function of the I Ks potassium current leads to the prolongation of cardiac repolarization and LQTS. Numerous loss-of-function mutations in KCNQ1 have been identified in LQTS [START_REF] Herbert | KCNQ1 gene mutations and the respective genotype-phenotype correlations in the long QT syndrome[END_REF][START_REF] Morita | The QT syndromes: long and short[END_REF] . The study of a cohort of LQTS patients showed that 2% of them (especially KCNQ1-related LQT1) have early-onset AF (aged less than 50 years) compared to a background prevalence of 0.1% [START_REF] Johnson | Prevalence of early-onset atrial fibrillation in congenital long QT syndrome[END_REF] . In addition, it has been shown that some LQT1 mutations can lead to variable mixed clinical pictures of AF and LQTS [START_REF] Lundby | KCNQ1 mutation Q147R is associated with atrial fibrillation and prolonged QT interval[END_REF][START_REF] Bartos | R231C mutation in KCNQ1 causes long QT syndrome type 1 and familial atrial fibrillation[END_REF][START_REF] Henrion | Overlapping Cardiac Phenotype Associated with a Familial Mutation in the Voltage Sensor of the KCNQ1 Channel[END_REF] .

In addition to KCNQ1, mutations in genes encoding the auxiliary subunits (KCNE1, KCNE2, KCNE5) and the atrial natriuretic peptide (NPPA), that regulate the I Ks potassium current, have also been identified in patients with AF [START_REF] Hodgson-Zingman | Atrial natriuretic peptide frameshift mutation in familial atrial fibrillation[END_REF][START_REF] Abraham | Augmented potassium current is a shared phenotype for two genetic defects associated with familial atrial fibrillation[END_REF][START_REF] Olesen | mutations in the potassium channel subunit kcne1 are associated with early onset familial atrial fibrillation[END_REF][START_REF] Yang | Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation[END_REF][START_REF] Ravn | Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation[END_REF] . Functional studies of these mutations showed gain-of-function effects on the I Ks potassium current.

KCNH2

The KCNH2 gene encodes the Kv11.1 α-subunit underlying I Kr , involved in the repolarization phase of cardiac AP. The KCNH2 gain-of-function mutation N588K has been identified in families with SQTS and AF. Increased I Kr leads to shortening of AP duration and refractoriness, and enhancement of re-entrant arrhythmia [START_REF] Hong | Short QT syndrome and atrial fibrillation caused by mutation in KCNH2[END_REF][START_REF] Brugada | Sudden Death Associated With Short-QT Syndrome Linked to Mutations in HERG[END_REF] . In addition, a mutation in the KCNE3 gene encoding the MiRP2 accessory subunit has been identified in patients with lone AF, and a functional study showed a gain-of-function effect of this mutation on I Kr [START_REF] Lundby | KCNE3 mutation V17M identified in a patient with lone atrial fibrillation[END_REF] .

KCNA5

The KCNA5 gene encodes the K v 1.5 subunit responsible for the atrial specific repolarizing current I Kur . Both gain- [START_REF] Mann | Epistatic Effects of Potassium Channel Variation on Cardiac Repolarization and Atrial Fibrillation Risk[END_REF][START_REF] Christophersen | Genetic variation in KCNA5: impact on the atrial-specific potassium current IKur in patients with lone atrial fibrillation[END_REF] and loss-of-function mutations [START_REF] Christophersen | Genetic variation in KCNA5: impact on the atrial-specific potassium current IKur in patients with lone atrial fibrillation[END_REF][START_REF] Olson | Kv1.5 channelopathy due to KCNA5 loss-offunction mutation causes human atrial fibrillation[END_REF][START_REF] Yang | Novel KCNA5 loss-of-function mutations responsible for atrial fibrillation[END_REF][START_REF] Yang | Novel KCNA5 mutation implicates tyrosine kinase signaling in human atrial fibrillation[END_REF] in KCNA5 have been identified in patients with AF, which further supports the hypothesis that shortening as well as prolongation of the AP duration can increase susceptibility to AF.

KCND3

The KCND3 gene encodes the pore-forming K + channel subunit K V 4.3 that interacts with the K + channel-interacting protein (KChIP2) underlying the transient outward K + current (I to ). Some gain-of-function mutations in the KCND3 gene have been involved in Brugada syndrome [START_REF] Giudicessi | Transient Outward Current (Ito) Gain-of-Function Mutations in the KCND3-Encoded Kv4.3 Potassium Channel and Brugada Syndrome[END_REF][START_REF] Giudicessi | Novel Mutations in the KCND3-Encoded Kv4.3 K+ Channel Associated with Autopsy-Negative Sudden Unexplained Death[END_REF] . Recently, a new gain-of-function mutation in KCND3 (A545P) was identified in a proband with early onset persistent AF at the age of 22. The proband did not have Brugada syndrome even after flecainide challenge. Computer simulation showed that the mutant is associated with a reduction of the AP duration [START_REF] Olesen | A novel KCND3 gain-of-function mutation associated with early-onset of persistent lone atrial fibrillation[END_REF] .

KCNJ2

The KCNJ2 gene encodes the Kir2.1 channel protein that underlies the inward rectifier potassium currents I K1. The I K1 is mainly involved at the end of the repolarization phase and in the resting membrane potential. In atrial cells, Kir2.1 has lower expression and smaller current density and conductance with shorter opening times compared to the ventricles. These differences lead to a more depolarized resting membrane potential and highly excitable atrial cells 8,9 . As of today, three gain-of-function mutations in KCNJ2 have been identified in patients with AF [START_REF] Xia | A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation[END_REF][START_REF] Kharche | Atrial proarrhythmia due to increased inward rectifier current (I(K1)) arising from KCNJ2 mutation--a simulation study[END_REF][START_REF] Hattori | A novel gain-of-function KCNJ2 mutation associated with short-QT syndrome impairs inward rectification of Kir2.1 currents[END_REF][START_REF] Deo | KCNJ2 mutation in short QT syndrome 3 results in atrial fibrillation and ventricular proarrhythmia[END_REF] . The increase in I KI accelerates the repolarization phase and the return to resting membrane potential, leading to shortening of AP duration and creation of a favourable substrate for re-entry, a mechanism involved in AF.

KCNJ8

The KCNJ8 gene encodes the cardiac K ATP channel Kir6.1 which facilitates a nonvoltage-gated inwardly rectifying potassium current, leading to a shortening of the AP duration under conditions of metabolic stress 11 . One gain-of-function mutation has been identified in KCNJ8 gene in lone AF [START_REF] Delaney | A KCNJ8 mutation associated with early repolarization and atrial fibrillation[END_REF][START_REF] Medeiros-Domingo | Gain-offunction mutation S422L in the KCNJ8-encoded cardiac K(ATP) channel Kir6.1 as a pathogenic substrate for J-wave syndromes[END_REF] .

ABCC9

The ABCC9 gene encodes the regulatory SUR2A subunit of the cardiac K ATP channel.

One loss-of-function mutation has been identified in a female patient with early-onset adrenergic AF originating from the vein of Marshall 87 .

Mutations in genes encoding sodium channel subunits

The SCN5A gene encodes the α-subunit of the cardiac sodium channel Na v 1.5, which is responsible for the depolarization phase of the cardiac AP. Many mutations in SCN5A have been identified in several arrhythmic cardiac diseases like Brugada syndrome, long QT syndrome, sick sinus syndrome and atrial fibrillation.

In AF, some studies have reported SCN5A mutations associated with gain-of-function effects [START_REF] Makiyama | A novel SCN5A gain-of-function mutation M1875T associated with familial atrial fibrillation[END_REF][START_REF] Li | Gain-of-function mutation of Nav1.5 in atrial fibrillation enhances cellular excitability and lowers the threshold for action potential firing[END_REF] , loss-of-function effects [START_REF] Laitinen-Forsblom | SCN5A mutation associated with cardiac conduction defect and atrial arrhythmias[END_REF][START_REF] Mcnair | SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia[END_REF][START_REF] Olson | Sodium channel mutations and susceptibility to heart failure and atrial fibrillation[END_REF][START_REF] Ellinor | Cardiac sodium channel mutation in atrial fibrillation[END_REF] , or gain-and loss-of-function with increased late sodium current (current that inactivates very slowly or does not inactivate at all, I NaL ) [START_REF] Olesen | High Prevalence of Long QT Syndrome-Associated SCN5A Variants in Patients With Early-Onset Lone Atrial FibrillationClinical Perspective[END_REF][START_REF] Darbar | Cardiac sodium channel (SCN5A) variants associated with atrial fibrillation[END_REF] .

The M1875T mutation was identified in a Japanese family with autosomal dominant AF [START_REF] Makiyama | A novel SCN5A gain-of-function mutation M1875T associated with familial atrial fibrillation[END_REF] . The majority of the patients had a similar clinical picture of palpitation due to PACs, starting in their teens, then progressing to paroxysmal and persistent AF, without QT prolongation [START_REF] Makiyama | A novel SCN5A gain-of-function mutation M1875T associated with familial atrial fibrillation[END_REF] . The K1493R mutation was discovered in a woman and her son with lone atrial fibrillation [START_REF] Li | Gain-of-function mutation of Nav1.5 in atrial fibrillation enhances cellular excitability and lowers the threshold for action potential firing[END_REF] . Both mutations prolonged the time constants of both fast and slow inactivation, and caused positive shift of the steady-state inactivation, suggesting a gain-of-function effect.

In addition, the mutant M1875T increased the peak sodium current I Na . Neither of these mutations increased the late sodium current. The gain-of-function of the cardiac sodium channel could lead to repolarization failure, or early after-depolarizations inducing triggered activities. Also, it might increase the conduction velocity and facilitate the maintenance of fibrillation waves. All of these conditions favor the occurrence of AF. In addition, functional study of the K1493R mutant in HL-1 atrial cardiomyocytes showed enhancement of the window current, lower threshold for AP firing, and spontaneous depolarizations, suggesting a cellular hyperexcitability, a phenomenon that can lead to AF [START_REF] Li | Gain-of-function mutation of Nav1.5 in atrial fibrillation enhances cellular excitability and lowers the threshold for action potential firing[END_REF] .

The loss-of-function SCN5A mutation D1275N has been reported in two families presenting with atrial arrhythmias (AF, cardiac conduction disease, and sick sinus syndrome)

as well as dilated cardiomyopathy [START_REF] Mcnair | SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia[END_REF][START_REF] Olson | Sodium channel mutations and susceptibility to heart failure and atrial fibrillation[END_REF] . This mutation was also found in a third family with atrial arrhythmia and intra-cardiac conduction defects but without prominent ventricular dilatation [START_REF] Laitinen-Forsblom | SCN5A mutation associated with cardiac conduction defect and atrial arrhythmias[END_REF] . Another mutant, N1986K, was identified in a proband with AF, and his father who presented with AF and sick sinus syndrome requiring pacemaker implantation [START_REF] Ellinor | Cardiac sodium channel mutation in atrial fibrillation[END_REF] . Both mutations, D1275N and N1986K, have a loss-of-function effect by positive shift of activation and negative shift of steady-state inactivation, respectively.

The third mechanism by which Na v 1.5 could play a role in AF is the generation of the late sodium current (I NaL ). In a cohort study of patients with early-onset lone AF [START_REF] Olesen | High Prevalence of Long QT Syndrome-Associated SCN5A Variants in Patients With Early-Onset Lone Atrial FibrillationClinical Perspective[END_REF] , eight nonsynonymous mutations and two rare variants were identified in SCN5A; five of these variants and mutations had previously been associated with LQTS. However, all mutation carriers had a normal or borderline QT c , but some of them showed QT c interval prolongation after a flecainide challenge, performed to rule out Brugada syndrome. Functional studies of these variants demonstrated electrophysiological parameters of gain-and loss-of-function effects.

Nevertheless, five of them increased I NaL . In another cohort of patients with either lone AF or AF associated with heart disease, variants associated with increased I NaL have also been identified [START_REF] Darbar | Cardiac sodium channel (SCN5A) variants associated with atrial fibrillation[END_REF] . This high prevalence of long QT variants in patients with AF indicates that I NaL could play a role in the genesis of AF by providing a substrate through AP prolongation, and a trigger by early and delayed after-depolarizations [START_REF] Lemoine | Arrhythmogenic left atrial cellular electrophysiology in a murine genetic long QT syndrome model[END_REF][START_REF] Song | An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes[END_REF] . In agreement with this study, patchclamp experiments performed in isolated right atrial myocytes from patients undergoing heart surgery for permanent AF, showed that permanent AF was associated with decreased Na v 1.5

expression and peak I Na density, and that I NaL was significantly greater [START_REF] Sossalla | Altered Na+ Currents in Atrial Fibrillation: Effects of Ranolazine on Arrhythmias and Contractility in Human Atrial Myocardium[END_REF] . In addition, in the ∆KPQ LQTS mouse model, which had a three-fold increased I NaL , prolonged atrial action potentials, EADs and triggered activity were found [START_REF] Lemoine | Arrhythmogenic left atrial cellular electrophysiology in a murine genetic long QT syndrome model[END_REF] . Since EADs and triggered activity in ∆KPQ mice atria were due to enhanced I NaL , ranolazine, a selective I NaL blocker, suppressed

EADs and triggered activity in ∆KPQ hearts [START_REF] Lemoine | Arrhythmogenic left atrial cellular electrophysiology in a murine genetic long QT syndrome model[END_REF] .

In the heart, the α-subunit Na v 1.5 interacts with β-auxiliary subunits that modulate I Na .

Mutations in genes encoding these subunits (SCN1B, SCN2B, and SCN3B) have also been identified in patients with AF [START_REF] Watanabe | Mutations in sodium channel β1and β2-subunits associated with atrial fibrillation[END_REF][START_REF] Olesen | SCN1Bb R214Q found in 3 patients: 1 with Brugada syndrome and 2 with lone atrial fibrillation[END_REF][100][101][102] . These mutations decrease the I Na current, which further supports the implication of the loss of sodium current in the pathogenesis of AF.

o Non-ion channel mutations

In addition to mutations in ion channel genes, mutations in other genes encoding gapjunction proteins (GJA1 103 , GJA5 [104][105][106][107] ) and transcription factors (GATA4 [108][109][110][111] and GATA6 [112][113][114] ) involved in cardiac development have been identified in patients with AF [START_REF] Olesen | Atrial fibrillation: the role of common and rare genetic variants[END_REF] .

Despite the fact that many genes have already been involved in AF, such rare variants are present in less than 1% of patients with AF [START_REF] Xiao | The genetics of atrial fibrillation: from the bench to the bedside[END_REF] , and cannot explain all the heritability aspects of AF. Nevertheless, identification of these mutations gives new insights into the genetic aspects of the disease, and the underlying mechanisms of AF caused by these genetic variations. Gain-of-function effect on I Na current [START_REF] Makiyama | A novel SCN5A gain-of-function mutation M1875T associated with familial atrial fibrillation[END_REF][START_REF] Li | Gain-of-function mutation of Nav1.5 in atrial fibrillation enhances cellular excitability and lowers the threshold for action potential firing[END_REF][START_REF] Darbar | Cardiac sodium channel (SCN5A) variants associated with atrial fibrillation[END_REF] Loss-of-function effect on I Na current [START_REF] Laitinen-Forsblom | SCN5A mutation associated with cardiac conduction defect and atrial arrhythmias[END_REF][START_REF] Mcnair | SCN5A mutation associated with dilated cardiomyopathy, conduction disorder, and arrhythmia[END_REF][START_REF] Olson | Sodium channel mutations and susceptibility to heart failure and atrial fibrillation[END_REF][START_REF] Ellinor | Cardiac sodium channel mutation in atrial fibrillation[END_REF] Mixed functional effect on I Na current [START_REF] Olesen | High Prevalence of Long QT Syndrome-Associated SCN5A Variants in Patients With Early-Onset Lone Atrial FibrillationClinical Perspective[END_REF] ATFB 13 SCN1B 19q13.1 ß1 I Na Loss-of-function effect on I Na current [START_REF] Watanabe | Mutations in sodium channel β1and β2-subunits associated with atrial fibrillation[END_REF][START_REF] Olesen | SCN1Bb R214Q found in 3 patients: 1 with Brugada syndrome and 2 with lone atrial fibrillation[END_REF][100] ATFB 14

SCN2B 11q23 ß2 I Na Loss-of-function effect on I Na current 98 SCN3B 11q23.3 ß3 I Na Loss-of-function effect on I Na current 101,102
Polymorphisms associated with non-familial AF (Table 3)

Single-nucleotide polymorphisms (SNPs) represent the most common type of inherited genetic variations. Genome-wide and candidate gene association studies have identified SNPs that could modulate disease susceptibility and physiological traits such as PR interval and QRS duration. Most of the identified SNPs are located in non-coding regions of the genome, and some of them can affect gene transcription by modulating regulatory factors, such as cis-regulatory elements and transcription factors. Nevertheless, most SNPs should only be considered as genetic markers of a region of interest (locus), putatively co-inherited with nearby functional SNPs (in linkage disequilibrium). In addition, some SNPs, especially those that are nonsynonymous, could directly alter protein functions.

GWAS performed in general AF populations led to the identification of variants that could contribute to AF susceptibility. In 2007, the first GWAS performed in three European and one Chinese populations showed that two variants on chromosome 4q25 (rs2200733, rs10033464) were linked to AF 115 . Association of these variants with AF was stronger in younger patients and in those with lone AF, but was also significant in older patients. These two variants were confirmed by several other studies, especially the rs2200733 [116][117][118][119] . In a recent meta-analysis study, an additional SNP, rs6817105, at the same locus was associated with AF with the highest score 120 . The closest gene to this locus is the transcription factor PITX2 which plays an important role in the determination of cardiac right-left asymmetry 121- 123 . PITX2 suppresses the formation of a left-atrial sinus node and plays an important role in the formation of the pulmonary vein myocardium 124,125 . These variants may dysregulate PITX2 expression, leading to an abnormal structure or function of the left atrium and pulmonary veins predisposing to AF. GWAS allowed the identification of several other loci linked to AF 120,[126][127][128][129][130] . The variants in these loci are not only in genes encoding ion channels (KCNN3 128 , HCN4 120 ), but also near or within genes involved in cardiopulmonary development (PRRX1) and signal transduction (CAV1) 120 . Moreover, in general populations, multiple loci modulating the PR interval length have been identified by GWAS, and some of them were found to be associated with AF 129,130 . However, most of these loci have not been confirmed by the recent metanalysis in AF 120 . Nevertheless, these studies suggest that studying the intermediate phenotype of AF, as PR interval duration 131 , may help in the identification of new variants linked to AF.

Candidate gene association studies in AF patients identified a variety of SNPs that may affect AF susceptibility 132 . These polymorphisms are located in genes encoding cardiac ion channels (Table 3), and regulatory proteins (NPPA 133 , SLN 134 , eNOS 135,136 , Table 3), a gap junction protein (GJA5 137,138 ), circulatory hormones (ACE, AGT 139,140,136,[141][142][143] ), an inflammatory mediator (interleukin-6 144 ), matrix metalloproteinases (MMP2 145 , MMP9 146 ) and others 132 .

Functional studies of some of these variants highlight a probable link between these SNPs and AF. The polymorphism E145D (rs12621643, minor allele frequency 33.6%) in the KCNE4 gene, which encodes the β-subunit (MiRP3) of the K v LQT1 potassium channel, was associated with AF in the Chinese population 147 . This variant showed a gain-of-function effect on the I Ks potassium current 148 . Study of the G38S polymorphism (rs1805127, minor allele frequency 33.1) in KCNE1 encoding the β subunit (minK) of K v LQT1, and the incidence of AF showed that the presence of the G variant increased the risk of developing AF. Whereas, when having two GG alleles, the risk increased further 135,149,150 . In contrast to KCNE4 E145D, the G allele of KCNE1 G38S reduced the I Ks current, due to decreased K v LQT1 membrane expression, and computer simulations demonstrated atrial AP prolongation, especially in the presence of a reduced repolarization reserve, and the occurrence of early afterdepolarization under specific conditions 151 . However, variants identified by the candidate gene association studies were not reported by the recent metaanalysis study 120 .

How these common genetic variants, or their combination with other factors, determine AF susceptibility remain to be investigated. The molecular pathways underlying AF could be the same in both sporadic and familial AF. Future association studies in large cohorts will be helpful in identifying new SNPs, especially those with a strong effect but which may have been missed due to their low allele frequencies, or specific haplotypes in which multiple SNPs are inherited in blocks, in order to have a thorough understanding of the genetic aspects of AF.

II.3 Bradyarrhythmias and conduction blocks

Bradyarrhythmias are defined as a heart rate below 60 bpm, and can be divided into two major categories depending on the site of conduction abnormalities: sinus node dysfunction (SND) and atrio-ventricular (AV) block. Sinus bradycardia is a physiological condition predominantly caused by increased vagal tone; it is commonly seen in athletes, or in young people at rest and during sleep. In addition, it can be caused by drugs such as betablockers and calcium-channel blockers. Persistent symptomatic sinus bradycardia with heart rate below 40 bpm is a pathological condition and usually represents the sick sinus syndrome that will be discussed later. On ECG, P-waves and QRS complexes are normal, but the heart rate is very slow (Figure 20). First-degree AV block (Figure 21A)

First-degree AV block corresponds to a conduction delay of atrial impulses to the ventricles, characterized by constant abnormal prolongation of the PR interval on ECG (PR > 0.2 s) with every P-wave normally followed by a QRS complex (Figure 21A). The conduction delay may occur within the atrium, AV node (the most common), or the His-Purkinje system.

Patients with first degree AV block are usually asymptomatic. However, patients may become symptomatic when there is a severe prolongation of PR interval, or during exercise as the heart rate increases but the PR interval does not shorten accordingly.

Second-degree AV block (Figure 21B&C)

Failure of conduction of some atrial impulses to the ventricles results in second degree AV block. According to the ECG criteria, it is divided into two types: type I (Mobitz I or Wenckebach) and type II (Mobitz II).

In Mobitz I (Figure 21B) there is a progressive prolongation of the PR interval (indicated by arrows) before the occurrence of a non-conducted P-wave. The first conducted P-wave after the non-conducted one has the shortest PR interval. In Mobitz II (Figure 21C)

there is a constant PR interval before and after the non-conducted P-wave.

Third-degree or complete heart block (Figure 21D andE)

Third-degree or complete heart block corresponds to a failure of conduction of each atrial impulse to the ventricles, resulting in complete AV dissociation with a higher atrial rate than ventricular one. It may be congenital or acquired, with either the AV node, bundle of His, or the right and left bundle branches as the sites of block. Having a narrow QRS complex (Figure 21D) with ventricular escape rhythm of 40-60 bpm indicates that the block is located at the AVN level, which is usually the case in congenital AV-block. However, wide QRS complex (Figure 21E) and/ or a ventricular rate of 20 to 40 bpm point out a block in the His-Purkinje system, which is often seen in acquired AV blocks.

Acquired AV block may be due to extrinsic causes such as drugs, electrolyte disturbances and others, or intrinsic causes like ischemic, inflammatory or infectious heart diseases 158 . However, in approximately 50% of AV block cases, idiopathic progressive degenerative fibrosis of the cardiac conduction system, named as Lenègre or Lev disease, is the leading cause 158 . The treatment of choice in patients with AV block is permanent cardiac pacing. Indication for cardiac pacing depends on the type and location of the AV block, symptoms, and concurrent diseases. Modified from http://www.unm.edu/~lkravitz/EKG/avblocks.html

II.3.1 Sick sinus syndrome (SSS)

SSS is characterized by abnormalities in the SAN impulse formation and propagation leading to atrial rates that are inadequate for physiological requirements. These abnormalities may include sinus bradycardia, sinus arrest, sinoatrial exit block, and chronotropic incompetence. SSS can be caused by an intrinsic disease of the sinus node, or by extrinsic causes 158 . SSS occurs more frequently in the elderly with a peak incidence in the seventh and eighth decades of life 159 , the idiopathic degenerative fibrosis of nodal tissue being the main cause 160 . However, it can occur in fetuses, infants, and children [161][162][163][164] , especially after cardiac surgery for congenital heart disease. SSS may also occur in young adults without structural abnormalities, and present a familial form, suggesting that genetic factors could be involved in the genesis of the SND 163,[165][166][167][168] .

II.3.1.1 Automaticity of SAN

The sinus node was discovered in 1907 by Keith and Flack while examining human heart histology 169 . Early on, it was described as a small condense area of tissue in the right atrium, where the superior vena cava enters the atrium 170,171 , but further studies showed that the SAN is more diffuse, and extends down the inferolateral aspect of the crista terminalis in a cigar shape 172 Normally, AP is initiated in a small area of the node called the leading pacemaker site, also known as the center of SAN. Then, it is conducted to atrial muscle through the transitional and peripheral region, where cells are larger and more organized 177 . The leading pacemaker site is not static and can be shifted in response to external factors such as sympathetic and parasympathetic stimulation 177 . The AP is slow and small in the center of the SAN compared to its periphery and the surrounding atrial cells. Differences in ion channels and gap junction expression between the SAN and atrial cells account for the automaticity of the SAN and the shape of nodal AP (Figure 23).

Outward currents

While Kir2.1 underlies the I K1 current responsible for the stable resting membrane potential in atrial and ventricular cells, it is absent in nodal cells 173,178 . This leads to an unstable resting membrane potential, and is responsible for the automaticity of nodal cells.

During AP, the delayed rectifier potassium channels underlying I Ks and I Kr are activated to account for the repolarization phase, and the decay of these channels allows the inward currents to depolarize the cells. So, it is believed that I K decay accounts for the earliest part of the pacemaker potential 163,179 (Figure 23).

Inward currents

The funny current

The hyperpolarization-activated or funny current I f is the major determinant of diastolic depolarization in the SAN. The hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are responsible for the I f current; there are four HCN channels encoded by different genes (HCN1-HCN4). HCN1 and HCN4 are the predominant cardiac isoforms that are present mainly in the human SAN 173 . The HCN channels have unique characteristics including permeability to K + and Na + , activation at hyperpolarized membrane potential and modulation by cAMP. These properties make them ideal for modulating pacemaker potential not only during normal rhythm generation but also during heart regulation by the autonomic nervous systems 180 . The importance of I f in pacemaker activity is supported by HCN4 mutations associated with SSS that will be discussed below.

Sodium and calcium currents

Early studies have suggested that Na v 1.5 plays a minor role in the depolarization phase of the SAN, since the maximum diastolic potential (MDP) is moderately negative, and the SAN has a long diastolic depolarization phase, and as such, most sodium channels are inactivated 179 . Later on, several studies have showed the importance of Na v 1.5 in modulating the AP in SAN pacemaker cells, and SCN5A mutations have been reported in patients with inherited form of SSS.

Studies on the human SAN showed that the Na v 1.5 channel was absent from the center of the SAN, but was present in the periphery, at the junction with atrial cells 173,178 . A large inward current with I Na characteristics was recorded in human SAN cells 181 . Despite that most Na + channels may reside in the inactivated state in SAN cells, it was argued that I Na may contribute to human SAN pacemaker activity, and that the atrium exerts an effective hyperpolarizing load on the periphery of the SAN, thereby bringing the peripheral cells to a more hyperpolarized potential where not all Na + channels are steady-state inactivated (window current) 181 . Consequently, the AP upstroke is slow in the center of the SAN, and it is the L-type Ca 2+ current I Ca,L that is responsible for the depolarization phase. In contrast to atrial and ventricular cells, where the Ca v 1.2 channel is the major isoform contributing to I Ca,L , nodal cells express Ca v 1.3 173 which has a more negative threshold potential than Ca v 1.2 163 , a feature that makes this channel more appropriate for pacemaker activity, as it is activated earlier in the pacemaker AP. Blocking I Ca,L abolishes AP generation in the center of the SAN, while blocking Na v 1.5 slows pacemaker activity at the periphery. This evidence suggests that the presence of Na v 1.5 at the periphery is important to provide sufficient inward current for atrial muscle stimulation, resulting in AP conduction out of the nodal cells 178 . In addition to the sodium and L-type Ca 2+ currents, the T-type Ca 2+ current, I Ca,T , may contribute to the generation of the pacemaker potential of the SAN, mainly the last two thirds of diastolic depolarization (Figure 23) 178 .

Moreover, the Na + -Ca 2+ exchanger contributes to pacemaker activity elicitated by outward movement of Ca 2+ during diastole, generating an inward current. It has been suggested that the Na + -Ca 2+ exchanger accounts for the last exponential phase of the pacemaker potential 163 .

Gap junctions

The electrical coupling in the center of the SAN is weak, mainly because of low connexin expression (Cx43 and Cx40). Instead, Cx45, which forms small conductance channels, is expressed in the center. However, electrical coupling increases at the periphery of the SAN with the expression of Cx43 and Cx40. This organisation of connexin favours the exit of the electrical signal from the node center through the periphery, but at the same time protects the cells in the center from the inhibitory hyperpolarizing influence of atrial muscles 163,173,182 .

II.3.1.2 Genetics of SSS

As the automaticity of the SAN has been modelled as membrane voltage and calcium clocks, it is not surprising that mutations affecting the membrane voltage clock (SCN5A and HCN4, Table 4 and5), the calcium clock (RYR2 and CASQ2) [183][184][185] , or both of them (ANKB) 186 have been identified in patients with SND. Mutations affecting the membrane voltage clock are discussed below.

SSS and SCN5A genetic mutations

Both loss-and gain-of-function mutations in the SCN5A gene have been identified in patients with SSS. In 2003, Benson et al screened the SCN5A gene in a series of ten pediatric patients with congenital SSS treated with pacemaker implantation 187 . In this pioneering study, four cases were diagnosed in utero, where SSS was associated with congenital heart defects such as aortic and pulmonary valve stenosis, and no SCN5A mutations were found. For the six other patients, SSS was diagnosed between two and nine years of age, in the absence of heart disease, and transmitted as an autosomal recessive trait with complete penetrance. SCN5A molecular analysis led to the discovery of compound heterozygous mutations in five patients from three different families. The three probands exhibited the following compound heterozygous mutations: G1408R-P1298L, T220I-R1623X, and delF1617-R1632H.

Functional studies showed that G1408R and R1623X expressed alone produced no current, and the others showed mild to severe channel dysfunction. Each patient harbored one nonfunctional or severe mutation on an allele, in combination with a milder mutation on the other allele. Interestingly, the subjects who carried only one of these mutations were either asymptomatic, or experienced latent cardiac conduction system disease, such as first degree AV block. However, previous studies have reported that two of these mutations, G1408R and delF1617, led to BrS or conduction abnormalities (G1408R 188 ), and long QT syndrome (delF1617 189 ), suggesting the role of additional factors in the phenotypical expression of these diseases. Later on, familial SSS was widely studied, and several SCN5A loss-of-function mutations have been identified [START_REF] Olson | Sodium channel mutations and susceptibility to heart failure and atrial fibrillation[END_REF][188][189][190][191][192][193][194][195][196][197] (Table 4). These mutations lead to no or reduced I Na , due to non-functional channel or altered biophysical properties. As shown in Telemetric ECG studies and isolated hearts showed sinus bradycardia, slowed SA conduction, and sino-atrial exit block 203 . These findings directly implicate the role of sodium current in the conduction of AP in the SAN as well as between the SAN and the surrounding atrial cells, and are in line with the SCN5A loss-of-function mutations identified in patients with SSS.

Bradycardia and sinus pause have also been reported in LQTS patients with SCN5A mutations (LQT3) [204][205][206][207][208][209][210][211][212][213] (Table 4). In most of the LQT3 mutations, incomplete or slowed channel inactivation induces a small persistent inward sodium current, which accounts for the delay in the repolarization and the prolongation of the QT interval. As shown in

SSS and HCN4 genetic mutations

Six HCN4 mutations have been identified in patients with SSS (Table 5), all of which are loss-of-function mutations. The decrease in I f is due to multiple mechanisms like altered biophysical properties [220][221][222][223][224] , trafficking defects 222,225 , synthesis defects 222,223 , and decreased sensitivity of the channel to cyclic adenosine monophosphate (cAMP) 220 . In addition, two mutations have dominant-negative effect on the WT channels 220,225 . These mutations are associated with variable clinical manifestations such as asymptomatic sinus bradycardia

II.4 Ventricular arrhythmias

Premature ventricular contractions (PVC)

Like PACs, PVCs are ectopic impulses occuring when a region of the ventricle depolarizes leading to premature beats. PVCs are usually a benign arrhythmia relatively frequent in healthy persons, but they can be caused by cardiac or noncardiac diseases.

Prognosis depends on the morphology and frequency of PVCs, as well as on the type and severity of the associated heart diseases. In general, polymorphic PVCs have poorer prognosis than those that are monomorphic, since they can be transformed into ventricular tachycardia and fibrillation. On ECG, PVC is characterized by an abnormally wide QRS complex, abnormal T wave, and an absence of the P wave followed by a compensatory pause (Figure 24).

Ventricular tachycardia (VT)

VT is a rapid heart beating entity originating in the ventricles, occuring with or without structural heart disease. Reentry is the main cause of such arrhythmia. On ECG, it is demonstrated by atrio-ventricular dissociation with a rapid ventricular rate, and wide QRS complexes (QRS > 120 ms) (Figure 25).

According to QRS morphology, VT is divided into monomorphic or polymorphic forms. In monomorphic VT, ventricular activation starts either in a single focus, or due to a re-entrant circuit caused by a stable substrate. On ECG, QRS complexes have the same form. TdP is a stereotyped polymorphic ventricular tachycardia, usually associated with LQTS, with a 2-to 3-fold increased frequency in females compared to males 226 . Transmural dispersion of depolarization with EAD as a common tigger is the underlying mechanism of TdP. On ECG, it is characterized by fluctuating amplitude and twisting of QRS complexes around the isoelectric line (Figure 27). In most circumstances, TdP terminates spontaneously, however degeneration into VF may occur resulting in sudden death. The main treatment for LQTS is beta-blockers. In some cases, an implantable cardioverter defibrillator (ICD) may be needed. In addition, patients with LQTS should avoid all medications that cause prolongation of QT interval. The list of such drugs is available on the following Website (www.qtdrugs.org).

o Catecholaminergic polymorphic ventricular tachycardia (CPVT)

CPVT is a rare disease characterized by normal ECG at rest with adrenergic-mediated polymorphic ventricular tachyarrhythmias during effort or emotional stress, and with a high mortality rate of up to 50% in severe untreated cases up to the age of 20 years 239,240 . It is mainly caused by mutations in genes involved in calcium homeostasis, the cardiac ryanodine receptor gene (RYR2, autosomal dominant) 241 , and the gene encoding the cardiac calsequestrin (CASQ2, autosomal recessive) 185,242,243 . Recently, mutations in the transmembrane sarcoplasmic reticulum protein triadin (TRDN, autosomal recessive) 244 , another protein involved in calcium homeostasis, have been reported. These mutations increase the cytosolic Ca 2+ concentration, leading to delayed after-depolarizations that trigger arrhythmias. Beta-blockers are the mainstay of treatment for CPVT, and in certain cases ICD can be used.

o Short QT syndrome (SQTS) SQTS is a hereditary disease described for the first time in 2000 245 , characterized by short QT interval (QTc<320 ms) with syncope and unexplained atrial or ventricular arrhythmia (atrial fibrillation, polymorphic ventricular tachycardia, and ventricular fibrillation) that could lead to sudden death 246,247 . In contrast to LQTS, SQTS is a very rare disease caused by gain-of-function mutations in the genes KCNH2 (SQT1), KCNQ1 (SQT2), and KCNJ2 (SQT3) [START_REF] Morita | The QT syndromes: long and short[END_REF] affecting the potassium repolarizing currents (I Kr , I Ks , I K1 ). Treatment options are limited; ICD can be used in cases with a high risk of sudden death.

II.4.1 Brugada syndrome (BrS)

Brugada syndrome is an autosomal inherited cardiac channelopathy with incomplete penetrance 228 . It is characterized by a structurally normal heart with a typical ST segment elevation in the right precordial leads of the ECG 248 . The Brugada brothers, in 1992, were the first to describe BrS as a clinical entity 249 . Patients with BrS have an increased risk of sudden cardiac death by ventricular fibrillation 250 .

II.4.1.1 Epidemiology

As the ECG pattern of BrS can be intermittent, often concealed and dependent on ECG lead position, it is difficult to ascertain the true prevalence of BrS in the general population. It has been estimated that the prevalence of a Brugada type 1 ECG pattern is approximately 0.05%, with a mean age of sudden death of 41±15 years 250 . However, this prevalence varies worldwide with the highest prevalence in Asia, followed by Europe and the United States 251 . Despite the fact that BrS is generally considered to be an autosomal dominant disease, its prevalence is up to 10 fold higher in men than women with increased severity 252,253 . BrS typically occurs in adulthood with mean age of diagnosis between 40 and 45 years 250 , an age at which most arrhythmic events happen, mainly during sleep or at rest 252- 255 . However, BrS can occur in children 250,256 . According to the consensus document, BrS represents 4% of all sudden deaths and at least 20% of sudden deaths in patients with structurally normal hearts 250 . In addition, in 2002, BrS and sudden unexplained nocturnal death syndrome (SUNDS) have been identified as the same entity 193 . SUNDS, which has the same ECG manifestation as BrS, is the most common cause of death in the young healthy Asian population (usually male) during sleep, highly present in Thailand, Japan, the Philippines and Cambodia 193,257,258 . Besides, some mutations in the BrS-related genes have been identified in sudden infant death syndrome (SIDS), an unexplained sudden death of an infant usually during sleep in the first year of life 259,260,[START_REF] Giudicessi | Novel Mutations in the KCND3-Encoded Kv4.3 K+ Channel Associated with Autopsy-Negative Sudden Unexplained Death[END_REF]100 .

II.4.1.2 Electrocardiographic characteristics

ECG abnormalities represent important criteria in the diagnosis of BrS. There are three different ECG patterns, however only type-1 ECG is diagnostic (Figure 29):

1. Type 1 is characterized by an ST-segment elevation equal to or greater than 2 mm in one or more right precordial leads V1, V2, positioned in the 2 nd , 3 rd or 4 th intercostal space. The ST-segment elevation has a coved type appearance and is followed by a negative T-wave. This ECG pattern can be observed spontaneously or after the administration of sodium channel blockers like flecainide and ajmaline, and it represents the gold standard in BrS diagnosis.

2. Type 2 has an ST-segment elevation equal to, or greater than 1 mm, followed by positive or biphasic T-wave with saddleback appearance.

II.4.1.3 The pathophysiology of BrS

Two main hypotheses have been reported to explain the pathophysiology of BrS: the repolarization and the depolarization hypotheses.

Repolarization hypothesis

Yan and Antzelevitch first proposed the repolarization hypothesis in 1999 based on results from an arterially perfused wedge preparation of the canine right ventricle 265 . In accordance to this hypothesis, there is a net enhancement of the repolarizing current at the end of phase one of the action potential that leads to the augmentation of the AP notch in the right ventricular epicardium.

In normal heart, there is a gradient expression of the transient outward potassium (I to ) channels through the ventricular wall, which are highly expressed in the epicardium compared to the endocardium 12,[266][267][268][269] , mainly in the right ventricle 270 . This gradient accounts for the notch and dome morphology during phase 1 of cardiac AP of the epicardium (Figure 30A). In BrS, reduction of the inward depolarizing currents, I Na or I CaL , or enhancement of the repolarizing potassium current I to , by mutations in the genes encoding these ion channels, increases the net repolarizing currents, and leads to an enhancement of the right ventricular notch and exaggeration of the transmural voltage gradient manifested by the elevation of the ST-segment on the ECG (Figure 30B). If the repolarization of the epicardium precedes that of the myocardium and endocardium, the T wave is positive resulting in a saddleback morphology on the ECG (Figure 30B). If the enhancement of the notch is accompanied by epicardial action potential prolongation, the myocardium and the endocardium will repolarize before the epicardium, reversing the transmural gradient and leading to the inversion of the Twave and the elevation of coved ST-segment, the typical ECG features seen in BrS (Figure 30C). Further enhancement in the net repolarizing currents could lead to loss of the AP dome in some but not all epicardial cells and not in the endocardium (Figure 30D). This dispersion of repolarization within the epicardium and also transmurally, creates an arrhythmic substrate that may cause phase-2 re-entry triggering polymorphic ventricular tachycardia and ventricular fibrillation (Figure 30E) 271,272 . In human, simultaneous recording of monophasic AP from the epicardium and endocardium in patients with BrS showed spike-and-dome morphology in the epicardial sites, which was absent in the endocardium and in control patients 273 .

The repolarization hypothesis is in agreement with the predominant occurrence of BrS in males, since in animals, a higher I to expression is seen in males compared to females 274 .

Moreover, higher testosterone levels are found in male patients with BrS compared to controls 275 , and disappearance of typical BrS-ECG after castration has been reported 276 .

Recent unpublished data by Antzelevitch and collaborators in human induced pluripotent stem cell-derived cardiomyocytes, showed that testosterone increases the expression of I to in these cardiomyocytes by augmenting the Kv4.3 mRNA expression 277 . In addition, Song et al showed that the estrogen hormone inhibits the Kv4.3 expression and trafficking in the myometrium of pregnant rats 278 . The depolarization hypothesis challenges the concept that BrS is a pure channelopathy without structural heart disease. In addition to the fibrosis and hypertrophy seen in the explanted heart of a BrS patient, other structural abnormalities were reported such as myocarditis, apoptosis, fibrofatty replacement of the RV free wall with RV enlargement, dilatation and RVOT enlargement 285,286 .

From all evidence mentioned above, we conclude that BrS is not just a monofactorial disease. Each of the above hypotheses alone cannot completely explain the disease, and it is certainly possible that BrS is a disease with multiple mechanisms. For type 2 and 3 ECG patterns; the appearance of these ECG changes in one or more right precordial leads V1, V2, positioned in 2 nd , 3 rd , or 4 th intercostal spaces under baseline conduction, with conversion to type 1 after using sodium channel blockers is considered diagnostic for BrS.

II.4.1.5 BrS treatment

Implantable cardioverter defibrillator (ICD)

According to the most recent consensus Report 287 , an ICD is the only effective treatment to prevent sudden death in patients with BrS.

According to this report, an ICD:

o is recommended for BrS patients who are survivors of a cardiac arrest and/or have documented spontaneous sustained VT with or without syncope.

o can be useful in patients with a spontaneous diagnostic type 1 ECG with history of syncope, which could be related to ventricular arrhythmias.

o may be considered in BrS patients with inducible VF.

o is not indicated in asymptomatic BrS patients with a drug induced type 1 ECG and on the basis of a family history of sudden cardiac death alone.

Drug therapy

Until now, limited support is present for the use of medication in the treatment of BrS, since there is a shortage in large randomized studies on the effectiveness of proposed drugs in these patients. However, based on the cellular mechanisms responsible for the Brugada phenotype, drugs that reduce I to or enhance I CaL could be therapeutic options for BrS. The most studied medication in BrS is quinidine, which blocks the I to and I Kr potassium currents.

It has shown efficacy in the treatment of electrical storm and in the prevention of spontaneous or induced ventricular arrhythmias. Some studies suggest that it can be used with an ICD or as an alternative, especially in patients who are asymptomatic but experience drug-induced arrhythmias [288][289][290][291][292][293][294] . The problem with this medication is the high rate of side effects like diarrhoea, and thrombocytopenia 291 . Isoproterenol, which increases I CaL , is another medication that has been used in treating an electrical storm 295 . Other medications have also been evaluated for their efficacy in BrS treatment 296 .

Ablation

Endocardial ablation of ventricular ectopic beats (mainly in the RVOT), which trigger VF, has been effective in treating VF storms [297][298][299][300] . However, as ablation is an invasive procedure, it is usually used after the failure of other therapies. According to the recent consensus report 287 , catheter ablation may be considered in BrS patients with a history of arrhythmic storms or repeated appropriate ICD shocks 287 .

Recently, another approach for ablation has been reported by Nademanee el al 301 who showed that the ablation of the region of conduction delay in the right ventricle, mainly the anterior aspect of RVOT epicardium, in nine patients with recurrent episodes of VF leads to VF suppression and normalization of the ECG patterns in 8/9 patients after two years of follow-up.

Additional recommendations

Patients with BrS should avoid all medications that could induce a type 1 ECG pattern and/or ventricular arrhythmia, such as psychotropic drugs, antihistamines and anaesthetics.

The list of these drugs is available on the following website (www.brugadadrugs.org) 302 .

II.4.1.6 Genetics of BrS

BrS is an autosomal dominant disease with incomplete penetrance and variable expression. It is considered as a channelopathy since, until now, 19 genes encoding ion channel and ion-channel regulatory proteins have been associated with BrS 303-305 (Table 6).

However, mutations in these genes have been identified in less than 30% of affected individuals 250,306 .

Mutations in the SCN5A gene is the most common genotype, accounting for approximately 18 to 30% of the BrS cases 250 , with a higher frequency in familial than in sporadic cases 307 . The first mutations were identified by a candidate-gene approach in 1998 in families with idiopathic VF 308 . Since then, an increasing number of SCN5A mutations have been reported, and up to now there are nearly 300 published SCN5A mutations 306 . The reported mutations include missense, nonsense, frame-shift, splice-site, and in-frame deletion/insertion mutations. Functional studies of some of these mutations in heterologous systems showed loss-of-function effects on the sodium current by different mechanisms including 309 :

Decreased surface expression of Na v 1.5 protein due to:

• Retention and premature degradation of the mutant proteins by the ER quality control system 310-314 . • Failure of the mutant to interact with β-subunits or regulatory proteins like ankyrin-G 315 , SAP97 316 , syntrophin 316 , and FHF1 317 , all which have an important role in stabilising channel expression at the surface as well as targeting Na v 1.5 to special domains in cardiomyocytes.

Here, mutations may be in the SCN5A gene, like the mutation E1053K 315 that abolishes the interaction with ankyrin-G, or in the genes encoding regulatory proteins, as the mutation Q7R 317 in FGF12, which reduces the binding of FHF1 to Na v 1.5.

Expression of non-functional channels 188,318 Some mutant channels have a normal membrane expression but they conduct either very small or no sodium currents, as the mutations are located in the channel pore.

Altered gating properties 210,[319][320][321][322][323][324] Delayed activation (activation at more positive potential), earlier inactivation (inactivation at more negative potential), faster inactivation, enhanced slow inactivation and delayed recovery from inactivation, all lead to decreased I Na .

Autosomal transmission of SCN5A mutations is not always observed. This is not only due to partial penetrance, but also to the fact that some BrS patients do not carry the familial mutation, suggesting another genetic origin. Indeed, in a study of 13 large families carrying SCN5A mutations 263 , type-1 Brugada ECG pattern was not observed even after drug challenge in nearly half of the mutation carriers. However, it was seen following drug challenge in five mutation-negative patients, and spontaneously in three others. In the same study, mutation carriers were found to have longer PR intervals and QRS durations than non-carriers, consistent with other studies 261,262,264 , suggesting that mutant channels have loss-of-function effects but do not cause BrS. These findings suggest that the simple model of autosomal dominant inheritance of BrS cannot completely explain the disease, and SCN5A mutations alone are sometimes not sufficient to induce the disease (although most mutation carriers have conduction problems indicating the loss-of-function effects of these mutations), but they may act as factors favouring BrS in the presence of a specific genetic background. Moreover, the existence of genotype-negative BrS patients indicates that other unknown rare or common genetic variants could play a role in the disease, and that negative genetic testing cannot exclude the disease.

In addition to SCN5A, mutations in the genes encoding the β-subunits and the regulatory proteins of Na v 1.5 have been involved in BrS, and all lead to Na v 1.5 loss-offunction effects (Table 6). Moreover, loss-of-function mutations in the gene encoding the components of L-type calcium channels (CACNA1C, CACNB2B and CACNA2D1) and private gain-of-function mutations in the outward potassium current have also been reported (Table 6). However, these mutations are rare, and account for less than 1% of all the BrS cases.

Although several genes have been reported in BrS, mutations in these genes cannot completely explain the disease phenotype. Meanwhile, other factors like genetic modifiers, drugs, gender and imbalance in autonomic tone could influence the BrS phenotype.

Moreover, the presence of structural heart disease in some cases of BrS has challenged the concept that BrS is a pure channelopathy. So, it is likely that genetic susceptibility along with other modulating factors lead to BrS, in combination but probably not in isolation. These abnormalities were more frequent when a BrS-ECG type 1 pattern was induced by a class 1 drug. Moreover, several studies showed that BrS patients carrying SCN5A mutations have longer PR intervals and QRS durations than non-carriers, suggesting that these ECG abnormalities may be used to predict the presence of SCN5A mutations [261][262][263][264] . These ECG abnormalities could also be used in the future for risk stratification of patients, since some of them have been linked to specific outcomes. Such abnormalities include life-threatening ventricular arrhythmias with QRS prolongation [341][342][343] , SD in women with longer PR interval 344 , and SD and/or ICD therapy with a first degree AVB 264 .

These conduction abnormalities are similar to those seen in the Lenègre disease 158,309 , a progressive cardiac conduction disease (PCCD), which is characterized by progressive prolongation of the conduction parameters (P wave, PR interval, QRS) and right or left bundle branch block. PCCD may result in complete heart block leading to syncope or sudden death, but is not associated with ventricular arrhythmias. It is most often caused by fibrosis affecting the cardiac conduction system. In the heritable form, loss-of-function mutations in the SCN5A gene or Na v 1.5 regulatory proteins have been reported 188,327,345,346 . These mutations reduce I Na by the same mechanisms mentioned previously for BrS. However some of these mutations cause PCCD alone or PCCD with BrS (overlap syndrome), indicating that the I Na reduction is sufficient to cause conduction abnormalities either in PCCD or in BrS.

However, in some cases, other factors in addition to I Na loss are required for the development of ST segment elevation, the standard ECG sign of BrS.

The conduction abnormalities observed in Lenègre disease are progressive with age, requiring a pacemaker implantation in the older patients. Similar progressive conduction abnormalities were also reported in some SCN5A BrS mutations carriers 262 . Nevertheless, the two diseases remain two distinct clinical entities, as the ST-segment elevation and the lifethreatening ventricular arrhythmias, two main BrS features, are absent in PCCD.

III. The cardiac sodium channel Na v 1.5

The cardiac sodium channel forms a large protein complex in the cardiomyocyte sarcolemma. The main pore-forming Na v 1.5 α-subunit is responsible for the large inward sodium current (I Na ) underlying the depolarization phase of the cardiac action potential. The

during channel activation and inactivation

Depending on the variations of the membrane potential, the sodium channel can have three conformational states (closed, open, and inactivated). The transition between these states relies on the status of the activation and inactivation gates (Figure 33).

Closed but capable of opening. In this state only the activation gate is closed.

Open allowing the entrance of Na + . In this state both activation and inactivation gates are opened.

Closed and not able to open. In this state only the inactivation gate is closed. Under prolonged depolarization, sodium channels enter into the slow inactivation state; a more stable nonconducting state from which channels need more time to reactivate, compared to the fast inactivation. In addition, the molecular mechanism underlying slow inactivation is less well understood, and it does not require the inactivation particle (the linker III-IV). However, it was proposed that conformational changes in the S5-S6 P-loops, which form the channel pore, could underlie this process 347 .

III.2 Expression and localization of Na v 1.5 in the cardiomyocytes

After synthesis in the ER, Na v 1.5 α-subunits undergo post-translational modification.

They are anchored to the cytoskeleton and possibly stored in the subcellular compartments, before being finally targeted to the membrane to carry out their function. Then, from the membrane, they are internalized into endocytic vesicles and degraded. However, little is known about the the detailed processes underlying the synthesis and trafficking of Na v 1.5 αsubunits.

Despite the fact that Na v 1.5 α-subunits alone can act as voltage-gated sodium channels, it has been demonstrated that these channels are part of multiprotein complexes that modulate biological and/or biophysical properties of the channels. Immunocytochemical studies in cardiomyocytes have shown the presence of the Na v 1.5 α-subunit at the intercalated discs, lateral membranes, and T-tubules [351][352][353] . At these sites, the Na v 1.5 α-subunit interacts with various proteins, creating different protein complexes that are localized to specific regions of the cell membrane. According to the localization of these complexes, two distinct functional pools of Na v 1.5 channels are present, one at the lateral membranes and the other at intercalated discs 354,355 (Figure 34). At the lateral membrane, Na v 1.5 interacts with the syntrophin-dystrophin complex, whereas at the intercalated discs, it interacts with the synapse-associated protein 97 (SAP97). These partner proteins will be discussed later in detail. These two pools are not only different in their composition, but also in their biophysical properties. Macropatch studies have showed that sodium channels at the lateral membranes have decreased peak sodium current density with a negative shift of steady-state inactivation, and slower recovery from inactivation, compared to channels located at the intercalated discs 356 . A possible third pool of Na v 1.5 may also be present at the T-tubules 355 .

The exact role of these pools in the (patho)-physiology of cardiomyocytes remains to be elucidated, and future studies are needed to explore their functional roles.

In addition to these distinct pools, Na v 1.5 shows heterogeneous expression across the ventricular wall, with a transmural gradient increasing from the epicardium toward the endocardium 7,357 . In cardiomyocytes, Navβ subunits are mainly localized at the T-tubules/Z-lines, and intercalated discs 352,360,361 .

Navβ subunits associate with the Na v 1.5 α-subunit and modulate its subcellular localization, surface expression as well as its biophysiological properties. Moreover, Navβ subunits play a role in the adhesion of Na v 1.5 to the cytoskeletal proteins and extracellular matrix, and in the recruitment of enzymes, signalling molecules, and cytoskeletal adaptor proteins 359 . However, current data concerning the effects of Navβ subunits on Na v 1.5 are sometimes contradictory, probably owing to the various models used, or to differences in experimental conditions. In addition, the specific site(s) of interaction between Navβ subunits and Na v 1.5 remains unknown.

Until now, several mutations in the β-subunits have been reported in different hereditary cardiac arrhythmias, supporting the role of Navβ in normal cardiac electrical activity. Such arrhythmias include cardiac conduction diseases (CCD) (SCN1B 327 ), AF (SCN1B [START_REF] Watanabe | Mutations in sodium channel β1and β2-subunits associated with atrial fibrillation[END_REF][START_REF] Olesen | SCN1Bb R214Q found in 3 patients: 1 with Brugada syndrome and 2 with lone atrial fibrillation[END_REF] , SCN2B 98 , SCN3B 101,102 ), BrS (SCN1B [START_REF] Olesen | SCN1Bb R214Q found in 3 patients: 1 with Brugada syndrome and 2 with lone atrial fibrillation[END_REF]100,327 , SCN2B 339 , SCN3B 329,362 ), idiopathic ventricular fibrillation (IVF) (SCN3B 363 ), and LQTS (SCN4B 364 ). All of these mutations associated with CCD, BrS, IVF, and AF have a loss-of-function effect on I Na , which is a typical mechanism underlying this kind of SCN5A channelopathy. In contrast, the mutation in Navβ4 increases the late sodium current, the same effect as reported for SCN5A mutations linked to type III LQTS.

Most of these mutations are localized within the extracellular N-terminal region of the β-subunits, suggesting the importance of this region in the interaction with and regulation of Na v 1.5.

Navβ1

The SCN1B gene encodes two splice variants, called β1 and β1B, both expressed in the human heart 7,365 . The β1B subunit does not contain the transmembrane domain, and is a secreted protein 366,367 . The Navβ1 transcript is highly expressed in the human heart compared to Navβ2 and Navβ3 subunits, with two-fold expression in the atrium compared to the ventricles 7 . Moreover, higher expression of Navβ1 transcript in the endocardium than the epicardium has been observed 7 . The β1-subunit co-immunoprecipitates with Na v 1.5 360 , and co-localizes with it within the ER 368 , suggesting a role for the β1-subunit in the transport of Na v 1.5 to the membrane. Several studies suggested that the interaction between Navβ1 and Na v 1.5 could occur at several sites, between the extracellular domain of β1 and the extracellular loops S5-S6 of DI, DIV 369,370 , but also between the carboxyl termini of the two subunits 371,372 .

In cardiomyocytes, phosphorylation of the amino acid tyrosine 181 in the C-terminal domain of the β1-subunit determines its subcellular localization. The phosphorylated β1subunits co-localize with Na v 1.5, connexin-43 and N-cadherin at the intercalated discs, whereas the non-phosphorylated subunits are present at the T-tubules 361 .

The effects of the β1-subunit on Na v 1.5 in heterologous expression systems are variable, depending on the cellular model used. However, most studies have demonstrated an increase in peak I Na density [373][374][375][376] , positive shift of steady-state inactivation 360,376,377 , and decreased I NaL 377,378 .

In contradiction, in dissociated SCN1B-null cardiomyocytes 379 , increased peak I Na and I NaL have been observed, with slowed repolarization. These results are consistent with the observed increase in mRNA and Na v 1.5 protein expression, and the prolonged QTc interval and slow heart rate seen in this model. However, no change in Na v 1.5 gating properties was observed. In addition to its cardiac features, this model has neurological abnormalities like ataxic gate and seizures, resulting from the dysregulation of the neuronal sodium channels (Na v 1.1, 1.3, 1.6) 380 .

Discrepancies between the results obtained in the heterologous systems and in the animal model highlight the limitations of the two approaches, and suggest that the heterologous systems may not be the ideal model to mimic the physiological environment of the channels in cardiomyocytes, especially considering the existence of multiple partners.

Mutations in the SCN1B gene have been reported in patients with CCD 327 , BrS [START_REF] Olesen | SCN1Bb R214Q found in 3 patients: 1 with Brugada syndrome and 2 with lone atrial fibrillation[END_REF]100,327 , or AF [START_REF] Watanabe | Mutations in sodium channel β1and β2-subunits associated with atrial fibrillation[END_REF][START_REF] Olesen | SCN1Bb R214Q found in 3 patients: 1 with Brugada syndrome and 2 with lone atrial fibrillation[END_REF] . Co-expression of all these mutant β1-subunits with Na v 1.5 in heterologous systems showed decreased peak I Na compared to the co-expression with the WT β1-subunit, suggesting a role for β1-subunits in sarcolemmal expression of Na v 1.5. These results are consistent with the I Na reduction observed for all BrS and CCD SCN5A mutations, and some SCN5A mutations linked to AF.

Navβ2

The Navβ2 subunit co-immunoprecipitates and co-localizes with Na v 1.5 at the intercalated discs in cardiomyocytes 352,360 . The Navβ2 subunit is present in human heart, but at lower levels compared to Navβ1 7 . The functional effects of Navβ2 are variable. In several studies, co-expression of Navβ2 with Na v 1.5 had no effect on the sodium current 360,368,381 , while in one study the modulation of Na v 1.5 by Navβ2 depended on the sialylation status of Navβ2 382 . When completely sialylated, Navβ2 caused a negative shift of activation of Na v 1.5.

Mutations in the SCN2B gene have been reported in patients with AF [START_REF] Watanabe | Mutations in sodium channel β1and β2-subunits associated with atrial fibrillation[END_REF] or BrS 339 . The BrS mutation is a C-terminal mutation that decreased the peak I Na by decreasing the surface expression of Na v 1.5, without affecting its gating properties 339 . The AF mutations are located in the extracellular N-terminal domain and are also associated with decreased I Na peak but with a positive shift in steady-state-activation [START_REF] Watanabe | Mutations in sodium channel β1and β2-subunits associated with atrial fibrillation[END_REF] . Since the Navβ2 subunits do not co-localize with Na v 1.5 in the ER 368 , the reduced peak I Na density and surface expression of Na v 1.5 with the Navβ2 mutants suggest a role for Navβ2 in the stability of Na v 1.5 at the membrane.

Navβ3

Like the other Navβ subunits, there is a discrepancy in the functional effects of Navβ3 on Na v 1.5, depending on the expression systems utilized. In Xenopus oocytes, Navβ3 increased the peak I Na density, caused a positive shift of steady-state inactivation, and enhanced the recovery from inactivation 374 . However, co-expression of Navβ3 in CHO and HEK cells showed no effect on the peak I Na , with negative shift of steady-state inactivation 363,378 , decreased the late current, and slowed recovery from inactivation in CHO cells 378 .

SCN3B knockout mice showed results that are concordant with those observed in Xenopus oocytes. The isolated cardiomyocytes from these mice present with decreased peak I Na density and negative shift of steady-state inactivation 383 .

Mutations in Navβ3 have been reported in BrS 329,362 , IVF 363 , SIDS 384 and AF 101,102 .

Most of these mutations are located in the extracellular amino terminus of Navβ3, and all lead to decreased I Na density, with alteration in gating properties for some mutations. Moreover, a reduced surface expression of Na v 1.5 induced by the Navβ3 mutants has been found 329,362,363 .

These results implicate a role for Navβ3 in the regulation of surface expression and the gating properties of Nav1.5, as well as the importance of its N-terminal domain in these functions.

Navβ4

The Navβ4 subunit co-immunoprecipitates with Na v 1.5 364 . Co-expression of the Navβ4 subunit with Na v 1.5 does not modify the peak I Na , but leads to a negative shift in the steady-state inactivation 364,384 . The contribution of Navβ4 in cardiac diseases was revealed by the identification of SCN4B mutations linked to LQTS 364 , and SIDS 384 .

The first SCN4B mutation (L179F) is located in the transmembrane domain of the Navβ4 subunit, and was identified in a family with LQTS and a history of unexpected and unexplained sudden death 364 . Another C-terminal SCN4B mutation (S206L) was linked to SIDS 384 . Co-expression of these mutants with Na v 1.5 in HEK293 cells resulted in an increase in I NaL with a positive shift in the steady-state inactivation 364,384 . Adenovirus infection of adult rat ventricular myocytes with the mutant S206L confirmed the results obtained in HEK293 cells 384 . The S206L infected cardiomyocytes displayed increased I NaL with longer AP duration compared to WT infected cardiomyocytes 384 . These results are consistent with the underlying mechanism of LQTS.

III.3.2 Anchoring-adaptor proteins

α1-Syntrophin

The cardiac sodium channel Na v 1.5 is a part of the dystrophin multiprotein complex 385 . It has been shown by pull-down studies that Na v 1.5 interacts with dystrophin and utrophin by the adaptor protein syntrophin 386,387 . This interaction occurs via the three last Cterminal amino acids of Na v 1.5, Serine-Isoleucine-Valine, which form the PDZ binding motif of Na v 1.5 386 (Figure 35). The dystrophin-syntrophin-Na v 1.5 complex is mainly localized at the lateral membrane of cardiomyocytes (Figure 34) 353 . In the cardiomyocytes of dystrophindeficient mice, both total Na v 1.5 protein expression and sodium current were decreased 386 .

Moreover, immunostaining studies showed reduced lateral membrane expression of Na v 1.5, without affecting the Na v 1.5 expression at the intercalated discs 353 . These loss-of-function effects on Na v 1.5 are in line with the conduction defect seen in this model, demonstrated by the prolongation of the QRS complex 386 . Recently, J. S. Rougier et al demonstrated that the reduction in total protein expression is due to a proteosomal degradation of Na v 1.5 388 .

Interestingly, in the heart of dystrophin-deficient mice, increased expression of utrophin was observed, suggesting a compensating mechanism. Moreover, mice lacking both dystrophin and utrophin showed more pronounced loss-of-function effects of Na v 1.5 compared to dystrophin deficient mice 387 .

The importance of the interaction between Na v 1.5 and syntrophin is further emphasized by the identification of LQTS mutations (LQT12) in the SNTA1 gene encoding α1-syntrophin. These mutations increased peak I Na and I NaL 389,390 .

SAP97

SAP97 (Synapse Associated Protein 97) is one of the MAGUK proteins (Membrane-Associated Guanylate Kinase) that are characterized by the presence of multiple domains for protein-protein interactions including PDZ domains 391 . MAGUK proteins have an important role in the organization of several ion channels through the formation of multi-protein complexes. MAGUK proteins are involved in the targeting, trafficking and localisation of ion channels in the brain 392 and in the heart 353,[393][394][395][396] .

In cardiomyocytes, SAP97 and ZO-1 (zonula occludens) are the major MAGUK proteins 394 . Petitprez et al have demonstrated that the PDZ domains of SAP97 interact with the PDZ binding motif of Na v 1.5, and that Na v 1.5 and SAP97 are co-localized at the intercalated discs of cardiomyocytes, but not at the lateral membrane like the syntrophindystrophin complex 353 (Figure 34). Silencing of SAP97 leads to decreased surface expression of Na v 1.5 and I Na current in HEK cells and cardiomyocytes, suggesting a role for SAP97 in the regulation of Na v 1.5 surface expression 353 .

Moreover, SAP97 interacts with the K v 4.2/3 393 (I to current), K v 1.5 394,395 (I kur current), and Kir2.x 396 potassium channels. These interactions enhance channel expression at the plasma membrane of cardiomyocytes.

Recently, our team has identified mutations in the DLG1 gene encoding SAP97 in BrS patients. Functional studies of these mutations showed loss-of-function effects on I Na current, and gain-of-function effects on I to current, resulting in a net increased repolarizing current, a mechanism that underlies BrS (Kattygnarath et al, unpublished data). These results, along with the fact that multiple channels interact with SAP97, suggest that Na v 1.5 is part of multichannel protein complexes, and that SAP97 is a key partner in the formation of these complexes, regulating the targeting of ion channels to specific compartments in cardiomyocytes. Moreover, SAP97 becomes a new susceptibility gene for BrS.

Ankyrin-G

Ankyrins are membrane-cytoskeleton adaptor proteins that anchor membrane proteins to the actin and spectrin cytoskeleton. There are three ankyrin genes (ANK1, ANK2, and ANK3), encoding three different ankyrin proteins (ankyrin-R, ankyrin-B and ankyrin-G).

Ankyrin-B and ankyrin-G are present in the heart 397 . Mutations in the ANK2 gene have been linked to LQT4 237 . Nevertheless, there is no data suggesting a direct regulation of Na v 1.5 by ankyrin-B. However, there is a direct interaction between ankyrin-G and the ankyrin-binding motif in the cytoplasmic linker II-III of Na v 1.5 315,398,399 . In cardiomyocytes, ankyrin-G colocalizes with Na v 1.5 at intercalated discs and in T-tubules 315 . A mutation in the ankyrin-G binding-motif of Na v 1.5 (E1053K) has been identified in a patient with BrS 315 . E1053K

abolishes the Na v 1.5-ankyrin-G interaction, and disrupts the localization of Na v 1.5 at the cardiomyocyte membrane 315 .

Silencing of ankyrin-G in cardiomyocytes leads to a decrease in total and surface Na v 1.5 expression, localization of Na v 1.5 in the perinuclear region, and a decrease of total I Na

399

. So far, no mutations in the ANK3 gene have been reported.

MOG1

The RANGFR gene encodes MOG1 (multicopy of suppressor gsp1), a small protein of 28-kDa. High mRNA levels of this ubiquitously expressed gene was observed in cardiac tissue. This protein plays a role in nuclear protein trafficking via its interacion with GTP binding nuclear protein Ran 400 . By yeast two-hybrid assays, Wu et al demonstrated that MOG1 is a partner of Na v 1.5 401 . The interaction between MOG1 and Na v 1.

α-Actinin-2

Four α-actinin isoforms are encoded by four distinct genes; they are members of the Factin cross-linking protein family which includes spectrin and dystrophin. These proteins are involved in many physiological functions, including binding of the transmembrane proteins to the actin filament network. α-Actinin-2 interacts with the linker III-IV of Na v 1.5, and increases the I Na density in a heterologous expression system by enhancing the surface expression of Na v 1.5, without affecting the biophysical properties of the channel.

Immunostaining studies on human ventricular cardiomyocytes showed that α-actinin-2 colocalizes with Na v 1.5 at the sarcomeric Z-lines 403 . Thus, α-actinin-2 could stabilize Na v 1.5 channels at specific cellular membrane compartments by its direct interaction with the channels or through other α-actinin-2-associated proteins.

III.3.3 Enzymes interacting with and modifying the channel

Ubiquitin-protein ligases of the Nedd4/Nedd4-like family

The ubiquitin-protein ligase enzymes (E3s enzymes) covalently link ubiquitin, a small protein of 76 amino acids, to target proteins. Ubiquitination of membranous proteins leads to their internalization, then to their degradation by the lysosome or proteasome systems, or to their de-ubiquitination by specific proteases and recycling back to the membrane 404 . The Nedd4/Nedd4-like proteins are a family of nine different enzymes, where Nedd4-2 is the isoform with the highest expression level in human heart 405 .

The Nedd4-like ligases bind specifically to the PY motif (proline, tyrosine (xPPxY))

in the C-terminal domain of Na v 1.5 [406][407][408] , as well as those motifs in other cardiac ion channels 405,408,409 . By yeast-two hybrid and GST-pulldown 407 , it has been demonstrated that the ubiquitin-protein ligase Nedd4-2, a member of Nedd4/Nedd4-like family, binds to Na v 1.5.

Nedd4-2 and Na v 1.5 co-expression leads to channel internalisation without affecting total Na v 1.5 protein expression, and decreased I Na current [406][407][408] . Moreover, it has been shown that under basal conditions, a fraction of Na v 1.5 channels is ubiquitinated, both in HEK293 cells and in mouse heart 407 . These results suggest that the Nedd4-2 ligase modulates the surface expression of Na v 1.5, however, the exact mechanism underlying this modulation remains to be elucidated.

Ca2 + /calmodulin-dependent protein kinase II (CaMKII)

CaMKII is a serine/threonine protein kinase, which has an important role in intracellular Ca +2 signaling, by transducing the increased intracellular Ca +2 into multiple proteins phosphorylation, including the cardiac ion channels 410 . Na v 1.5 is regulated by CaMKII, which co-immunoprecipitates with and phosphorylates Na v 1.5, resulting in alteration of its gating properties 411,412

Protein tyrosine kinase Fyn and protein tyrosine phosphatase

PTPH1

The tyrosine kinase Fyn, which belongs to Src family tyrosine kinase, phosphorylates the tyrosine Y1495 located in the linker III-IV, beside the IFM-motif involved in Na v 1.5 inactivation 414 (Figure 35). Phosphorylation of Y1495 leads to a positive shift in the steadystate inactivation, and enhanced recovery from inactivation. However the site of interaction of tyrosine kinase Fyn on Na v 1.5 is unknown. Tyrosine phosphatase PTPH1 interacts directly with Na v 1.5 via the PDZ domain 415 . In HEK293 cells, co-expression of PTPH1 leads to negative shift of steady-state inactivation, this effect depending on the integrity of the SIV motif of Na v 1.5 415 . The effect of PTPH1 on Na v 1.5 is opposite to that of the tyrosine kinase Fyn, suggesting that the balance between tyrosine kinases (such as Fyn) and phosphatases (such as PTPH1), may regulate the degree of Na v 1.5 phosphorylation, and by this way the channel inactivation.

III.3.4 Proteins that modulate the biophysiological properties of Na v 1.5 upon binding

Calmodulin (CaM)

The C-terminal domain of Na v 1.5 has an IQ motif (IQxxxRxxxxR), which forms the binding motif of the CaM, a ubiquitous Ca +2 -sensing protein that has a role in several cellular processes [416][417][418] . CaM interacts also with the linker III-IV of Nav1.5 419,420 (Figure 35).

However, there is a discrepancy in the functional effects of CaM on Na v 1.5. Steady-state inactivation and entry into slow inactivation have been shown to be either decreased, increased, or not changed 416,417,[419][420][421] . Another study showed that CaM results in negative shift of the voltage dependence of activation without affecting the inactivation 422 . It has been proposed that, at basal Ca +2 concentration, CaM binds to the IQ motif of Na v 1.5. When the Ca +2 level increases, the CaM binds to Ca +2 , resulting in CaM conformational changes that weaken the interaction between CaM and the IQ motif. The dissociated CaM can then bind to CaMKII or to the other CaM binding domain in the linker III-IV 423 . The interaction with the linker III-IV will inhibit channel inactivation 419,420 . Moreover, when CaM is released, the IQ motif can interact with EF-hand Ca +2 binding domain located in the proximal C-terminal region of Na v 1.5, and this interaction enhances the affinity of EF domain to Ca +2 423 .

Telethonin

Telethonin is a small sarcomeric protein shown to be a partner of Na v 1.5 by immunofluorescence and co-immunoprecipitation studies in mouse heart tissue, but its interaction site on the channel remains unknown 424 . Telethonin is mainly expressed in striated muscles 425 , but expression in smooth muscles of the gastrointestinal tract was also observed 424 . Mutations in the TCAP gene, encoding telethonin, have been reported in limb girdle muscular dystrophy (LGMD2G) and hypertrophic and dilated cardiomyopathies 426,427 .

Another mutation in TCAP, R76C, was found in a patient with intestinal pseudoobstruction 424 . In the human gastrointestinal tract, Na v 1.5 is expressed in the smooth muscle cells and in the interstitial cells of Cajal (ICC) 428 , which generate active pacemaker currents responsible for smooth muscle contraction. Interestingly, expression of the R76C mutant with the Na v 1.5 channel in a heterologous expression system caused a negative shift of the voltagedependent activation and increased the window sodium current. Moreover, silencing of telethonin in HEK293 cells stably expressing Na v 1.5, led to a positive shift in voltagedependent activation of I Na 424 , suggesting that the R76C mutation enhances the effect of telethonin on Na v 1.5 424 . However, no cardiac phenotype was observed in the patient carrying the R76C mutation.

Glycerol phosphate dehydrogenase 1-like protein (GPD1L)

The GPD1L, encoded by the GPD1L gene, is homologous to glycerol phosphate dehydrogenase (GPD1) that catalyzes the reversible conversion of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP), however the function of GPD1L is unknown.

This gene on chromosome 3p24 has been linked to BrS by linkage analysis in a large multigenerational family, and a mutation cosegregating with a BrS phenotype in this family has been identified 429,430 . Since then, mutations in GPD1L have been reported in subjects from a cohort with SIDS 431 . Functional analysis of these mutations showed a loss-of-function effect on Na v 1.5, by decreasing I Na through the reduction of Na v 1.5 surface expression. Two more recent studies have demonstrated that the modulation of I Na by GPD1L occurs via the protein kinase C (PKC) 432,433 . In HEK293 cells, when co-expressing the GPD1L mutant and Na v 1.5, the decrease in the surface expression of Na v 1.5 and in I Na was dependent on the phosphorylation of serine 1503 by PKC 432 , a process already known to decrease I Na 434 . In addition, it has been shown that there is an interaction between Na v 1.5 and GPD1L, and that the mutations did not affect this interaction, but the enzymatic activity of GPD1L 432 .

However, the site of interaction of GPD1L on Na v 1.5 remains unknown. Another study showed that the reduction of I Na caused by the mutant GDP1L can be reproduced by adding an activator of PKC or NADH, and inhibited by an inhibitor of PKC or of NAD + 433 . The phosphorylation of the channel by the PKC/GPD1L could thus constitute a link between the metabolic state of the cell and its excitability by modulating the sodium current.

14-3-3η protein

The 14-3-3 dimeric cytosolic adaptor proteins interact with a wide variety of other proteins and are involved in the regulation of cell signaling pathways and ion channels 435 . An interaction between the 14-3-3η channel and Na v 1.5 was revealed by yeast two-hybrid assay and confirmed by co-immunoprecipitation studies 436 . This protein binds to the cytoplasmic loop DI-DII of Na v 1.5 (Figure 35) and co-localizes with Na v 1.5 at the intercalated discs of cardiomyocytes. Co-expression of 14-3-3η with Na v 1.5 in COS cells led to a negative shift in steady-state inactivation and slowed recovery from inactivation. However, the peak current density was not affected. These results indicate that 14-3-3η could alter the biophysical properties of Na v 1.5 but not its trafficking, at least in COS cells 436 . Different isoforms of the 14-3-3 protein family are present in the heart, thus further studies are needed to define the exact role of these cardiac isoforms and their possible implication in heart diseases 436 .

Plakophilin-2 and desmoglein-2

Plakophilin-2 and desmoglein-2 are desmosomal cardiac proteins encoded by the genes PKP2 and DSG2, respectively. The association of plakophilin-2 with the Na v 1.5 channel was demonstrated by GST-pulldown assay and co-immunoprecipitation studies 437 .

Immunostaining analysis showed co-localization of the two proteins at the intercalated discs of cardiomyocytes. Silencing of plakophilin-2 by shRNA in adult rat cardiomyocytes significantly decreased the sodium current density, negatively shifted the steady-state inactivation, increased the time needed for the recovery from inactivation of Na v 1.5, and slowed the action potential propagation 437 . These results were confirmed in the heterozygous knockout mouse model of PKP2, where isolated ventricular cardiomyocytes showed a decrease in the sodium current and an accelerated inactivation of Na v 1.

Caveolin-3

The function of Na v 1.5 is regulated by β-adrenergic stimulation. Stimulation of the βadrenergic receptor by isoproterenol increases I Na . It has been proposed that this increase in I Na is due to two mechanisms: one is protein kinase A-dependent (PKA-dependent), the other is PKA-independent.

PKA stimulation induces phosphorylation of the serine at the position 526 and 529 in the linker I-II of Na v 1.5 [441][442][443] (Figure 35). In addition to these phosphorylation sites, the linker I-II has three ER specific retention motifs (RXR) that could interact with ER chaperon proteins preventing the trafficking of Na v 1.5. Elimination of these various motifs either reduces or abolishes the effect of PKA on Na v 1.5. This suggests that the phosphorylation of Na v 1.5 by the PKA and the presence of these motifs are required for the PKA effects, and that phosphorylation could hide these retention motifs and allow the transport of Na v 1.5 to the membrane 444 .

Isoproterenol is also able to increase I Na in the presence of a PKA inhibitor 445 . The time needed for this effect is short (less than 10 minutes), and the effect is reversible upon washing of the β-agonist, suggesting that Na v 1.5 proteins could be in a storage compartment and could be released following adrenergic stimulation. Then, it was suggested that caveolae could underlie this PKA-independent mechanism 446 Mutations in the CAV3 gene, encoding caveolin-3, have been reported in patients with LQTS 448 and SIDS 449 . Functional studies of these mutations showed increased late current, which is in accordance with the phenotypes.

Fibroblast growth factor homologous factors (FHFs)

The fibroblast growth factor homologous factors (FHF) constitute a subset of the fibroblast growth factor (FGF) family, however they are unable to stimulate the FGF receptors, and lack the N-terminal secretion signal sequences. Consequently, they are not secreted, but remain intracellular, and bind to the C-terminal domain of the voltage-gated sodium channels (VGSCs), and to islet-brain-2 (IB2), the neuronal mitogen activated protein (MAP) kinase scaffold protein 450 . The genes encoding the four FHFs (FHF1 to FHF4) are officially named FGF12, FGF13, FGF11, FGF14, respectively. They are mainly expressed in excitable cells, especially in the nervous system, and to a lesser extent in the heart 450 . Each FHF has multiple isoforms resulting from alternative promoter usage and 5′ alternative splicing, leading to 10 FHF isoforms in humans that differ in the amino-terminal region 451 (Figure 36). Each isoform has N-terminal, core, and C-terminal regions (Figure 36). The core and the C-terminal regions of FHFs show high degrees of similarity between the various FHFs. In heterologous expression systems, differences in the localization of the two A and B isoforms have been observed. The A isoforms mainly have a nuclear localization, while the B isoforms are cytoplasmic and nuclear 451 . This is due to the presence of a nuclear localization signal in the A isoforms 452 (Figure 36).

o

Interaction of FHF with the C-terminal domain of the VGSCs

A yeast-two-hybrid screen for partners of Na v 1.9 was the first to demonstrate that FHF1B interacts with the C-terminal domain of Na v 1.9, a sodium channel expressed in nociceptive neurons 453 . Then further studies have showed that FHF1A, FHF1B, FHF2A, and FHF3A interact with the cardiac sodium channel Na v 1.5 [454][455][456] , FHF2B with the neuronal sodium channel Na v 1.6 457 , and other FHFs with other VGSCs 455 .

The exact interaction site of of FHF1B on Na v 1.5 is debated, located either in the proximal (amino acids 1773-1832) 454 , or in the proximal and the distal parts of Na v 1.5 Cterminus (amino acids 1773-1908) 456 . Moreover, differences in the effects of FHF1B on Na v 1.5 were also observed 454,456 . In HEK293 cells, co-expression of FHF1B with Na v 1.5 leads to a negative shift of steady-state inactivation 454 , or a positive shift of the steady-state inactivation of Na v 1.5 456 .

Crystal structure of FHF2A demonstrates a conserved surface on the FHF core domain involved in the binding of VGSCs to the FHFs 455 . Thirteen residues at different positions in the protein sequence, conserved among the different FHF isoforms but not in FGF, constitute the VGSC binding site (Figure 36) 455 . Moreover, the alternatively spliced N-termini of FHFs have been excluded as binding sites with VGSCs, since the truncation of the N-terminal region of FHF isoforms FHF2A, FHF3A and FHF4B does not affect their interaction with the VGSCs 455 . Recent study has showed that the human FGF12 polymorphism rs17852067 (P149Q), which is located in the core region but not in the previously reported binding sites to Na v 1.5, could also modulate the interaction of FHFs with the VGSCs 456 , suggesting that multiple FHF sites could be involved in the VGSC interaction. o

Expression of the FHF isoforms in the heart

The cardiac expression of the different FHF isoforms was relatively unknown, until a recent study by GS Pitt's group 458 , showing that FGF13 is the most expressed FHF in adult mouse ventricular myocytes. By a quantitative reverse-transcriptase polymerase chain reaction (qPCR), using isoform-specific primers, they demonstrated that FGF13-VY is highly expressed in adult mouse cardiomyocytes. The FGF13-V and FGF13-Y isoforms are expressed at low levels, the other isoforms nearly undetectable (Figure 37A). However, at the protein level, only FHF2 (most probably FHF2-VY) could be detected. Very recent results from the same group 304 , using qPCR on RNA from nonfailing human ventricular tissue, showed that in the human heart, FGF12-B is the main isoform, whereas FGF12-A was nearly undetectable (Figure 37B). The FFG13-Y and/or FGF13-VY (qPCR was unable to distinguish between these two isoforms) were the second most highly expressed isoforms (Figure 37B). Silencing of FGF13 in rat and mouse ventricular myocytes led to reduced peak I Na , a negative shift of steady-state inactivation 304,458 , and delayed recovery from inactivation of Na v 1.5 458 .

Cell surface biotinylation of isolated cardiomyocytes showed decreased surface expression of Na v 1.5 458 , accounting for the decreased I Na , along with the altered biophysical properties.

o FHF genetics

The role of the FHFs has been mainly studied in the nervous system, as the FHFs are highly expressed in the central and peripheral nervous systems 452 , and several loss-of-function mutations have been identified in the FGF14 gene in spinocerebellar ataxia 27 (SCA27), a hereditary neurodegenerative disease [461][462][463][464] . Functional studies of the first FGF14 mutation identified in SCA27, F145S, in rat hippocampal neurons showed decreased neurological sodium current density, reduced Na + channels localization at the axon initial segment, and decreased neuronal excitability in a dominant-negative way 464 . A mouse model lacking FGF14 showed an ataxia phenotype, which is in line with decreased Na + channel function and decreased neuronal excitability 465 . Moreover, it has been shown that double knockdown of both FGF12 and FGF14 leads to a severe ataxia phenotype 466 .

The role of FHFs in heart disease is less well known, however, a very recent study reported a mutation in FGF12-B (Figure 36) in a patient with BrS 304 . The missense mutation Q7R was identified in a 61 years old male patient who had a suspicious ECG of BrS type during flecainide treatment for AF. Flecainide test to unmask BrS induced type 1 ECG pattern of BrS. The mutant Q7R has a reduced binding affinity for the C-terminal domain of Na v 1.5 compared to the WT. However, it does not affect the interaction of FHF1B with the junctophilin-2. In order to study the effects of this mutation on the sodium and calcium currents in the native environment where all the partners of Na v 1.5 are present, an FGF13

shRNA strategy was used to supress the endogenous FGF13 of adult rat cardiomyocytes.

Then, FGF13 was replaced by a WT or mutant FGF12-B human variant. In this system, knockdown of FGF13 decreased the sodium and calcium current densities, with mislocalization of the Ca v 1.2, and negative shift of steady-state inactivation of Na v 1.5, in harmony with previous studies 458,459 However, FGF12-B mutations affecting both the calcium and sodium channels could also be present.

Mutations in SCN5A, the gene encoding the Na v 1.5 α-subunit, have been implicated in many inherited cardiac arrhythmias, which were firstly considered as separate clinical entities with distinct phenotypes. However, a wide spectrum of mixed disease phenotypes was recently reported in SCN5A-related arrhythmias, referred to as "overlap syndromes" of cardiac sodium channelopathy. In addition to their variable expression, they are characterized by incomplete penetrance. The reasons behind these two features remain unknown, but this suggests that other genetic and environmental factors, combined with specific altered biophysical properties of a given SCN5A mutation could play an important role in the modulation of the clinical phenotype. The main aim of my thesis work was to study such modulating factors. Moreover, since no mutation was found in 75% of BrS patients, I aimed to identify a new candidate gene in BrS.

The detailed objectives of my thesis work were around the following two axes:

1.

By the functional characterization of two SCN5A mutations, and the screening analysis of common polymorphisms associated with AF and BrS in SCN5A mutation carriers, I tried to demonstrate how the specific alteration of the biophysical properties of a given mutation, combined with the genetic background of mutation carriers, could modulate the clinical picture.

2.

By candidate gene approach in a group of patients with BrS, I searched for new mutations in a gene encoding a partner of Na v 1.5.

To carry out this project, I used several methodological approaches including different techniques of biochemical analysis and molecular biology, transfection in heterologous expression systems, and genotyping.

These methods, in addition to patch-clamp analysis, allowed us to demonstrate the involvement of three factors in modulating the phenotypic expression caused by SCN5A mutations: the interaction between the Na v 1.5 α-subunits (article 1), the mutant biophysical defects associated with the differences in the electrical properties between the atrial and ventricular cardiomyocytes, and the genetic background of the patients (article 2).

MATERIALS & METHODS

I. Functional studies I.1 Expression vectors and site-directed mutagenesis

The plasmids used in our study are expression vectors designed for mammalian

Site-directed mutagenesis

Na v 1.5 mutants R104W, R104K, R121W, R878C, R1860Gfs*12 and L1821fs*10, and FHF1B mutant V127M were prepared using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, USA) according to the manufacturer's instructions and were verified by sequencing. The principle of this technique is to introduce a mutation into the gene encoding the protein of interest. Briefly, each wild-type plasmid was amplified by PCR in its entirety with a pair of primers bearing the desired mutation in their center. Then the PCR products were subjected to enzymatic digestion with DpnI. This endonuclease enzyme is specific for methylated DNA, and is used to digest the parental WT DNA template, which is methylated due to its production in bacteria, leaving only the newly PCR-synthesized mutated plasmid (not methylated). The primers used for each mutagenesis are listed in Table 7.

Table 7: Forward (-F) and reverse (-R) primers used for site-directed mutagenesis *Mutated nucleotides are bolded, enlarged and in red; deleted nucleotides are in blue.

Bacterial transformation

The newly synthesized mutated plasmids were transformed into the XL10-Gold strain of E. coli. The DNA was added to the competent bacterial cells and incubated on ice for 30 min. Bacteria were then transformed by a heat shock for 30 sec at 42°C. After 500 µL super optimal broth (SOC) medium was added to the bacteria, they were grown for 1h at 37°C with shaking at 225 rpm. This outgrowth step allows the bacteria to generate the antibiotic resistance proteins encoded on the plasmid backbone. The transformation mixture was then spread onto agar plates containing the appropriate antibiotic and cultured overnight at 37°C.

Only the bacteria that received the plasmid were able to grow and form colonies. The following day, five colonies were selected and grown overnight at 37°C with 225 rpm shaking in liquid lysogeny broth (LB) medium containing the selection antibiotic.

Plasmid extraction

From this bacterial culture, the extraction of the plasmid was carried out using the PureLink® Quick Plasmid Miniprep Kit (Life Technologies, USA). Bacterial cells were lysed using an alkaline/SDS procedure and the lysate was then applied to a silica membrane column that selectively binds the DNA. The plasmid was then eluted in water, and sequenced over the entire gene of interest, in order to verify the presence of the desired mutation and to ensure that no other mutation was inserted.

I.2 Heterologous expression systems and cell culture

The cell line HEK293 (Human Embryonic Kidney) was used as the main heterologous expression system for patch-clamp and biochemical analysis. Indeed, these cells are human and they do not express endogenous sodium channels. HEK cells were cultured in DMEM medium (Gibco, Life Technologies, USA) containing 10% fetal calf serum (FCS), 50 IU/ml penicillin, and 50 µg/ml streptomycin at 37°C in an incubator with 5% CO2. We also used a stable HEK293 cell line expressing the cardiac sodium channel Na v 1.5, which was a gift from Dr H. Abriel (Bern, Switzerland). This cell line was maintained in the same culture medium as previously described above, but also complemented with 25 g/ml of the selective antibiotic Zeocin (Life Technologies) in order to select for cells expressing the gene of interest. Cells were not kept in culture for more than 20 passages to maintain homogeneity of the results.

I.3 Transient transfection of HEK293 cells

The day before transfection, cells were plated into 6-well plates for patch-clamp analysis or into T25 flasks (25 cm 2 ) for biochemical analysis. When the cells reached 70-80% confluence, they were transfected using the cationic lipofectant JetPEI ® (Polyplus Transfection, USA) according to the manufacturer's recommendations. The amounts of transfected plasmids are specified in each article. For patch-clamp recordings, 24h after transfection, cells were rinsed with phosphate buffered saline (PBS; Life Technologies), detached using 200 µl Trypsin-EDTA per well, resuspended in fresh medium and re-plated at a lower density on 35-mm petri dishes (suitable for the patch-clamp set up) to obtain single cells the next day. Biochemical analyses were performed 36 h after transfection.

In order to inhibit proteasome activity, cells were incubated with 1 µM of MG132, which is a specific, reversible and cell-permeable inhibitor of the 26S proteasome subunit, or with 0.1 % dimethyl sulfoxide (DMSO) as a control for 24 h before protein extraction.

I.4 Biochemical analysis

I.4.1 Total protein extraction

Forty-eight hours after transfection, HEK293 cells were washed twice with PBS and lysed in a RIPA lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 1% NP40, 0.1% SDS, 0.5% deoxycholate), and complete protease inhibitor cocktail (Roche, Germany) for 1 h at 4°C with rotation. The soluble fractions from two subsequent 4°C 30-min 14 000 g centrifugations were then used for the experiments. To load each lane of the SDS-PAGE with equivalent amounts of total proteins, the protein concentration of each lysate was measured in duplicate by the Bradford assay using a BSA (Bovine Serum Albumin) standard curve.

I.4.2 Cell surface biotinylation

Cell surface biotinylation is a powerful tool designed to study cell surface protein expression. Biotin is a small vitamin efficiently reacting with primary amino groups to form stable amide bonds, and is subsequently used as a marker of labeled molecules. Since biotin has a small size, it does not disrupt the properties of the target molecule. Binding of biotin to primary amino groups is conferred by its ester group N-hydroxysuccinimide (NHS). Several different NHS esters of biotin are available, such as the Sulfo-NHS ester reagent that we used in our experiments (Figure 38). Since it is a water-soluble molecule, biotinylation experiments can be performed in the absence of organic solvents. In addition, as it is a charged molecule, thus unable to cross biological membranes, only primary amines present on the cell surface will be biotinylated. To purify the biotinylated proteins, we used neutravidin agarose resins. Neutravidin is a deglycosylated form of avidin, which is a tetramer with a strong affinity to biotin. The interaction between biotin and avidin represents one of the strongest existing non-covalent bonds. Finally, to elute the biotinylated proteins from the neutravidin resins, the biotin intramolecular disulfide bridge is reduced by Dithiothreitol (DTT) (Figure 39).

Forty-eight hours after transfection with WT or mutant constructs, HEK293 cells were washed twice with ice-cold PBS and incubated for 60 min at 4°C with PBS containing 1.5 mg/mL of EZ Link Sulfo-NHS-SS-Biotin (Pierce, USA). Plates were washed twice and then incubated for 10 min at 4°C with PBS with 100 mM glycine to quench unlinked biotin, and washed twice with PBS. Cells were then lysed for 1 h at 4°C with the lysis buffer described in the previous paragraph. After centrifugation at 14 000 g for 30 min at 4°C, supernatants were incubated with immobilized neutravidin beads (Pierce, USA) overnight at 4°C and pelleted by centrifugation at 14 000 g for 1 min at 4°C. After three washes with lysis buffer, the biotinylated proteins were eluted with the Laemmli sample buffer complemented with 50 mM DTT at 37°C for 30 min. Biotinylated proteins were then analyzed by western blot.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a negative control of biotinylation, and transferrin receptor (TfnR), as a positive control.

Immunoprecipitation

This technique aims to purify a protein of interest and its partners from a cell lysate.

The total cell lysate containing the protein X is incubated with magnetic beads (Dynal, Norway) on which has been adsorbed an anti-X antibody allowing the specific precipitation of the protein X and its partners if X is part of a protein complex (Figure 40). protease inhibitor cocktail). Cell pellets were pipetted up and down 20 times, flushed 20 times through a 22-gauge needle, incubated on a rotating wheel for 1 h at 4°C, and finally centrifuged for 10 min at 16 000 g. Magnetic beads were washed twice with PBS-tween 0.02%, incubated with the antibody against the protein of interest for 2 h at room temperature, washed twice again with PBS-tween 0.02%, and incubated with the pre-cleared lysates.

Samples were incubated overnight at 4°C on a rotating wheel. After washing the beads four times with PBS-tween 0.02%, proteins were eluted with the Laemmli sample buffer at 37°C

for 30 min with agitation, and analyzed by western blot. protein detection was performed using the Odyssey infrared imaging system (LI-COR Biosciences). Signals were quantified using the ImageJ software and normalized to WT levels.

Antibody

II. Molecular biology II.1 Total RNA extraction

Total mRNA was extracted from frozen mouse or human hearts using TRIzol (Life technologies, USA) and the Pure link TM RNA mini kit (Life technologies, USA). A small piece of frozen heart was homogenized in 500 µL of TRIzol, incubated 5 min at room temperature while vortexing to allow complete dissociation of nucleoprotein complexes. Then 200 µL of chloroform was added and samples were mixed vigorously by hand for 15 sec and incubated at room temperature for 1-2 min. Lysates were then centrifuged at 15 000 g for 10 min at 4°C. This step separates the RNA in the upper aqueous phase, from the DNA and protein in the middle and lower organic phases. RNAs were then precipitated in 70% ethanol and transferred to a spin column. After two washes, RNAs were eluted with RNase-free water.

II.2 RNA reverse transcription

This technique synthesizes complementary strand DNA (cDNA) from an RNA template using an RNA-dependent DNA polymerase (reverse transcriptase). The cDNA contains only the coding sequence of a gene and can be then amplified by PCR.

Here, we reverse-transcribed total RNA using oligo-dT primers, which are short sequences of deoxy-thymine nucleotides that bind to the RNA polyadenylate tail, providing a free 3'-OH end that can be extended by the reverse transcriptase enzyme to create the complementary DNA strand. Then the RNAse enzyme was added to remove the original template mRNA, leaving single stranded cDNA (sscDNA) (Figure 41). This sscDNA could then be converted into a double stranded DNA (dscDNA) by the DNA polymerase.

We used the reverse-transcription kit SuperScript® III First-Strand Synthesis System (Life Technologies, USA). We utilized 1µg of total RNA per 20 µL reaction. At the beginning, we prepared the primer/RNA mix (1µg RNA, 1µL 50 µM Oligo dT, 1µL 10 mM dNTPs, and water to a final volume of 10µL), which was denatured at 65°C for 5 min and then cooled on ice for 1 min. Then we added 10 µL of cDNA synthesis mix (2µL 10X RT buffer, 4µL 25 mM MgCl2, 2µL 0.1 M DTT, 1µL RNAse out, 1µL super script III enzyme)

and the reverse transcription reaction was incubated at 50°C for 50 min. We terminated the reaction by heating the sample to 85°C for 5 min. Finally, RNA removal was performed by treating the sample with 1µL of RNase H and by heating to 37°C for 20 min.

RNase H enzyme

RNase H enzyme starting point of the exponential phase is called the threshold cycle (Ct). It corresponds to the number of cycles at which the fluorescence intensity differs from background noise (threshold line), and it is directly related to the amount of matrix originally present in the sample. For example if we have two samples X and Y, and the CtX < CtY, this means that the initial amount of template of X is more than Y (Figure 42). The third and last phase is the plateau phase representing the saturation of the reaction caused by the accumulation of PCR products.

Primers used for qPCR

For our study of the relative expression of the FGF12 and FGF13 isoforms in human and mouse hearts, we designed primers located in the homologous region between human and mouse FHF transcripts, as well as primers specific to each isoform. Moreover, in order to selectively amplify the cDNA and not the genomic DNA, primers were designed spanning adjacent exons with large introns in between (Figure 43 and Table 9). ---------------------------------------------------------------- Table 9: qPCR primer pairs used for detecting FGF12 and FGF13 transcripts in adult human and mouse hearts * Ribosomal protein L32 used as a normalizer.

------------------------------------------------------------------Baseline -
------------------------------------------------------------------Baseline ------------------------------------------------------------------ Threshold CtX=18

qPCR

The cDNA reverse-transcribed from 1 µg of total RNA was used to perform the qPCR. The primer effectiveness was determined by quantitative PCR using a range of successive cDNA dilutions. Quantitative PCR was performed in 96-well plates. Samples were run in triplicate, each well contained 5 µl of total cDNA solution, 1 µl of each sense and antisense 10 µM primer, 10 µl of 2X SYBR Green buffer (LightCycler ® 480 DNA SYBR Green I Master, Roche). Negative controls were realized under the same conditions but with

E2 E3 E4 E5 E2 E3 E4 E5 E1 E1 FGF12A R1 FGF12B R1 E2 E3 E4 E5 V E2 E3 E4 E5 Y V E2 E3 E4 E5 Y R2 F F F FGF13V FGF12VY FGF12VY/Y R2 R2 F F
no template. The qPCR was performed using the LightCycler 480 machine (Roche, Germany) with the following PCR program: (i) denaturation 10 min at 95°C, (ii) 45 cycles of amplification (denaturation at 95°C for 10 s, hybridization of primers at 60°C for 20 s, and extension at 72°C for 20 s), (iii) melting step (denaturation at 95°C for 30 s, hybridization at 68°C for 30 s, then the samples were heated gradually (0.06°C/sec) from 65°C to 95°C). This increase in temperature led to dsDNA dissociation (or melting) into single strands, releasing the dye, and causing a change in fluorescence. The result is a melting curve profile characteristic of the amplicon (Figure 44).

To assess the relative expression of FHF isoforms in human and mouse hearts, we calculated the difference between the Ct of each isoform. First of all, to normalize any differences due to the amount of nucleic acids placed in each well, we used the Ct of a reference gene encoding the ribosomal protein L32 (RPL32) to calculate the ∆Ct = Ct of RPL32 minus the Ct of each FHF isoform. Then, we calculated the ∆∆Ct = ∆Ct of each FHF isoform minus the ∆Ct of the most highly expressed FHF isoform (FGF12-B in human heart, FGF13-VY in mouse heart). Then the relative expression of each isoform (Q) was calculated using the formula Q = 2 ∆∆Ct , where ∆∆Ct of the highly expressed isoform is = 0 and Q = 1.

The other isoforms have ∆∆Ct < 0 and Q < 1, so each FHF isoform was compared to the highly expressed one.

III. Genotyping

Our laboratory has a large collection of DNA from patients with inherited cardiac arrhythmias. For the screening analysis of FGF12, 182 BrS probands, without mutations in the SCN5A, MOG1, DLG1, KCNE3 and SCN1B genes, were included in our study. Diagnosis of BrS was based on the accepted criteria of the recent consensus statement in 2013 287 : the presence of a spontaneous type-1 ECG pattern (ST segment elevation ≥2 mm in one or more right precordial leads) spontaneously or following the administration of a sodium channel blocker. Structural heart disease was excluded by echocardiography.

Blood samples were obtained for genetic analyses after signed written informed consent was obtained, and following approval by the local ethics committee of the Pitié-Salpêtrière Hospital. The study was conducted according to the principles of the Helsinki declaration.

III.1 The genomic regions analyzed

All sequences were obtained from the reference databases "NCBI" and "Ensembl genome browser". PCR primers (Table 10) were designed to amplify all exons within their flanking intronic regions (approximately 50 base pairs upstream and downstream).

FGF12-B exons

Forward primer (5' -3') Reverse primer (5' -3') Product length (bp) Before starting the sequence analysis, the concentration and the purity of each genomic DNA sample were evaluated using a spectrophotometer (Nanodrop ®, Thermo Scientific, USA). Nucleic acids have an absorbance wavelength of 260 nm. The DNA concentration is given in ng/µl. The purity of the sample is determined by a 260/280 ratio (DNA/protein). A ratio of ~1.8 is generally accepted as "pure" for DNA.

III.3 Sequence analysis of FGF12-B

All FGF12-B exons and intronic junctions were amplified by PCR. PCR products were purified using the P100 column (BioRad ®) and the sequencing reaction was realized using the Sanger method with Big Dye Terminator v.3.1 kit (Applied Biosystems ®), followed by a G50 (Sephadex®) purification. The Sanger method depends on the incorporation of chainterminating dideoxynucleotides by the DNA polymerase during the sequence reaction. The sequence reaction mixture contains the DNA template, forward or reverse primer, DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation (Figure 45). These chain-terminating nucleotides do not carry the 3'-OH group needed for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to stop the extension of DNA when a ddNTP is incorporated. These ddNTPs are fluorescently labelled to be detected in automated sequencing machines. At the end of the sequencing reaction, fragments of various sizes were generated. They were then separated, according to their size, by migration via a 16 capillary 3130 sequencer (Applied biosystems ®, Life technologies, USA). When the fragments pass through the detection sensor, a laser beam excites their fluorescence, which is in turn captured by a camera coupled to a charger that converts this fluorescence into electrical data transferred to the computer in the 3130 Data Collection software (Figure 45). Sequence analysis was then performed using the CodonCode Aligner v3.

RESULTS

suggesting the degradation of the mutants by the ubiquitin-proteasome system. However, the ∆Nter was slightly degraded and normally adressed to the membrane. The retention of the mutant channels in the ER and their subsequent degradation could explain their loss-offunction effects in the homozygous state, while the retention of the WT in the ER in the presence of the mutant could account for the dominant-negative effect, and suggests a cooperation between the Na v 1.5 α-subunits. Co-immunoprecipitation study in HEK293 cells transfected with two different Na v 1.5-tag constructs demonstrated that the WT α-subunits interacted with each other, as well as with the mutant R104W. As the mutant was able to inhibit the trafficking of the WT, most probably through the interaction between Na v 1.5 αsubunits, we hypothesised that the WT subunits could be able to transport a small part of the mutant subunits with it to the membrane. To test this hypothesis, the N-terminal mutants R104W and R121W were co-transfected with the trafficking-competent but non-functional Na v 1.5 mutant, R878C. Patch-clamp analysis showed that the R878C subunits were able to restore a very small sodium current density with a positive shift of the activation curve. This partial rescue confirmed the interaction between the Na v 1.5 α-subunits during their transport to the membrane, while the positive shift of activation suggests a possible cooperation between Na v 1.5 α-subunits at the membrane. Finally, expression of the Nter with the WT Na v 1.5 increased the sodium current density by two-fold compared to the WT.

Discussion

The dominant-negative effect of mutant ion channel subunits on the WT ones has been described mainly for ion channels consisting of multiple α-subunits, like the potassium channels K v LQT1 467 , which form homotetramers, but Na v 1.5 α-subunits were not known to oligomerize. Thus, the dominant-negative effect of the N-terminal mutants observed in our study was unexpected, while it has been reported once, for the trafficking-defective mutant L325R 468 . In this study, the authors hypothesized that the mutant channels could exert the dominant-negative effect by affecting the biosynthesis or the trafficking of the WT. In our study, we showed for the first time that an interaction between Na v 1.5 α-subunits exists, and that it is not affected by the presence of the mutant R104W. This interaction could underlie the dominant-negative of the mutant. Under normal condition, the WT α-subunits interact with each other, most propably in the ER, and traffic together to the plasma membrane.

However, the mutant R104W was retained in the ER and degraded by the ubiquitinproteasome system, and through the interaction with the WT α-subunits, it retained the WT with it in the ER. Consequently, few WT α-subunits interacting with each other reached the membrane, accounting for the dominant-negative effect. The mechanism of mutant channel retention remains to be elucidated. It is possible that the N-terminal mutatnts caused conformational changes preventing the proper folding of the Na v 1.5 protein, resulting in its degradation by the ER-associated degradation (ERAD) system. In contrast to the traffickingdefective N-terminal mutants, the R878C mutant and the ∆Nter-Na v 1.5, which were normally expressed at the membrane, did not have dominant-negative effect on the WT. The partial rescue by the trafficking-competent R878C on the N-terminal mutants further supports the cooperation between Na v 1.5 α-subunits.

By studying the N-terminal mutants, the ∆Nter-Na v 1.5 and the Nter, our study highlights the important role of the N-terminal region of Na v 1.5. The proper expression of the ∆Nter-Na v 1.5 at the membrane, with its inability to conduct sodium current suggest that the N-terminal domain is not essential to the trafficking of Na v 1.5, but it is important for channel opening. Moreover, the positive shift of activation when the N-terminal mutants were expressed with the WT or with R878C, suggests the involvement of the N-terminal domain in channel activation, and a possible functional cooperation between Na v 1.5 α-subunits at the membrane.

From a clinical point of view, the loss-of-function effects of the mutant R104W in the heterozygous state are most probably responsible for the BrS in this patient.

Conclusion

Our study demonstrated for the first time a physical interaction between Na v 1.5 αsubunits, likely occuring in the ER, accounting for the dominant-negative effects of the Nterminal mutants, and its partial rescue by the R878C mutant. In addition, our results highlight the role of the N-terminus of Na v 1.5 in the channel activation and opening.

I.3

The N-terminal domain is not the site of interaction between Na v 1.5 α α α αsubunits Our previous study demonstrated for the first time that Na v 1.5 α-subunits interact with each other. So, our aim was then to search for the site of interaction between Na v 1.5 αsubunits. As the N-terminal mutant R104W has a dominant-negative effect on the WT by retaining it in the ER, we thought that the N-terminal domain of Na v 1.5 could be the site of interaction between Na v 1.5 α-subunits. To test this hypothesis, co-immunoprecipitation studies were performed in HEK293 transfected with either HA-WT + GFP-WT or HA-WT + ∆Nter-GFP, and HA-WT alone as a negative control. Immunoprecipitations were performed with the anti-GFP antibody, and western blot revealed with the anti-HA and anti-GFP antibodies. In cells expressing HA-WT + GFP-WT or HA-WT + ∆Nter-GFP, a specific band at the expected size of HA-WT was present (Figure 46), indicating that the WT and ∆Nter αsubunits interacted with each other. Our results suggest that the interaction between Na v 1.5 αsubunits is not N-ter-N-ter. However, we cannot exclude that the interaction could occur for example between the N-terminus and the C-terminus, or between the C-termini. To test this hypothesis further constructs are needed where both N-and C-terminal domains are deleted.

In addition, it is possible that the interaction occurs at different sites along the channel, involving the P-loops and the cytoplasmic linkers. In this study we characterized the C-terminal mutation R1860Gfs*12, which is a frame-shift mutation changing the amino acid arginine at position 1860 into glycine and causing premature termination of Na v 1.5 protein at position 1871, resulting in a loss of interaction with several C-terminal-partner proteins. R1860Gfs*12 was identified in a family with a mixed clinical picture of SSS and AF or flutter, and atrioventricular-block.

Spontaneous BrS ECG pattern or ventricular arrhythmias were not reported in this family. In addition, ajmaline test in one mutation carrier ruled out BrS but enhanced the conduction abnormalities.

Methods and Results

Patch-clamp analysis of HEK293 transfected with the C-terminal mutant R1860Gfs*12 alone showed that the mutant induced a drastic reduction of I Na density, a positive shift of activation, a negative shift of steady-state inactivation and an increase in persistent current compared to WT. The total protein expression of the mutant was decreased compared to the WT, which was consistent with the reduction in I Na density. In addition, treating the cells with MG132, a 26S-subunit ubiquitin-proteasome inhibitor, increased the total protein expression of the mutant, suggesting its partial proteasomal degradation. At the heterozygous state, the mutant did not have a dominant-negative effect on the WT channel, but the steady-state inactivation remained importantly left-shifted, and a persistent current was still present. Since the clinical phenotype in this family was mostly atrial, we thought that differences in the electrophysiological properties between atrium and ventricles could be the underlying cause. By measuring I Na of the WT and heterozygous state in HEK293 cells at holding potentials mimicking the resting membrane potentials of atrial or ventricular cardiomyocytes, we showed that the mutant has more pronounced loss-of-function effect at the atrial potential compared to the ventricular one. Moreover, the biophysical properties of Na + current for the WT and the heterozygous state were included into a computer model simulation of atrial and ventricular membrane action potentials, which showed that the mutant induced a more marked decrease in AP maximum upstroke velocity, with a marked lengthening of the action potential duration in atrial cells compared to ventricular ones.

Since we demonstrated in our previous study that Na v 1.5 α-subunits interact with each other, we tested whether the R1860Gfs*12 truncated mutant still interacted with the WT. By co-immunoprecipitation studies, we showed that the mutant α-subunits interacted with the WT as well as with themselves.

The proband in this family developed the disease at a young age compared to her father and uncle, who carry also the mutation. She had severe SSS requiring the implantation of a pacemaker at 13, and AF at 20. This suggests that the genetic background of the proband could have a role in modulating the disease phenotype. We screened in this family 17 SNPs linked to AF, and we showed that the proband was the only mutation carrier who had one atrisk allele located upstream of PITX2, a gene widely associated with AF.

Discussion

The mixed clinical picture caused by a single SCN5A mutation raised the possibility of the involvement of other factors in modulating the disease phenotype. In this study, by characterizing the C-terminal truncating mutation R1860Gfs*12, we showed that the differences in the electrophysiological properties between the atrium and ventricles and the altered biophysical properties of the mutant, combined to the genetic background of patients could be some of these factors.

The R1860Gfs *12 is the most distal truncating SCN5A mutation identified so far. It is located in the C-terminal region, which has an important role in the channel function through the interaction with many partner proteins 469 . R1860Gfs*12 mutation carriers presented with a mixed clinical picture, mostly atrial with variable severity (SSS, artial fibrillation or flutter with atrioventricular block).

In the heterozygous state mimicking the heterozygosity of the patients, the altered biophysical properties, mainly the significant negative shift of steady-state inactivation, and the persistent sodium current could explain the clinical phenotype. The increased persistent Na + current might cause early after depolarizations (EADs), and delayed after depolarizations (DADs), thereby inducing triggered activities accounting for AF [START_REF] Lemoine | Arrhythmogenic left atrial cellular electrophysiology in a murine genetic long QT syndrome model[END_REF][START_REF] Song | An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes[END_REF] . The negative shift of steady-state inactivation combined to the persistent current could be responsible for the SSS and the conduction abnormalities through the reduction of Na + channel availability at the normal resting potential of cardiac cells and by the prolongation of the AP 211 . However, these finding cannot explain why the clinical presentation in this family was mostly atrial and why no ventricular arrhythmias were observed. I Na recording in HEK293 cells at holding potential mimicking the constitutively different resting membrane potentials of atrial and ventricular cells, and the computer model stimulation demonstrated a more pronounced effect of the mutant in atrial conditions compared to the ventricular ones, that could explain the prominence of atrial arrhythmias in this family. Indeed, it has been demonstrated that resting membrane potential of atrial cells is more depolarized, and the critical depolarization and current threshold for action potential initiation are smaller in atrial cells than in ventricular cells 470,471 , indicating that atrial cells are more readily excitable, and are constitutively more susceptible to develop arrhythmia. These findings suggest that the altered functions of Na v 1.5 channel caused by some SCN5A mutations, like R1860Gfs*12, could have a greater impact in the atria than in the ventricles, depending on the mutation biophysical properties, and thus predisposing to the development of atrial arrhythmias more readily than to ventricular ones.

By screening 17 SNPs linked to AF, we showed that the proband of this family was the only mutation carrier to carry an AF at-risk allele located upstream of the PITX2 gene, which encodes a transcription factor involved in the development of the sinus node and the pulmonary vein 124,125 . PITX2 variants have been widely associated with AF development, mainly early onset AF 115,120 . This association has also been reported to contribute to postcardiac surgery AF 472 and to recurrence of AF after catheter ablation 473 and cardioversion 474 .

In atrial-specific PITX2 knockout mouse model, reduced mRNA and Na v 1.5 protein levels were observed 475 . We hypothesized that that PITX2 variant could lead to early morphological changes and further loss-of-function of Na v 1.5 channels in the proband's atrium compared to her father and uncle, accounting for the occurrence of severe SSS and early onset AF in the proband.

Conclusion

The constitutive differences in the resting membrane potential between atrium and ventricle combined with the altered biophysical properties of Na v 1.5, and the genetic background of patients are factors that could modify the phenotype caused by SCN5A mutations, and most probably responsible for the predominance of the atrial arrhythmias in these patients carrying the R1860Gfs*12 mutation.

Introduction

Coordinated activity of multiple ion channels tightly controls generation and propagation of cardiac action potentials (AP) 1 . Mutations in the SCN5A gene, encoding the Na v 1.5 αsubunit of the cardiac sodium channel, have been involved in numerous inherited cardiac arrhythmias including long QT syndrome (LQTS), Brugada syndrome (BrS), and rare cases of sick sinus syndrome (SSS) and atrial fibrillation (AF) 2 . Atrial arrhythmias are being increasingly diagnosed in patients with BrS (incidence of 6-38%) 3 , as well as LQTS 4 .

Originally, the various SCN5A-related arrhythmias were considered separate clinical entities with distinct phenotypical characteristics. Recently, a wide spectrum of mixed disease phenotypes was reported in these arrhythmias, referred to as overlap syndrome of cardiac Na + channelopathy 2 . The reasons why the same SCN5A mutation can result in different phenotypes remain unknown, but it raises the possibility that the disease expressivity is influenced by altered biophysical properties and genetic modifiers 5 .

In this study, we characterized the Na v 1.5 C-terminal truncating mutation R1860Gfs*12 identified in a family presenting with a complex clinical picture of SSS and AF or atrial flutter. Heterologous expression of the mutant channels alone or with wild-type (WT) channels led to a reduction in I Na density, a persistent current and a drastic alteration of the inactivation properties. Interestingly, because of the constitutively different resting membrane potentials in atrial and ventricular tissues, the atrium of the patients might be more susceptible to the altered biophysical properties of the mutant channels and, thus, more prompt to develop arrhythmias. Moreover, the proband carries at-risk polymorphisms upstream of PITX2, a gene widely associated with AF development. Altogether, our results could explain the mixed clinical phenotype of this family.

Statistical analysis

Data are presented as means ± SEM. Statistical significance was estimated with SigmaPlot® software by Student's t-test or ANOVA, as appropriate. P< 0.05 was considered significant.

Results

Identification of a truncating mutation in SCN5A

The proband (III.2) was a 26-year-old female diagnosed at 13 with severe SSS associated with severe sinus bradycardia, symptomatic sinus pauses and syncope (Figure 1A andB of the PR interval (300 ms) and the QRS complex (240 ms). In addition, sinus node dysfunction with junctional escape rhythm, and supraventricular premature contractions were observed (Supplemental Figure 1). Echocardiograms ruled out structural heart disease except for mild atrial dilatation in the proband's father. The mother and the proband's sister were asymptomatic (Figure 1A).

We sequenced the coding regions of fourteen genes involved in cardiac arrhythmias and identified a unique mutation in SCN5A, a deletion of one base pair (A) at the position 5578 in exon 28 (c.5578delA). This deletion induced a frameshift mutation, p.R1860Gfs*12, which changed the amino acid arginine at position 1860 into glycine followed by 10 frame-shifted amino acids before a premature stop codon (Supplemental Figure 2). The proband, her father and uncle carried this mutation, whereas her mother and sister did not (Figure 1A). This variant has never been described and is absent from publicly available databases.

Suspecting the possible contribution of additional genetic factors for AF development, we genotyped the family members for 17 SNPs that alter AF susceptibility (Supplemental Table 1). Interestingly, the proband, who experienced severe SSS and early onset AF, is the only mutation carrier to have one at-risk allele of rs6817105 and rs2200733 located upstream of the PITX2 gene, which she received from her mother (Figure 1A and Supplemental Table 1). In addition, the father had 2 copies of the protective allele of rs3853445, another SNP Indeed, the current reduction was 17% for WT channels, 25% for WT + R1860Gfs*12, and 40% for, R1860Gfs*12 at a HP of -83 vs -86 mV.

Differences between WT and heterozygous channel characteristics were included into computer-model simulations of single atrial and ventricular cell membrane action potentials (Supplemental methods). The AP maximum upstroke velocity ([dV/dt] max ) of the heterozygous state exhibited an important decrease in the atrial (68%) compared to the ventricular myocyte (33%), as well as a marked lengthening of the AP duration in the atria (Figure 4).

The R1860Gfs*12 mutant was partially degraded by the ubiquitin-proteasome system

Western blots of total lysates from transfected HEK293 cells showed a significant decrease of ≈70 % in the total protein expression of the mutant channels when compared to WT, in accordance with the reduction of the current density (Figure 5A). Moreover, incubation of the cells with the 26S-subunit ubiquitin-proteasome inhibitor, MG132, significantly increased the mutant total expression, suggesting a proteasomal degradation of the R1860Gfs*12 mutant (Figure 5A).

The R1860Gfs*12 mutant interacted with WT α-subunits

We have previously demonstrated by co-immunoprecipitation that Na v 1.5 α-subunits interact together 8 . To assess if the R1860Gfs*12 truncating mutant still interacted with the WT channel, we performed co-immunoprecipitation in HEK293 cells transfected with either Flag-WT + GFP-WT or Flag-WT + GFP-R1860Gfs*12, and Flag-WT alone as a negative control. Immunoprecipitations were performed with the anti-GFP antibody, and western blot revealed with the anti-Flag antibody. In cells expressing Flag-WT + GFP-WT or Flag-WT + GFP-R1860Gfs*12, a specific band at the expected size of Flag-WT was present (Figure 5B), indicating that the WT and the R1860Gfs*12 truncated α-subunits interacted with each other.

To test whether the R1860Gfs*12 α-subunits could interact with each other, coimmunoprecipitation was performed in HEK293 cells transfected with Flag-R1860Gfs*12 + GFP-R1860Gfs*12. Figure 5B shows that the interaction between truncated α-subunits still occurred. Thus, our results suggested that the last 156 amino acids of the C-terminal domain of Na v 1.5 were not essential for the interaction between Na v 1.5 α-subunits.

Discussion

We functionally characterized a novel frameshift deletion in SCN5A inducing the most distal truncation of the C-terminus of Na v 1.5 reported so far, identified in a family with early SSS and atrial arrhythmias. We report that the R1860Gfs*12 mutant showed a loss-and gainof-function phenotype, which, combined with the variable genetic background of the mutation carriers, may explain the patients' clinical features.

Functional characteristics of R1860Gfs*12 channels

Mutations in SCN5A are known to cause various types of arrhythmias including BrS, LQTS, sinus node dysfunction, AF, and overlap syndrome 2 . In general, SCN5A mutations leading to a decrease in I Na (loss-of-function) have been associated with conduction slowing and BrS. Gain-of-function mutations in SCN5A are typical of LQTS. Nevertheless, numerous reports have shown that a single Na v 1.5 mutation may induce various combinations of clinical phenotypes 2 . Here, the R1860Gfs*12 mutant channel expressed alone was mostly degraded, causing a severe reduction in I Na density. Moreover, it induced a 7-mV positive shift of activation, which could delay the action potential, and a dramatic -25 mV shift of the steadystate inactivation, which is likely due to the location of the mutation within a region involved in the inactivation of Na v 1.5 and would decrease I Na and affect the action potential velocity. In addition, we showed an inactivation defect delaying the fast and slow inactivation. This lossof-function phenotype was associated with gain-of-function characteristics since the mutant produced a significant persistent Na + current. When co-expressing this mutant with WT channels in order to mimic the heterozygosity of the patients, the steady-state inactivation was also importantly left-shifted, and a persistent current was still present. These parameters have been shown to be a substrate for atrial fibrillation by causing repolarization failure, early after-depolarizations and delayed after-depolarizations [12][13][14] . Moreover, regarding the important role of Na v 1.5 channels in the sino-atrial node function 15,16 , the negative shift of inactivation and the persistent current could account for the sinus node dysfunction in this family 17 .

We have recently shown that Na v 1.5 α-subunits were able to oligomerize, leading to the dominant-negative effect of some BrS SCN5A mutations through the retention of WT channels by mutant channels 8 . Here, we showed that the truncated R1860Gfs*12 mutant channels were still interacting with the WT channels. This result suggests that the last 156 amino acids of the C-terminal domain of Na v 1.5 are not the main site for α-subunit interaction. In addition, the significant negative shift of the steady-state inactivation in cells co-expressing mutant and WT channels suggests that Na v 1.5 α-subunits also function as oligomers. Alternatively, the C-terminus of Na v 1.5 is a site of interactions with several proteins that have important roles, not only in channel function but also in the stabilization of the channels within the plasma membrane 18 . We verified that the truncated R1860Gfs*12 channel did not interact with FHF1B (Supplemental methods, and Supplemental Figure 5). A similar loss of interaction with partners such as SAP97 and syntrophin could contribute to a loss of stability and expression of the truncated channels at the cell membrane 18 .

R1860Gfs*12 gain-and loss-of-function features underlie a complex clinical phenotype

Since Na v 1.5 channels are expressed in both the atrium and the ventricle, it is unclear why the R1860Gfs*12 mutation carriers showed only atrial but no ventricular arrhythmias. One hypothesis is that the constitutively different electrophysiological properties of atrial and ventricular cells might be an underlying cause. Indeed, it has been shown that the resting membrane potential is more depolarized 10,11 , and the I Na density is more important with a negative shift in the steady-state inactivation in the atrium compared to the ventricle 10 .

Moreover, atrial cells are more readily excitable than ventricular cells 11 . This suggests that the atrium is constitutively more susceptible to develop arrhythmias than the ventricle. This led us to explore the R1860Gfs*12 mutant current density at the atrial vs the ventricular resting membrane potential. Here we showed that a 3-mV difference in resting membrane potentials led to a significant decrease of the peak current by 40% in the atria vs the ventricle for the mutant alone, and by 25% when the mutant was co-expressed with WT channels. These results were confirmed by computer-model simulations showing that co-expression of mutant and WT channels led to a more important decrease in the maximum upstroke velocity ([dV/dt] max) in the atrial compared to the ventricular myocytes, as well as a significant increase in the AP duration. Therefore, this SCN5A mutation could have different tissue consequences, and could contribute to the predominance of an atrial phenotype in this family.

A very few C-terminal truncating mutations have been identified in Na v 1.5 19,20 , but their functional consequence have not been studied, except for the C-terminal truncating mutation L1821fs*10. This mutation has been detected in a patient with a complex clinical phenotype of congenital SSS, cardiac conduction disorder with recurrent monomorphic ventricular tachycardia, but no BrS 20 . Functional characterization of this mutant evidenced biophysical properties very similar to the ones of R1860Gfs*12, displaying a gain-and loss-of-function phenotype. Nevertheless, Tan et al only reported the absence of a dominant-negative effect of the mutant on the WT channel I Na density 20 , while we observed an alteration of the inactivation biophysical parameters and the presence of a persistent current, when R1860Gfs*12 was co-expressed with WT channels. Altogether, these observations suggest that other parameters, such as additional genetic factors, might influence the clinical expression of the R1860Gfs*12 mutation.

Genetic background of the R1860Gfs*12 mutation carriers

The early occurrence of sinus node dysfunction in the proband at the age of 13 years, followed by atrial fibrillation at 20 was suggestive of a specific genetic background in this patient. Thus, we explored the possibility that polymorphisms associated with AF could modulate the phenotypic expression of the R1860Gfs*12 mutation. Interestingly, the proband is the only mutation carrier who received the PITX2 at-risk alleles for AF (rs6817105, rs2200733) 6,7 . Furthermore, she received only one PITX2 protective allele (rs3853445) 9 , while her father had 2 copies. The role of the PITX2 transcription factor has been reported in the development of the sinus node and pulmonary vein myocardium 21 , which is an important site for AF initiation. Among the numerous variants associated with AF, the 4q25 variants upstream of the PITX2 gene have the largest effect in early and late AF 6,7 . Reduced PITX2c expression, the main cardiac isoform, was found in AF patients and was shown to induce atrial electrical and structural remodeling in a murine model with a reduction of mRNA and Na v 1.5 protein levels 21 . Thus, it is tempting to speculate that the presence of 4q25 variants could induce early atrial morphological changes and further loss of function of Na v 1.5 channels in the atrium in the proband, and may thus account for the occurrence of severe SSS with AF at young age compared to her father who had asymptomatic SSS, and her uncle who developed SSS later in his life. Of course, additional factors may also contribute to the clinical phenotypes.

Conclusion

In conclusion, we showed that a C-terminal truncated Na v 1.5 α-subunit interacts with WT α-subunits. Their co-expression induced only a mild reduction of the I Na density but a marked shift in inactivation and gave rise to a persistent current, compatible with SSS and AF. The mutant biophysical defects associated with the difference in resting membrane potentials between atrium and ventricle combined with genetic factors might explain the predominantly atrial expression of this mutant. A better knowledge of the functional effects of mutations in SCN5A and the genetic background of the mutation carriers should help in the future to explain the large phenotypical variability of the sodium channelopathies. 1.

.5 x G Na-fast WT, h ∞ (V m ) hetero: -16.5mV shift of h ∞ (V m ) WT m ∞ (V m ) hetero: -4.0mV shift of m ∞ (V m ) WT τ(V m ) hetero: 1.8 x τ(V m ) WT.

Co-immunoprecipitation

For co-immunoprecipitation of Na v 1. Several partners interact with the C-terminus of Na v 1.5, including the Fibroblast Growth Factor Homologous Factor 1B (FHF1B), which modulates Na v 1.5 inactivation. Its interaction site on Na v 1.5 was debated 2,3 . It was first reported to be located in the proximal C-terminal domain (amino acids 1773-1832) 2 , and later not only in the proximal but also in the distal part of the Na v 1.5 Cterminus (amino acids 1773-1908) 3 . By co-immunoprecipitation, we demonstrated that R1860Gfs*12 mutant did not interact with FHF1B, even after increasing the total protein expression of R1860Gfs*12 with the proteasome inhibitor MG132, indicating the involvement of both the proximal and distal parts of Na v 1.5 C-terminus in the interaction with FHF1B.

HEK293 cells were transfected with GFP-WT, GFP-WT or GFP-mutant with FHF1B (C-terminal His 6 -tag). Cells were treated with or without MG132. Immunoprecipitations were performed with anti-His 6 antibody (Ab), and blots were revealed with the anti-His 6 antibody or with the anti-GFP antibody. The results demonstrated that only the WT channel was precipitated with the FHF1B and not the mutant, even after treating the cells with MG132.

II.3 Study of the recently BrS-associated SNPs in the family with the

R1860Gfs*12 mutation

In the family with the R1860Gfs*12 mutation, in addition to the SNPs linked to AF, we explored the possibility that reported BrS associated SNPs could modulate the phenotypic expression. We screened the three SNPs recently shown to be associated with BrS 476 (Table 11). All R1860Gfs*12 mutation carriers have three risk alleles, while in the pioneer study, most of the BrS patients have two to four risk alleles, and controls have one to three risk alleles. The role of these SNPs remains unclear since 1.5% of European population is expected to carry more than four risk alleles. (A) FHF1B was immunoprecipitated using an anti-His 6 antibody, and the bound proteins were then detected by western blot analysis with anti-GFP and anti-His 6 antibodies. Only the WT GFP-Na v 1.5 was immunoprecipitated with FHF1B even after increasing the total protein expression of the two mutants with MG132. (B) Reciprocal co-immunoprecipitation performed using the anti-GFP antibody for immunoprecipitation of GFP-Na v 1.5, and the anti-His 6 and anti-GFP antibodies for western blot analysis. FHF1B protein was precipitated with the WT channels but not with the two mutants.

III.2 FGF12-B is the most highly expressed FHF isoform in the human ventricles

The expression of the different FHF isoforms in the heart was relatively unknown, until a recent study which demonstrated by qPCR that FGF13-VY is the most expressed FHF isoform in the adult mouse ventricular myocytes 458 , but no study was done at that time in human. So, we aimed to study the expression of FHF isoforms in human and mouse ventricles. RNA extraction from human and mouse ventricles and reverse transcriptase were performed as described in the Material and Methods. Then, we performed qPCR using isoform-specific primers for FGF12 isoforms A and B, and FGF13 isoforms VY, VY/Y and V (Table 9 of Material and Methods). Primers are located in the homologous region between human and mouse FHF transcripts. All the data were corrected to a reference gene encoding the ribosomal protein L32 (RPL32), and then normalized to FGF13-VY in mouse and to FGF12-B in human. In the mouse heart FGF13-VY was the most highly expressed isoform, FGF13-V was expressed at low level, FGF12-B was detectable, but FGF13-Y and FGF12-A were undetectable (Figure 48A). These results are in line with the previous study in mouse heart 458 except that the FGF13-Y isoform in our study was undetectable. In the human heart, FGF12-B was the most highly expressed isoform, FGF13-VY was detectable, but FGF13-Y and FGF12-A were undetectable (Figure 48B). Our results are consistent with a very recent study, that was unpublished at the time we started our work, which showed that FGF12-B is most the highly expressed isoform in human heart 304 . In this study, the qPCR was unable to distinguish between the FGF13-VY and FGF13-Y isoforms, and these isoforms were expressed at approximately 40% of FGF12-B in human ventricles. In our study, FGF13-VY was detectable but at a very low level compared to FGF12-B, while FGF13-Y was undetectable.

Figure 48: Relative mRNA expression of FHF isoforms in mouse (A) and human (B) ventricular cardiomyocytes FGF13-VY was the most expressed isoform in mouse heart, while FGF12-B was the highly expressed isoform in human heart. For comparison with the previous studies see Figure 37.

III.3 Screening of FGF12-B in patients with BrS

Since no mutation was found in 75% of BrS patients, and FHF1B is a partner of

1,1 1,2 F G F 1 3 -V Y F G F 1 3 -Y F G F 1 3 -V F G F 1 2 -A F G F 1 2 -B mRNA relative expression mRNA relative expression 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1 1,2 F G F 1 3 -V Y F G F 1 3 -Y F G F 1 3 -V F G F 1 2 -A F G F 1 2 -B 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1 1,2 F G F 1 3 -V Y F G F 1 3 -Y F G F 1 3 -V F G F 1 2 -A F G F 1 2 -B mRNA relative expression mRNA relative expression 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1 1,2 F G F 1 3 -V Y F G F 1 3 -Y F G F 1 3 -V F G F 1 2 -A F G F 1 2 -B 1
All FGF12-B exons and intronic junctions (GenBank accession number NM_004113.5) were amplified by PCR (PCR primers are reported in Table 10 of Material and Methods), and the sequencing reaction was realized using the Sanger method with the Big Dye Terminator v.3.1 kit (Applied Biosystems) as described in the Material and Methods.

Sequence analysis was then performed using the CodonCode Aligner v3.7.1 software.

A c.379 G>A transversion in exon 4, predicting a valine-to-methionine substitution at position 127 (p.V127M), was identified in one patient (Figure 36 and49A). Alignment of the FGF12 amino acid sequences demonstrated that the valine acid in position 127 is highly conserved across species, and it is also conserved in the human FGF11, FGF13 and FGF14, suggesting the importance of this residue (Figure 49B). Moreover, this variant has never been described and is absent from the following publicly available databases:

III.4 Preliminary functional study of the variant V127M in FGF12-B

The plasmid containing the variant V127M in FGF12-B was prepared by site directed mutagenesis of the WT pIRES2-acGFP1-FGF12-B where the FGF12-B C-terminus was fused to a His 6 -tag. Patch-clamp analysis of HEK293 cells stably expressing Na v 1.5 transfected with the WT or mutant FGF12-B was performed in our team. In our experiments, expression of the WT FGF12-B did not affect the peak I Na and caused a negative shift of the steady-state inactivation of Na v 1.5. However, no difference was observed in the functional effects on the Na + current between the WT and the mutant FGF12-B (Figure 50). As the heterologous system does not represent the physiological environment of ventricular cardiomyocytes, and since it was recently reported that the effects of the coexpression of FHFs on Na v 1.5 or on the calcium channel Ca v 1.2 in HEK293 cells were different than in adult ventricular cardiomyocytes 304 , we plan to study the V127M variant in adult rat ventricular cardiomyocytes, where the endogenous FGF13 is knocked out. These experiments will be performed through a collaboration with the group of Geoffrey Pitt in the USA. In this thesis work, we addressed SCN5A mutations, which are involved in a wide spectrum of disease phenotypes including LQTS, BrS, PCCD, AF, SSS, and dilated cardiomyopathies. Moreover, a mixed clinical picture now known as "overlap syndrome" has been reported in SCN5A channelopathies 477 . Currently, a growing number of SCN5A variants have been identified, but few of them are characterized by electrophysiological studies. This further complicates the interpretation of genetic testing since some of these variants are benign and may not be the actual cause of the disease.

G A G C A A A A G G T G A G C A A A A G/A G T p.V127M c.379 G>A Control Human FHF FGF12 p.Val127 G A G C A A A A G G T G A G C A A A A G G T A G C A A A A G G T G A G C A A A A G/A G T A B Figure
By characterizing two SCN5A mutations, R104W and R1860Gfs*12, we demonstrated three possible mechanisms that could play a role in the determination of the phenotypic expression of SCN5A mutations. Moreover, we highlighted the role of the Na v 1.5

N-terminal domain in the channel function.

The interaction between Na v 1.5 α-subunits

We characterized the N-terminal mutant R104W, which was identified in a BrS patient. Using co-immunoprecipitation studies with two distinct Na v 1.5 α-subunit tagged constructs, we demonstrated for the first time that Na v 1.5 α-subunits interact with each other, and that this interaction was responsible for the dominant-negative effect of the R104W mutant on the WT channels. When expressed alone, the R104W was retained in the ER, degraded by the ubiquitine-proteasome system, and no I Na was detected . Co-expression of R104W with WT, led to WT retention the in the ER through the interaction between Na v 1.5

α-subunits, resulting in a dominant-negative effect. Few SCN5A mutations have been studied in conditions mimicking patient heterozygous state, since the large Na v 1.5 α-subunits were not thought to oligomerize, contrary to many potassium channels that form homotetramers 467,478,479 . Nevertheless, a dominant-negative effect caused by a Na v 1.5 mutant has been reported once for the trafficking-defective mutant L325R, located in the pore region (loop S5-S6) of domain I 468 . The authors hypothesized that the mutant channels exerted the dominant-negative effect by affecting the trafficking of WT channels. Another recent study has confirmed the interaction between Na v 1.5 α-subunits, which is responsible for the dominant-negative effect of the BrS SCN5A mutation R1432G 480 . This trafficking-defective mutant is located in the pore region (loop S5-S6) of domain III, and abolished I Na . In the heterozygous state, the mutant decreases WT surface expression, with a resultant dominantnegative effect. In contrast to the R104W N-terminal mutant, both L325R and R1432G mutants were not degraded by the ubiquitin-proteasome system. The dominant-negative effect observed for the three mutations, L325R, R1432G and R104W, is mutation specific and not a generalized mechanism. Indeed, for example we have shown that the trafficking competent but non-functional channels, R878C and ∆N-Na v 1.5, do not exert a dominant-negative effect on WT channels.

The three trafficking-defective mutants, L325R, R1432G and R104W, most likely caused the retention of WT channels due to the interaction between Na v 1.5 α-subunits.

However, the mechanisms underlying the retention of each of these mutants seem to be different. The mutant L325R caused an 80% reduction in I Na , while no I Na was recorded with R1432G and R104W mutants, suggesting that a small part of L325R was able to reach the membrane. In addition, incubating the cells at low temperature or with mexiletine, experimental conditions known to partially rescue some misfolded channels, rescue the mutant L325R 468 , but not the R104W and R1432G mutants 310 . Moreover, the N-terminal mutant R104W was degraded by the ubiquitine-proteasome system, but not the two pore mutants L325R 468 and R1432G 480 . These differences suggest that these mutants do not follow the same retention pathway, and that misfolding is not the only cause underlying the intracellular retention. Therefore, further studies are needed to explore the mechanisms underlying this mutant channel retention. We hypothesized that the R104W mutation caused conformational changes in the mutant Na v 1.5 protein, which then unmasked an ER retention motif leading to its retention. This hypothesis is in line with our experiments in neonatal rat cardiomyocytes showing a co-localization of the mutant R104W and CD4-KKXX, a construct containing the ER retention motif, KKXX.

Since the trafficking-defective mutant R104W was able to inhibit the trafficking of WT channels, most probably through the interaction between Na v 1.5 α-subunits, it is possible that trafficking-competent channels could be able to transport a small amount of the mutant channels with them to the membrane. By expressing the N-terminal mutants R104W and R121W with the trafficking-competent but non-functional Na v 1.5 mutant R878C 195 , we showed that the mutant R878C restored a very small I Na . This mechanism of transcomplementation has been previously reported for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel 481,482 . Interestingly, it has also been demonstrated that the common SCN5A polymorphism, H558R, located in the linker I-II, is able to restore the trafficking-defective R282H mutant, located in the pore region (loop S5-S6) of domain I, when it was present in the second allele, but not in the same allele 483 . It is possible that the assembly of the Na v 1.5 α-subunits in the ER masks an ER retention signal in the misfolded protein, or it provides an export signal from the ER, as it has been suggested for the CFTR mutant 481,482 . Another possibility is that the interaction between the Na v 1.5 αsubunits could stabilize both proteins allowing proper folding. Shinlapawittayatorn et al 484 have recently shown that small peptides containing the polymorphism H558R are able to restore the trafficking-defective R282H mutant by promoting proper folding in the ER.

Moreover, in the same study, the small peptides were able to restore the trafficking of five out of nine mutant proteins with the mutations located in the pore region of domain 1, but did not have an effect on the WT, suggesting that the peptides specifically restored some mutants.

Interestingly, in this study acute expression of the peptides in the intracellular solution during the patch-clamp analysis did not rescue the mutant channels, suggesting that this peptide effect occurs mostly in the ER. In our study, co-expression of the WT Na v 1.5 and the Na v 1.5

N-terminus, Nter, induced a two-fold increase in I Na . The mechanism underlying this increase in I Na remains unknown. We hypothesize that the Nter could saturate the ER quality control machinery, allowing more WT channels to escape this system and reach the membrane. Other possible explanations are that the Nter occludes the retention signal or provides exit codes, enhancing channel export from the ER or other cellular compartments. Further studies are needed to understand the mechanisms responsible for this increase in I Na . We should invesigate whether the Nter increases the surface expression of the WT, and if it is able to rescue the N-terminal mutant R104W or other trafficking-defective mutants. Moreover, coimmunoprecipitation studies between Na v 1.5 α-subunits and the Nter or the small peptides containing the H558R are needed to see if these small peptides interact directly with Na v 1.5.

Altogether, these data strongly suggest that the interaction between Na v 1.5 α-subunits occurs in the ER before trafficking to the membrane, either impairing or enhancing it.

Nevertheless, an interaction at the membrane could also be occuring. The observed positive shift of activation of the N-terminal mutants in the heterozygous state and of the N-terminal mutants rescued by R878C, as well as the negative shift of steady-state inactivation of the Cterminal mutant R1860Gfs*12 in the heterozygous state, highly suggest that interaction between Na v 1.5 α-subunits also exists at the membrane, and that Na v 1.5 α-subunits function as oligomers. Our results are in line with a previous study demonstrating double and triple simultaneous openings and closings of the sodium channels, and the authors hypothesized that there is a tight cooperation in channel gating 485 . Interestingly, a recent study has demonstrated that one mutant is able to completely restore the gating defects of the another mutant located in the second allele 486 . The compound heterozygous SCN5A mutations, D1690N located in loop S5-S6 of domain IV, and G1748D located in the S6 of domain IV, were identified in a BrS patient 486 . Both mutants are trafficking-defective leading to a severe reduction in I Na , with altered biophysical properties for the G1748D mutant. When expressed with the WT channels, both mutants have a dominant-negative effect on WT channels. Co-expression of D1690N with G1748D, mimicking the heterozygous state of the patient, completely restored the gating defects of G1748D, while it partially restored its trafficking defects.

Detailed aspects of the interaction between Na v 1.5 α-subunits remain to be elucidated.

In the study of mutant R1432G 480 , the authors reported that co-transfection of the β1-subunit with Na v 1.5 is essential for the dominant-negative effect, as well as for the interaction between Na v 1.5 α-subunits. The experiments in this study were performed in HEK293, the same heterologous system used in our study. Nevertheless, in our study co-transfection with the β1-subunit was not required neither for the interaction between Na v 1.5 α-subunits, nor for the dominant-negative effect. We cannot exclude that the interaction between Na v 1.5 is mediated by the β1-subunit, since the β1-subunit is endogenously expressed in HEK293 cells 487 . So it remains to be verified if the β1-subunit is needed for the interaction between the α-subunits. Co-immunoprecipitation study in HEK293 cells, where the endogenous β1subunit is suppressed by RNA interference, or in another heterologous system where β1subunit is absent are needed to test this hypothesis. However, if the interaction is β1dependent, it remains unknown why, in the study of the R1432G mutant, the interaction was dependent on the co-expression of the β1 subunits since they are endogenously expressed. A possible explanation is that the mutant R1432G weakens the interaction between Na v 1.5 and the β1-subunit and over-expression of the β1-subunit is needed to overcome this problem.

However, further studies are needed to better understand the mechanism of interaction between Na v 1.5 α-subunits.

The site of Na v 1.5 involved in the interaction between Na v 1.5 α-subunits needs to be fully defined. Such information would be very useful in order to predict the potential effects of SCN5A mutations on this interaction as well as in determining the contribution of this interaction (dominant-negative effect or rescue) to the highly variable clinical manifestations of SCN5A mutations. By co-immunoprecipitation studies using the ∆Nter construct and the Cterminal truncating R1860Gf*12 mutation, we demonstrated that the N-and C-terminal truncated channels were still able to interact with WT Na v 1.5 channels. In addition, the R1860Gfs*12 α-subunits interacted with each other. These results showed that deletion of the N-terminal domain or the distal region of the C-terminal domain did not inhibit the interaction between α-subunits. These data combined with the results of the rescue effect of the small peptides around the H558R polymorphism, and the increase of I Na with the Nter, suggest that multiple sites of Na v 1.5 could be involved in this interaction. Further studies are needed to define these interaction sites.

In conclusion, the interaction between Na v 1.5 α-subunits is a new concept in the field of sodium channels that were not previously thought to oligomerize. This is a new mechanism that could modulate phenotypic expression, and highlight the importance of heterozygous expression when characterizing SCN5A mutations. Nevertheless, the dominant-negative and the rescue effects should be confirmed in cardiomyocytes, where all channel partners are physiologically present. Moreover, the rescue effects of the H558R peptides, and the increased I Na with Nter are interesting findings that should be confirmed in vivo. These small peptides may lead to the development of new treatment strategies for the sodium channelopathies. These peptides have the advantage of being small fragments of the target gene, which should reduce the difficulties usually encountered with gene therapy.

The role of the Na v 1.5 N-terminal domain in channel function

The role of the Na v 1.5 N-terminal domain in channel function was previously largely unknown, even though several N-terminal mutations associated with LQTS, conduction abnormalities or BrS have been reported 306 . By the characterization of the N-terminal mutants R104W, R121W 214,306 , and of the two constructs, ∆Nter, where the N-terminal region was deleted, and Nter, where only the N-terminal region was present, we highlighted the important role of the Na v 1.5 N-terminal domain in channel function. Our results demonstrated that the N-terminal domain is not necessary for the trafficking of Na v 1.5 to the membrane, since the ∆Nter Na v 1.5 was able to reach the membrane. Nevertheless, the missense mutations, R104W and R121W, led to channel ER retention and their subsequent degradation. Our results suggest that these missense mutations cause severe conformational changes in the channel proteins compared to ∆Nter, leading to the retention of the mutant channels by the quality control system, while most of the ∆Nter Na v 1.5 has escaped from this system and reached the membrane. However, the mechanism of mutant channel retention remains to be clearly defined. It is plausible that cells could use the ERAD system to quickly move the misfolded proteins from the ER to the cytosol, and then degrade them by the ubiquitin-proteasome system 488 . This would prevent the accumulation of misfolded proteins in the ER and the occurrence of reticular stress. This hypothesis correlates with our results showing that treating the transfected cells with MG132, a 26S-subunit ubiquitin-proteasome inhibitor, increased the total expression of the mutants, suggesting that they are degraded by the ubiquitineproteasome system. Moreover, we reinforced this hypothesis by demonstrating that the mutant proteins did not induce reticular stress in HEK293 cells. Similarly to the Na v 1.5 Nterminal mutants, the N-terminal mutant S21P in the neuronal sodium channel Na v 1.6 was retained in the Golgi apparatus and degraded 489 .

The ∆Nter Na v 1.5 was expressed at the membrane but was unable to conduct I Na , proposing a role for the N-terminal domain in channel opening, which has not previously been reported. Moreover, the positive shift of activation observed in the heterozygous state and for the small current rescued by R878C, further supports the involvement of the Nterminal domain in channel activation and opening. Our results are in line with recent data from Gutter et al 490 characterizing several N-terminal SCN5A mutations identified in LQTS and BrS. In this study, the expression of the BrS R104Q mutant in HEK293 results in the absence of I Na similar to the R104W mutant, suggesting an essential role for the amino acid 104 in Na v 1.5 channel function. However, expression of R104Q in Xenopus oocytes gives a small I Na with a negative shift of the steady-state inactivation, which decreases channel availability. This difference between the two heterologous systems could be due to differences in the cellular mechanisms between the two models. Interestingly, the other five N-terminal BrS mutants in this study conduct I Na similarly to the WT channel, but with a positive shift of activation for two of them. This positive shift of activation is similar to that observed in our study in the heterozygous state of R104W and in the rescued current.

Combined with the fact that the ∆Nter construct did not alter the voltage-activation of the WT Na v 1.5, this alteration in the activation is most probably due to the N-terminal domain of Na v 1.5. Surprisingly, in three of the six BrS mutations, and one of the three LQTS mutations ventricular fibrillation requiring 20 defibrillations over 72 hours. In addition, she had paroxysmal AF, and sinus bradycardia at basal conditions and after a flecainide test, however, no BrS ECG pattern was revealed. Interestingly, a flecainide test performed in one son and two sisters of the proband carrying the same mutation induced a type-1 BrS ECG pattern in the son and one sister, while it was negative in the second sister who had permanent AF.

Moreover, other asymptomatic mutation carriers were also present in the family. This high variability in the clinical picture observed with the C-terminal truncating mutations could be due to specific alterations in the sodium current properties. Interestingly, functional studies of these three mutants performed in heterologous systems showed both gain-and loss-offunction effects on Na v 1.5.

Gain-and loss-of-function effects of the C-terminal truncating mutations

When expressed in heterologous expression systems (HEK293 or CHO cells), the after-depolarizations (EADs) and delayed after-depolarizations (DADs) [START_REF] Lemoine | Arrhythmogenic left atrial cellular electrophysiology in a murine genetic long QT syndrome model[END_REF][START_REF] Song | An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes[END_REF][START_REF] Sossalla | Altered Na+ Currents in Atrial Fibrillation: Effects of Ranolazine on Arrhythmias and Contractility in Human Atrial Myocardium[END_REF]494 , thereby inducing triggering activities accounting for AF. This is supported by a study using isolated atrial myocytes from patients with permanent AF, which showed that permanent AF is associated with significantly greater I NaL , decreased peak I Na density and Na v 1.5 expression [START_REF] Sossalla | Altered Na+ Currents in Atrial Fibrillation: Effects of Ranolazine on Arrhythmias and Contractility in Human Atrial Myocardium[END_REF] .

In addition, in the study of a cohort of patients with early-onset lone AF, a high prevalence of SCN5A mutations previously associated with LQT3 have been identified, all leading to I NaL [START_REF] Olesen | High Prevalence of Long QT Syndrome-Associated SCN5A Variants in Patients With Early-Onset Lone Atrial FibrillationClinical Perspective[END_REF] .

However, it is important to emphasize that our study in a heterologous system or using a computer model, like other studies in the field, may not completely reflect what happens in the cardiomyocytes, due to the tissue specific variability of subunits and interacting proteins. mainly early onset AF. This association has been replicated in many studies [115][116][117][118][119][120] , and 4q25

variants have also been reported to contribute to post-cardiac surgery AF 472 and to AF recurrence after catheter ablation 473 and cardioversion 474 . These SNPs are located 150-kb upstream the PITX2 gene. PITX2 encodes a homeobox transcription factor of the paired type, which has a very important role in the left-right asymmetry [121][122][123] , in the normal development of the sinus node 124 and the pulmonary vein 125 , which is an important site for AF initiation 495 .

Loss of the PITX2c isoform expression in mice can lead to a right atrial isomerization, a failure to suppress the default pathway for sinus node development of the pulmonary myocardium 125,496 . In humans, the PITX2c isoform is highly expressed in the left atrium compared to the right atrium and ventricles 497 . It has been demonstrated that patients with sustained AF have decreased atrial PITX2c RNA expression compared to patients in sinus rhythm 475 . The PITX2c +/-mouse model has no obvious alteration in cardiac morphology, but does display shorter atrial AP duration, and an increased risk to develop inducible AF compared to WT 497 . In this model, a number of cellular and molecular pathways, including genes related to calcium ion binding, gap and tight junctions and ion channels were altered. In addition, in atrial-specific PITX2 deficient mice 475 , the ECG showed evidence of AVN block and absence of P waves, despite the presence of properly organized SA and AV nodes.

Electrophysiological studies showed that the left atrium of this mouse model had more depolarized resting action potential and smaller action potential amplitude. Moreover, both mRNA and protein levels of Kir2.1 and Na v 1.5 channels were decreased in the atrial chamber.

We hypothesized that the 4q25 variants could modulate the expression of the PITX2 gene, Since the diagnosis of the disease is often made only when a clinical threshold is exceeded, a large number of predisposing-variant carriers will be present in the healthy control population. GWAS of the intermediate phenotypes, which are traits associated with, or predisposing to specific diseases, have proven their efficacy in identifying modulating variants with greater statistical power. Then, these identified variants can be investigated for their association with the disease. GWAS on ECG parameters in the general population have identified genetic variants modulating QT c 501-503 , PR intervals 129,130 , QRS duration 129,504 and heart rate 129,505,506 . These variants are not only located in, or near genes encoding ion channels, but also in or near genes encoding transcription factors. These variants represent good candidates to be investigated for their contribution to specific diseases. Studies such as this are starting to become more popular. Indeed, the common genetic variants in NOS1AP

modulating the QT interval in the general population were found to modulate the QT interval duration and the risk of arrhythmias in long QT syndrome 507,508 . Variants associated with PR interval prolongation were also found to be associated with AF 129,130 .

GWAS have provided a huge number of SNPs that can modulate physiological traits or disease development. However, the major difficulty is to link these variants to specific genes, to detect the true functional variants, and to determine their underlying mechanisms of action. Despite these difficulties, the concept that a combination of rare and common variants contribute to disease development could have a significant impact on the management of the disease, through the identification of patients who are at a high risk to develop disease or to display severe phenotypes.

Expression of disease mutations is a complex process where many factors modulate the functional defects caused by a given mutation to lead to a clinical phenotype. In this thesis, we characterized three mechanisms that contribute to the variability of the phenotypes caused by mutations in SCN5A: interaction between the Na v 1.5 α-subunits, the differences in the constitutive electrical properties between atrial and ventricular cardiomyocytes, and the genetic background of the mutation carriers.

Many aspects of the regulation of the Na v 1.5 cardiac sodium channel remain to be elucidated, given the large number of partner proteins interacting with this channel, and our limited knowledge of its in vivo regulation. In addition, channel regulation may vary at the cellular level (intercalated discs vs lateral membranes), as well as at the tissue level, given differences in the expression of the channel and its partners in specific cardiac tissues (ventricle vs atrium vs Purkinje fibers, right vs left heart, epicardium vs endocardium). The classical view of sodium channelopathies, which links a single genetic mutation with a specific disease, does no longer exist. Obviously, the overall effects of any given SCN5A mutation should be considered in an integrated system combining the genetic background, the alteration caused by the mutation, and the physiological cardiac environment. Animal models or the new approach of human induced pluripotent stem cell cardiomyocytes could help us to better understand the complexity of the cardiac pathophysiology. Such cardiomyocytes from patients carrying common and/or rare genetic variants would be a useful model to explore how the genetic background of patients combined with the altered biophysical properties of a given ion channel mutation could modulate the disease phenotype.

  Clés : Arythmie, Syndrome de Brugada, Fibrillation auriculaire, Na v 1.5, SCN5A, PITX2, Polymorphisme Inserm-UPMC UMR_S 1166 Institut de recherche sur les maladies cardiovasculaires, du métabolisme et de la nutrition Faculté de médecine Pierre et Marie Curie 75634 mechanisms underlying the variable phenotypes caused by N-and Cterminal mutations in the cardiac sodium channel Mutations in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Na v 1.5, are implicated in different inherited cardiac arrhythmias. The incomplete penetrance observed in these diseases suggests the existence of other factors modulating the phenotype of these mutations. In this thesis work, we characterized two mutations identified in SCN5A.

Figure 1 :

 1 Figure 1: Anatomical structure of the heart and blood circulation ..................................

Figure 2 :

 2 Figure 2: Anatomical structure of cardiomyocytes.............................................................

Figure 3 :

 3 Figure 3: The cardiac conduction system ............................................................................

Figure 4 :

 4 Figure 4: Ion-channel genes expression in various cardiac regions ..................................

Figure 5 :

 5 Figure 5: Cardiac action potential (AP) ...............................................................................

Figure 6 :

 6 Figure 6: Gap junctions .........................................................................................................

Figure 7 :

 7 Figure 7: ECG of normal sinus rhythm ...............................................................................

Figure 8 :

 8 Figure 8: The twelve standard ECG leads ...........................................................................

Figure 9 :

 9 Figure 9: Representation of triggered activity ....................................................................

Figure 10 :

 10 Figure 10: Representation of a re-entry excitation .............................................................

Figure 11 :

 11 Figure 11: Typical atrial flutter ............................................................................................

Figure 12 :

 12 Figure 12: ECG signs of atrial flutter...................................................................................

Figure 13 :

 13 Figure 13: Ectopic atrial tachycardia...................................................................................

Figure 14

 14 Figure 14: AVNRT .................................................................................................................

Figure 15

 15 Figure 15: WPW Syndrome ..................................................................................................

Figure 16 :

 16 Figure 16: Premature atrial contraction ..............................................................................

Figure 17 :

 17 Figure 17: ECG signs of AF ..................................................................................................

Figure 18 :

 18 Figure 18: Management cascade for patients with AF .......................................................

Figure 19 :

 19 Figure 19: Generation sites of multiple electrical impulses in AF.....................................

Figure 20 :

 20 Figure 20: Sinus bradycardia................................................................................................

Figure 21 :

 21 Figure 21: AV conduction blocks..........................................................................................

Figure 22 :

 22 Figure 22: Five ionic currents involved in SAN pacemaking.............................................

Figure 23 :

 23 Figure 23: Typical APs recorded from atrial muscle (light gray) and the center of the SAN (black) ..........................................................................................................

Figure 24 :

 24 Figure 24: Premature ventricular contraction ....................................................................

Figure 25

 25 Figure 25: Ventricular tachycardia ......................................................................................

Figure 26

 26 Figure 26: Ventricular fibrillation........................................................................................

Figure 27

 27 Figure 27: Torsade de pointes ...............................................................................................

Figure 28 :

 28 Figure 28: Ion channels involved in hereditary cardiac arrhythmias...............................

Figure 29 :

 29 Figure 29: Examples of Brugada type 1, 2, and 3 ECG patterns.......................................

Figure 30 :

 30 Figure 30: The repolarization hypothesis ............................................................................

Figure 31 :

 31 Figure 31: The depolarization hypothesis............................................................................

Figure 32 :

 32 Figure 32: Schematic presentation of the Na v 1.5 α-subunit .............................................

Figure 33 :

 33 Figure 33: Conformational changes of Na v 1.5...................................................................

Figure 34 :

 34 Figure 34: Representative schematic of the Na V 1.5 multiple pools model......................

Figure 35 :

 35 Figure 35: Schematic representation of Na v 1.5 and its modulating proteins .................

Figure 36 :

 36 Figure 36: Structure-based sequence alignment of the 10 human FHF isoforms..........

Figure 37 :

 37 Figure 37: Relative mRNA expression of FHF isoforms in mouse (A) and human (B) cardiomyocytes ............................................................................................

Figure 38 :

 38 Figure 38: Structure of the EZ-Link TM SULFO-NHS-SS-Biotin ....................................

Figure 39 :

 39 Figure 39: Principle of cell surface protein biotinylation.................................................

Figure 40 :

 40 Figure 40: Principle of the immunoprecipitation technique ............................................

Figure 41 :

 41 Figure 41: Principle of reverse transcription ....................................................................

Figure 42 :

 42 Figure 42: Fluorescence curves detected during PCR amplification ..............................

Figure 43 :

 43 Figure 43: Primers used for the detection of FGF12 and FGF13 mRNA transcript levels in adult human and mouse hearts .........................................................

Figure 44 :

 44 Figure 44: Melting curve of a qPCR...................................................................................

Figure 45 :

 45 Figure 45: Principle of the Sanger method for sequencing ..............................................

Figure 46 :

 46 Figure 46: Co-imunoprecipitation study between WT Na v 1.5 and ∆ ∆ ∆ ∆Nter α-subunits ..

Figure 47 :

 47 Figure 47: Both proximal and distal regions of Na v 1.5 are involved in the interaction with FHF1B........................................................................................................

Figure 48 :

 48 Figure 48: Relative mRNA expression of FHF isoforms in mouse (A) and human (B) ventricular cardiomyocytes ..............................................................................

Figure 49 :

 49 Figure 49: The FGF12-B variant p.V127M .......................................................................

Figure 50 :

 50 Figure 50: Preliminary functional study of the FGF12-B variant V127M.....................

  The heart is a hollow striated muscular pump, which supplies the whole body with the requirements of nutrients and oxygen through the blood circulation. It is divided into right and left halves by a muscular wall that is called septum. Another muscular wall separates the upper parts of the heart from the lower parts, resulting in four chambers: right and left upper atria, right and left lower ventricles (Figure1A). Each one of these chambers has a specific role in the circulatory system. The right atrium receives the non-oxygenated, waste-rich blood from the whole body through two large veins; the superior and inferior vena cava. Then, by the contraction of the right atrium, the blood passes through the one-way tricuspid valve into the right ventricle. The right ventricle contracts in turn and the non-oxygenated blood passes through the pulmonary valve into the pulmonary artery up to the lung. Once oxygenated, blood returns to the left atrium through the pulmonary veins. During the contraction of the left atrium, blood passes through the one-way mitral valve into the left ventricle. Then, it is

Figure 1 :

 1 Figure 1: Anatomical structure of the heart and blood circulation http://babyheart.in/tag/normal-structure-of-heart/ http://www.tutorvista.com/content/biology/biology-iv/circulation-animals/blood-circulationmammalian-heart.php

  intercalated discs play an important role in the function of cardiac muscle, and consist of three types of membrane junctions: adherens junctions, desmosomes and gap junctions. Adherens junctions (containing N-cadherin, catenins, and vinculin), and desmosomes (containing desmoplakin, desmocollin, desmoglein, plakophilin, and plakoglobin), mediate cell-cell adhesion and anchor underlying cytoskeletal structures to the cell membrane. Gap junctions allow the conduction of electrical signals between cardiomyocytes and exchanges of metabolites.The transverse tubules (T tubules) are deep invaginations of the sarcolemma communicating with the sarcoplasmic reticulum (SR), an intracellular calcium (Ca 2+ ) reserve whose release is controlled by the ryanodine receptors (RyR2). The transmission of electrical signals through the T-tubules causes massive release of Ca 2+ from the SR, leading to contraction of the myofibrils (excitation-contraction coupling). This contractile activity of cardiomyocytes is generated by the fundamental contractile unit, the sarcomere, which consists of thin (actin) and thick (myosin) filaments (Figure2).

Figure 2 :

 2 Figure 2: Anatomical structure of cardiomyocytes Modified from http://antranik.org/myocardium/

  ejected blood into the ventricles before ventricular contraction. It is also important to protect the ventricles from excessive heart rate such as in cases of atrial arrhythmia. From the AVN down to the apex, the electrical signal propagates through specialized bundle of conduction fibers called the bundle of His. The bundle of His enters the inter-ventricular septum and divides rapidly into right and left branches that end in the cardiac apex as Purkinje fibers. This network of fibers ensures synchronized ventricular contraction.

Figure 3 :

 3 Figure 3: The cardiac conduction system http://mdmedicine.wordpress.com/2011/04/24/heart-conduction-system/

Figure 4 :

 4 Figure 4: Ion-channel genes expression in various cardiac regions The absolute expression levels of genes that are significantly more strongly expressed in a region of interest relative to the reference region indicated are provided by colour coding: genes expressed > 20-fold compared to the reference gene (HPRT: hypoxanthine guanine phosphoribosyl transferase) in the tissue/region of interest are indicated in bold and genes expressed at > 185 times the reference gene level are shown in bold red. Values shown are differences that were statistically significant for both right and left-sided comparisons for atrium versus ventricle and epicardium versus endocardium. Modified from Gaborit N. et al. J Physiol 2007 7

Figure 5 :

 5 Figure 5: Cardiac action potential (AP) (A) Phases and principal corresponding ion currents involved in cardiac AP. Inward (depolarizing) currents are in red, outward (repolarizing) currents are in blue. Modified from Michael G. et al. Cardiovasc Res 2009 13 . (B) Differences between nodal, atrial and ventricular cardiac APs. Modified from Giudicessi J. R. et al. Nat Rev Cardiol 2012 10 and Ravens U. et al. Europace 2008 14

  , which are four transmembrane domain proteins with N-and C-terminal cytoplasmic domains. They are arranged in groups of six to form hemichannels, or connexons, containing an aqueous pore in the center. Two connexons of two adjacent cells are aligned together to form a junctional channel (Figure6). In working myocytes, gap junctions are present at the intercalated discs with desmosomes and fasciae adherens junctions. In nodal cells, only small and dispersed gap junctions are present. Various connexins have been identified in the heart7,15 . Connexin 43 is highly expressed in the ventricles and atria, while connexin 40 is more expressed in the atria. Nodal cells contain connexin 45.

Figure 6 :

 6 Figure 6: Gap junctions http://en.wikipedia.org/wiki/Connexin

Figure 7 :

 7 Figure 7: ECG of normal sinus rhythm Modified from http://catatanmahasiswafk.blogspot.fr/2012/03/skill-lab-pemeriksaan-ekg.html

Figure 8 :

 8 Figure 8: The twelve standard ECG leads http://www.amperordirect.com/pc/help-ecg-monitor/z-what-is-ecg.html

Figure 9 :

 9 Figure 9: Representation of triggered activity (A) Phase 2 early after-depolarization. (B) Phase 3 early after-depolarization. (C) Delayed after-depolarization. Gaztanaga L. et al. Rev Esp Cardiol (Engl Ed) 2012 19

Figure 10 :

 10 Figure 10: Representation of a re-entry excitation http://www.cvphysiology.com/Arrhythmias/A008c.htm

Figure 11 :

 11 Figure 11: Typical atrial flutter This left anterior oblique view of the right atrium shows the orifices of the cavae and the coronary sinus and the crista terminalis (vertical strip). The arrows indicate the direction of activation. The green dotted lines mark the critical isthmus of each circuit, the target of ablation. Modified from Garcia-Cosio F. et al. Rev Esp Cardiol 2012 27

Figure 12 :

 12 Figure 12: ECG signs of atrial flutter Modified from http://www.dochemp.com/afib.html

Figure 13 :

 13 Figure 13: Ectopic atrial tachycardia http://www.emedu.org/ecg/searchdr.php?diag=SVT

Figure 14 :

 14 Figure 14: AVNRT P waves appears at the end of the QRS complex http://www.emedu.org/ecg/tachycardia.htm

Figure 15 :

 15 Figure 15: WPW Syndrome Short PR interval of less than 120 ms, normal P-wave, wide QRS complex, delta wave representing ventricular pre-excitation. Modified from http://paramedicine101.blogspot.fr/2009/05/wolff-parkinson-white-syndrome.html

Figure 16 :

 16 Figure 16: Premature atrial contraction Abnormal P-wave (arrow), followed by a longer RR interval, QRS complexes are normal. http://www.crkirk.com/thumbnail/arrhythmias/ectopics.htm

Figure 17 :

 17 Figure 17: ECG signs of AF

Figure 18 :

 18 Figure 18: Management cascade for patients with AF EHRA = European Heart Rhythm Association; ACEI = angiotensin-converting enzyme inhibitor; AF = atrial fibrillation; ARB = angiotensin receptor blocker; PUFA = polyunsaturated fatty acid; TE = thrombo-embolism. Modified from Camm A. J. et al. Eur Heart J 2010 31

Figure 19 :

 19 Figure 19: Generation sites of multiple electrical impulses in AF http://www.educationforhealth.org/data/files/resources/atrial-fibrilliation-part-1-pathophysiology-ccottrell.pdf

Figure 20 :

 20 Figure 20: Sinus bradycardia Normal P-waves and QRS complexes with a heart rate of 37 bpm. http://rrapid.leeds.ac.uk/ebook/05-circulation-06.html

Figure 21 :

 21 Figure 21: AV conduction blocks (A) First-degree AV block. (B and C) Mobitz I and Mobitz II second-degree AV blocks. (D and E) narrow and wide QRS complex third degree AV blocks.

  . In addition, Chandler et al identified in 2009 a paranodal area which has a molecular architecture intermediate between the SAN and atrial muscles 173 . It could facilitate the exit of the AP from the SAN into the atrial muscle. The SAN is the natural pacemaker of the heart since it generates impulses faster than other areas of the heart with pacemaker potential. Pacemaker activity of the SAN relies mainly on the spontaneous depolarisation thatoccurs during diastole (pacemaker potential). SAN Automaticity has been modelled as two clocks[174][175][176] , the membrane voltage clock and the calcium clock (Figure22). The membrane voltage clock represents the net disequilibrium between the decay of outward potassium currents and the activation of inward currents. The calcium clock contributes to SAN automaticity through Ca 2+ release from the ER, which can activate the Na + -Ca 2+ exchanger, generating an inward depolarizing current.

Figure 22 :

 22 Figure 22: Five ionic currents involved in SAN pacemakingA SAN myocyte is shown. Membrane clock: during the pacemaker potential, there is a voltage-dependent decay of outward currents (I K or outward rectifier K + current; 1) and a voltage-dependent activation of at least three inward currents, I f (funny current; 2), I Ca,L (L-type Ca 2+ current; 3) and I Ca,T (T-type Ca 2+ current; 4). Ca 2+ clock: during the final phase of the pacemaker potential, there is an activation of inward I NaCa (Na + -Ca 2+ exchange current; 5) in response to a spontaneous release of Ca 2+ from the sarcoplasmic reticulum via the ryanodine receptor (RyR2). Because the Na + -Ca 2+ exchanger exchanges one intracellular Ca 2+ ion for three extracellular Na + ions, it is electrogenic and generates an inward current (I NaCa ) on removing Ca 2+ from the cell. Ca 2+ release from the sarcoplasmic reticulum also occurs as a result of Ca 2+ -induced Ca 2+ release in response to Ca 2+ entry into the cell via I Ca,L and I Ca,T . The sarcoplasmic reticulum is replenished with Ca 2+ by reuptake of Ca 2+ into the sarcoplasmic reticulum via SERCA2 (Sarco/Endoplasmic Reticulum Ca 2+ ATPase). Store-operated Ca 2+ channels (SOCC) at the cell surface membrane may also help to replenish the sarcoplasmic reticulum with Ca 2+ .Monfredi O. et al. Pacing Clin Electrophysiol 2010 163

Figure 23 :

 23 Figure 23: Typical APs recorded from atrial muscle (light gray) and the center of the SAN (black) The temporal contributions of the main ionic currents to the pacemaker potential are shown by the black bars. Monfredi O. et al. Pacing Clin Electrophysiol 2010 163 .

Figure 24 :

 24 Figure 24: Premature ventricular contraction VPB: ventricular premature beat. http://www.medicine-on-line.com/html/ecg/e0001en_files/08.htm

  However, in polymorphic VT, they have different forms indicating an alteration in ventricular activating sites. Polymorphic VT is more dangerous since it can be transformed into ventricular fibrillation.

Figure 25 :

 25 Figure 25: Ventricular tachycardia http://www.medicine-on-line.com/html/ecg/e0001en.htm

Figure 26 :

 26 Figure 26: Ventricular fibrillation http://cardiac.northbayhealthcarenursing.com/ventricular-fibrillation.html

Figure 27 :

 27 Figure 27: Torsade de pointes http://lifeinthefastlane.com/ecg-library/tdp/

Figure 28 :

 28 Figure 28: Ion channels involved in hereditary cardiac arrhythmias Blue and green circles represent loss-and gain-of-function mutations. Solid and dashed lines indicate autosomal dominant and recessive inheritance. Modified from Giudicessi J. R. et al. Transl Res 2013 228

3 .

 3 Timothy syndrome (TS) is an extremely rare multisystem disorder, which manifests by the presence of variable degrees of autism spectrum disorder, syndactyly, and severe cardiac arrhythmias 234 . It is caused by some specific loss-of-function missense mutations in the CACNA1C gene 235 . 4. The Andersen-Tawil syndrome (ATS) is another rare multisystem disorder with mild QT prolongation and moderate risk of life-threatening ventricular arrhythmia. Patients with ATS have variable dysmorphic physical features (low-set ears, micrognathia, and clinodactyly), and periodic paralysis as extracardiac manifestations. It is caused by loss-of-function mutations in the KCNJ2 gene 236 . 5. The ankyrin-B syndrome (ABS) is caused by mutations in the ANK2 gene encoding the adaptor protein ankyrin B. Patients have QT prolongation, sick sinus syndrome and episodic atrial fibrillation 237 . 6. The recurrent infantile cardiac arrest syndrome is a recent multisystem form of LQTS caused by mutations in the genes encoding the Ca + binding protein, calmodulin. It is characterized by severe QT prolongation, 2:1 atrio-ventricular block, neurodevelopmental delay, seizures, and recurrent cardiac arrest during early infancy 238 .

3 .

 3 Type 3 has an ST-segment elevation of less than 1mm of saddleback type, coved type or both. Type 2 and 3 ECG patterns are not diagnostic of BrS. Signs of conduction abnormalities, like prolonged P wave duration and PR intervals, and right bundle branch block, are often seen in BrS ECGs, especially in patients with SCN5A mutations 261-264 . Importantly, through patient examination including personal and family history, ECG, echocardiography and coronary angiography, other causes of ST-segment elevation should be carefully ruled out, such as myocardial ischemia or infarction, structural cardiac abnormalities and arrhythmogenic right ventricular cardiomyopathy (ARVC).

Figure 29 :

 29 Figure 29: Examples of Brugada type 1, 2, and 3 ECG patterns (A) Brugada type 1 (coved) in leads V1 and V2 (arrows); (B) Brugada type 2 (saddleback) in leads V2 and V3 (arrows); (C) Brugada type 3 in lead V1. Modified from Li and Behr. Future Cardiology 2013 248

Figure 30 :

 30 Figure 30: The repolarization hypothesis (A) Normal transmembrane APs showing a prominent phase 1 notch in epicardium (Epi) and M cells (M) due to a high I to channel expression. (B) Reduction in I Na channel function increases phase 1 notch leading to a steep transmural voltage gradient, inscribing an elevated J-wave on the surface ECG. (C) Prolongation of epicardial repolarization results in a negative T-wave on the surface ECG. (D) Further reduction in I Na leads to loss of the AP dome in some epicardial cells but not others, leading to transmural and epicardial dispersion of repolarization. (E) Phase 2 re-entry leads to short coupled extrasystoles, triggering ventricular fibrillation. AP: Action potential; Endo: Endocardial. Modified from Li and Behr. Future Cardiol 2013 248

Figure 31 :II

 31 Figure 31: The depolarization hypothesis (A and B) Conduction delay in RVOT versus RV. (C and D) The electrical gradient between RV and RVOT results in an ST-segment elevation in V2 in the high intercostal lead configuration. (E and F) Delayed repolarization of the RVOT reverses electrical gradient from the RVOT to the RV leading to negative T-waves on the surface ECG. RA: Right atrium; RV: Right ventricle; RVOT: Right ventricular outflow tract; V2IC3: Surface ECG lead V2 in the third intercostal space. Modified from Li and Behr. Future Cardiol 2013 248

Na v 1 .

 1 5 α-subunit is a glycosylated protein of 2016 amino acids. It has four homologous domains (I-IV), each consisting of six transmembrane α-helical segments (S1-S6), with Nand C-terminal cytoplasmic domains (Figure32A). The four domains are connected to each other by cytoplasmic linkers, while the transmembrane segments are linked to each other by alternating extracellular and cytoplasmic loops (P-loops). The folding of the four domains enables the formation of the ion-conducting pore, which is lined by the extracellular loops between the S5 and S6 segments (Figure32B), that determine the selectivity for Na + . The S4 segments of domains I to IV are positively charged, and act as a voltage sensor to trigger channel activation. Depolarization of the cardiomyocytes by electrical stimuli leads to outward movement of the S4 segments, allowing the channel to activate and thus the opening of the pore. Channel activation is rapid compared to channel inactivation, allowing the channel to open for a short period of time 347 .

Figure 32 :

 32 Figure 32: Schematic presentation of the Na v 1.5 α-subunit Modified from Wilde and Brugada, Circ Res 2011 348

Figure 33 : 5

 335 Figure 33: Conformational changes of Na v 1.5 Modified from http://test.classconnection.s3.amazonaws.com/11/flashcards/399011/png/na_channel.p ng

Figure 34 :III. 3

 343 Figure 34: Representative schematic of the Na V 1.5 multiple pools model Shy D. et al. Biochim Biophys Acta 2013 355

Figure 35 :III. 3 . 1

 3531 Figure 35: Schematic representation of Na v 1.5 and its modulating proteins Rook M B. et al. Cardiovasc Res 2012 358

  5 was further confirmed by in vitro pull-down assays, and by co-immunoprecipitation studies in both HEK293 and cardiac cells. MOG1 interacts with the linker II-III of Na v 1.5 (Figure35), and the two proteins colocalize at the intercalated discs of cardiomyocytes. Over-expression of MOG1 in neonatal cardiomyocytes, or co-expression of MOG1 with Na v 1.5 in HEK293 cells increases the I Na density. The increased current density is due to enhanced surface expression of Na v 1.5. Recently, the MOG1 mutation E83D was reported in a patient with a history of syncope and cardiac arrest, who displayed a type 1 BrS ECG pattern after resuscitation402 . In HEK293 cells stably expressing Na v 1.5, expression of E83D mutant alone or with WT MOG1 failed to increase the peak I Na density, suggesting a loss-of-function effect of the mutant on Na v 1.5 and a dominant-negative effect on the WT MOG1. Immunostaining studies of adult rat cardiomyocytes transfected with the mutant MOG1 showed decreased surface expression of Na v 1.5 compared to the WT, indicating abnormal trafficking of the channel.

  5 438 . Mutations in PKP2 have been reported in arrhythmogenic right ventricular cardiomyopathy (ARVC) 439 . Recently, mutations in PKP2 have been identified in patients with BrS 305 . In the HL-1 derived cardiac cell line, knockdown of PKP2 led to decreased I Na and decreased localization of Na v 1.5 at the intercalated discs. Transfection of PKP2-deficient HL-1 cells with WT PKP2, but not the mutants, restores the functional defects of Na v 1.5 305 . The same rescue experiment was performed in human induced pluripotent stem cell cardiomyocytes from an ARVC patient with a homozygous loss-of-function PKP2 mutation 305 . These results confirm the role of PKP2 in the regulation of Na v 1.5, and add a new candidate gene to the list of BrS genes. Regarding desmoglein-2, a recent study by Rizzo et al showed an interaction between desmoglein-2 and Na v 1.5 by co-immunoprecipitation studies performed in mouse heart lysate 440 . Mutations in DSG2 also cause ARVC 439 . Using a mouse model of ARVC overexpressing the mutant desmoglein-2 N271S, reduced I Na was observed in cardiomyocytes without changes in biophysical properties 440 . The site(s) of interaction of these desmosomal proteins on Na v 1.5 is (are) still unknown, and the role of these proteins in the targeting or maintenance of channels in the desmosomes remains unclear. Nevertheless, these results indicate that the presence of plakophilin-2 and desmoglein-2 at the intercalated discs are necessary for the proper function of Na v 1.5.

  . Caveolae are small invaginations of the plasma membrane, rich in several proteins, including ion channels, and play an important role in cell signaling, protein trafficking and cholesterol homeostasis 447 . Caveolin proteins represent important components of caveolae, and caveolin-3 is the main caveolin expressed in cardiac myocytes447 . It has been shown that caveolin-3 co-immunoprecipitates with Na v 1.5 in rat cardiac tissue446 and in HEK293 cell lysates 448 , but its site of interaction on Na v 1.5 remains unknown. Immunostaining studies demonstated co-localization of Na v 1.5 and caveolin-3 at the membrane of rat 446 and human cardiomyocytes 448 . Along with Na v 1.5, the αsubunit of the stimulatory heterotrimeric G-protein (Gα s ) is localized in the caveolar membrane446 . The effect of Gα s on Na v 1.5 mimics β-adrenergic stimulation. Thus, it has been proposed that Na v 1.5 could be stored in the caveolae, and that activation of Gα s by adrenergic stimulation leads to the opening of the caveolae, resulting in increased expression of Na v 1.5 at the membrane. The inhibition of β-adrenergic effects on Na v 1.5 expression by a caveolin-3 antibody, supports this hypothesis 446 .

Figure 36 :FGF12B

 36 Figure 36: Structure-based sequence alignment of the 10 human FHF isoforms For comparison, the sequence of fibroblast growth factor FGF2 is included in the alignment. Secondary structure elements (β1-β12, g11) are indicated above and below alignments, and residues containing these elements for known secondary structures are boxed. β1 and β12 strands of FHF1 are indicated by dashed boxes. A slash in each sequence marks the junction between alternatively spliced N-terminal region and FHF homology core region. The FHF homology core region is shaded in light gray, and the 18-residue-long C-terminal extension not shared with FGFs is indicated by a box drawn around the sequences. Residues of the FHF homology region engaged in channel binding are shaded in darker gray. Underlined residues in the FHF1A isoform indicate the nuclear localization sequences. p.Q7R: FGF12B BrS mutation identified by Hennessey J A. et al 304 . p.V127M: FGF12B variant identified in a BrS patient in our laboratory. Modified from Goetz R. et al. J Biol Chem 2009 455 .

Figure 37 :

 37 Figure 37: Relative mRNA expression of FHF isoforms in mouse (A) and human (B) cardiomyocytes Modified from Wang C. et al. Circ Res 2011 458 , and Hennessey J A. et al. Heart Rhythm 2013 304

  of FGF13 does not affect the mRNA level or total protein expression of Na v 1.5, suggesting a role of FHF in the trafficking of Na v 1.5. Silencing of FGF13 in neonatal rat ventricular myocyte monolayers showed a slowed conduction velocity compared to control myocytes. The reduced I Na and delayed recovery from inactivation of Na v 1.5 could explain the conduction abnormality 458 .In addition to Na v 1.5, it has been shown that FHF2 regulates the L-type, voltage-gated Ca 2+ channel (Ca v 1.2) 459 . FGF13 knockdown decreases the Ca 2+ current density with alteration in the localization of the α 1C , the Ca v 1.2 pore-forming subunit. FHF2 modulates the targeting of α 1C to the T-tubules 459 , possibly through the protein junctophilin-2, a protein involved in the juxtaposing of the tubular Ca v 1.2 to the sarcoplasmic reticulum RyR2 460 .Silencing of FGF13 leads to decreased surface expression of α 1C and decreased Ca 2+ current density, without affecting the biophysical properties 459 . Moreover, decreased Ca 2+ release from the sarcoplasmic reticulum was also observed. Measured action potentials from FGF13 silencing adult rat ventricular cardiomyocytes showed decreased AP peak amplitude, with shortening of AP half-width, consistent with the loss-of-function effects on both Na v 1.5 and Ca v 1.2 459 .

  systems. Plasmids pcDNA3.1-SCN5A (no tag) and pcDNA3.1-GFP-SCN5A (N-terminal-GFP) were gifts from Dr H.Abriel (Bern, Switzerland). Plasmids pGFP-N3-SCN5A (Cterminal-GFP) and pRcCMV-FLAG-SCN5A (N-terminal-FLAG) were gifts from Dr F. Le Bouffant (Nantes, France) and Dr N. Makita (Nagasaki, Japan), respectively. The plasmid pcDNA3.1-HA-SCN5A (HA-WT) was a gift from Dr P. Mohler (Columbus, OH, USA). The plasmid pGFP-N3-SCN5A-∆Nter (C-terminal-GFP) was generated in our team by removing the first 381 nucleotides of SCN5A (127 amino acids), and replacing the residue 128 by a methionine. We created the plasmid pNter-IRES2-mRFP1 by cloning the sequence of the first 132 amino acids of Na v 1.5 into the pIRES2-mRFP1 vector carrying the red fluorescent protein (RFP) reporter gene. All plasmids contained the hH1a isoform of SCN5A. The plasmid pIRES2-acGFP1-FHF1B (C-terminal His 6 -tag) was a gift from Dr G. S. Pitt (Durham NC, USA). Plasmid pcDNA3-CD4-KKXX was a gift from J. Mérot (Nantes, France). This plasmid was designed to express the CD4 protein carrying the ER retention KKXX.

Figure 38 :Figure 39 :

 3839 Figure 38: Structure of the EZ-Link TM SULFO-NHS-SS-Biotin Modified from http://www.piercenet.com/product/ez-link-sulfo-nhs-ss-biotin-biotinylation-kits

  Forty-eight hours after transfection, HEK293 cells were washed with PBS and lysed in a specific lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH7.5, 1% Triton, and complete Covalent bond with primary amines exposed at the cell

Figure 40 :

 40 Figure 40: Principle of the immunoprecipitation technique

Figure 41 :

 41 Figure 41: Principle of reverse transcription

Figure 42 :

 42 Figure 42: Fluorescence curves detected during PCR amplification

Figure 43 :

 43 Figure 43: Primers used for the detection of FGF12 and FGF13 mRNA transcript levels in adult human and mouse hearts

Figure 44 :

 44 Figure 44: Melting curve of a qPCR

  7.1 software. Each obtained sequence was aligned to the reference sequence. Sequence variants were confirmed by sequencing a second DNA sample from the individual carrying the supposed variant.

Figure 45 :

 45 Figure 45: Principle of the Sanger method for sequencing

1 :Supplemental Figure 2 :

 12 Electrophysiological characterization of WT and R104K channels. To determine whether the absence of the positively charged nature of residue 104 is responsible for the loss of function of the R104W mutant, arginine 104 was replaced by another positive amino acid, lysine. (A) Representative Na + current traces of Na v 1.5 WT and R104K channels in HEK293 cells transfected with a total of 0.6 µg of plasmid. Solid lines indicate the zero current level. (B) Current density-voltage relationships of WT and R104K channels. R104K gave rise to a small I Na of 12% of WT I Na density (WT: 160±11 pA/pF, n=18 vs R104K: 19.4±2.3 pA/pF, n=9, P≤0.001), suggesting that arginine 104 has a more specific role than simply providing a positive charge. (C) V m -activation relationships of WT and R104K channels. R104K activation was rightward shifted by 8.6 mV compared to WT (WT: -44.2±1.6 mV, n= 17 vs R104K: -35.7±2.1 mV, n=8, P≤0.001). R104K V 1/2 of inactivation was not changed compared to controls (WT: -81.7±0.6 mV, n= 10 vs R104K: -79.2±0.6 mV, n=6, ns). Inactivation and recovery from inactivation of WT+R104Wchannels. A. Kinetics of activation. Time to peak obtained from current traces elicited with the standard I-V protocol, was the time delay between the beginning of the test pulse and the time to reach the current peak. No significant differences were measured in presence of R104W mutant channels compared to WT alone. B. Kinetics of inactivation. The fast (τ f ) and slow (τ s ) time constants of inactivation as determined by fitting decaying current curves obtained from the I-V protocol to a double-exponential function. In cells expressing R104W mutant, τ f of inactivation was faster compared to cells expressing WT alone, while τ s was unaffected (data not shown). This suggested that the presence of the R104W mutant channel affected inactivation kinetics of WT channels. C. Recovery from inactivation of I Na at holding potentials of -120 mV (left panel) or -100 mV (right panel). Test-and conditioning-pulses of 25 ms were separated by the recovery time ∆t from 0.5 ms to 1000 ms. Relative currents were calculated as peak I Na elicited by test-pulse versus that elicited by conditioning-pulse, for two different holding potentials: -100 mV and -120 mV. At a holding potential (HP) of -120 mV, τ fast of WT channels was significantly shorter (3.0±0.4 ms (n=10)) than τ fast of WT+R104W channels (4.7±0.5 ms (n=15); P<0.05). At a HP of -100 mV, τ fast was not different between WT and WT+R104W channels. τ slow was 43±17 ms, (n=10) for WT, and 60±14 ms, (n=15) for WT+R104W, and were not significantly different. * P<0.05, ** P<0.01, *** P<0.001.

Figure 46

 46 Figure 46: Co-imunoprecipitation study between WT Na v 1.5 and ∆ ∆ ∆ ∆Nter αsubunits

  ) which required a pacemaker implantation. At the age of 20, paroxysmal AF was documented in the memories of the pacemaker. She secondarily developed a first degree AV-block (PR = 280 ms, QRS = 80 ms, QTc = 360 ms). Her father (II.2) was diagnosed at 27 with an asymptomatic sinus node dysfunction. At the age of 42, he had one episode of atrial flutter after jogging, and was treated by cardioversion. Moreover, he developed first degree AVblock with left anterior hemi-block (PR = 240 ms, QRS = 80 ms, QTc = 400 ms) (Figures 1A and Ca-b). The proband's uncle (II.1) was diagnosed with SSS which required a pacemaker implantation at 30. None displayed spontaneous Brugada ECG pattern or developed ventricular arrhythmias. The proband's father underwent an ajmaline challenge that excluded BrS ECG pattern (Figure 1Cc-d). It induced bradycardia (40 beats/min), severe prolongation

  as means ± SEM. Peak current density was calculated at -20 mV. WT indicates wild type, ** P≤0.005, *** P≤0.001 and ns: not significant compared with WT channel. V 1/2 indicates half activation or inactivation value, k inverse slope factor, and τ f and τ s : fast and slow time constants of I Na inactivation.

Figure 1 :Figure 2 :

 12 Figure 1: Pedigree and ECG recordings of the family (A) Pedigree of the family. The proband (III.2) is indicated by an arrow. Squares represent males, circles females, + and -signs, mutation carriers and noncarriers. The haplotypes for 5 SNPs located upstream of the PITX2 gene are indicated under each subject. At-risk alleles are written in red and protective ones in blue. (B) ECG recordings of the proband. The pacemaker spikes are indicated by arrows. The proband's ECG showed prolongation of the PR interval, a sign of first degree AV block. (C) ECG recordings of the father (II.2) showing the atrial flutter that he developed at 42 (a), and his normal sinus rhythm after cardioversion with left axis deviation (b). (c and d) ECG recordings of II-2 during the ajmaline test (1mg/kg over 5 min) he underwent at 64. At basal condition (c, time 0), the patient has a regular sinus rhythm with a heart rate (HR) of 60 beats/min, a prolongation of the PR interval of 240 ms, and a QRS duration of 120 ms. After the injection of ajmaline (d, time 7 min), bradycardia was observed (HR= 40 beats/min) with a prolongation of the PR interval (300 ms) and of the QRS duration (240 ms), without sign of BrS. Rapid sodium-bicarbonate infusion was then immediately started.

Figure 3 :

 3 Figure 3: Electrophysiological characterization of Na v 1.5 channels (2) (A) and (B) Fast and slow time constants (τ f , and τ s , respectively) for WT, R1860Gfs*12 and WT + R1860Gfs*12 channels were plotted against the test potential. R1860Gfs*12 and WT + R1860Gfs*12 showed an overall slowing of the fast and slow rates of inactivation compared to the WT. (C) Three superimposed representative I Na traces corresponding to the above conditions. The R1860Gfs*12 and the WT + R1860Gfs*12 current traces were normalized to the peak of the WT current (≈ 1200 pA). (D) Ratios of the persistent Na + current amplitude (end of pulse) over the peak I Na amplitude for WT, WT + R1860Gfs*12 and R1860Gfs*12 channels, reported in percentage. (E) Ratios of the I Na loss between HPs of -86 and -83 mV for WT, WT+ R1860Gfs*12 and R1860Gfs*12. Peak current density was calculated at -20 mV. * indicates P≤0.05, ** P≤0.01, *** P≤0.001, and n the number of tested cells.

Figure 4 :

 4 Figure 4: Computer model simulations of single atrial cell and ventricular cell membrane action potentials Computed AP time courses and expanded time-scale inset exhibiting first time derivatives (dV/dt) of the AP upstroke, in A: atrium and in B: ventricle.Computed dV/dt time courses exhibit a higher decrease in [dV/dt] max for the "heterozygous atrium" condition than for WT (respectively: 68 V/s vs. 212 V/s) whereas the decrease was smaller for "heterozygous ventricle" condition than for WT (respectively: 147 V/s vs. 220 V/s).

Figure 5 :

 5 Figure 5: Biochemical analysis HEK cells were transfected with GFP-Na v 1.5 (A) and with GFP-Na v 1.5 and/or Flag-Na v 1.5 (B). Data are represented as arbitrary units. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as total protein loading control. (A) Western blots of total cell lysates show that the R1860Gfs*12 protein quantity is significantly lower than the WT. Incubation of transfected cells with MG132 prevented the degradation of the C-terminal mutant; ***P≤0.001. (B) Co-immunoprecipitation of Na v 1.5 α-subunits tagged with either Flag or GFP. Immunoprecipitation was performed with the anti-GFP antibody, and western blot revealed with the anti-Flag antibody. These results demonstrated an interaction between R1860Gfs*12 α-subunits, and between WT and R1860Gfs*12 α-subunits.

FiguresSupplemental Figure 1 : 2 :Supplemental Figure 3 :

 123 Figures

  associated SNPs in the family with the R1860Gfs*12 SCN5A mutation Risk alleles are in red.

Figure 47 :

 47 Figure47: Both proximal and distal regions of Na v 1.5 are involved in the interaction with FHF1B (A) FHF1B was immunoprecipitated using an anti-His 6 antibody, and the bound proteins were then detected by western blot analysis with anti-GFP and anti-His 6 antibodies. Only the WT GFP-Na v 1.5 was immunoprecipitated with FHF1B even after increasing the total protein expression of the two mutants with MG132. (B) Reciprocal co-immunoprecipitation performed using the anti-GFP antibody for immunoprecipitation of GFP-Na v 1.5, and the anti-His 6 and anti-GFP antibodies for western blot analysis. FHF1B protein was precipitated with the WT channels but not with the two mutants.

Na v 1 . 5 ,

 15 we screened the FGF12-B gene coding for the most highly expressed FHF isoform in human ventricles in a group of BrS patients. Our study population consisted of 182 unrelated patients diagnosed with BrS based on the presence of a spontaneous type-1 ECG pattern (ST segment elevation ≥2 mm in one or more right precordial leads) spontaneously or following the administration of a sodium channel blocker. Structural heart disease was excluded by echocardiography. Informed consent for genetic testing was given by each patient. All probands were negative for mutations in SCN5A, MOG1, DLG1, KCNE3 and SCN1B.

Figure 49 :

 49 Figure 49: The FGF12-B variant p.V127M (A) DNA sequence chromatogram showing the nucleotide change resulting in a valine (V) to methionine (M) substitution at position 127 compared to control. (B) Conservation of the valine 127 across species and among the human FHFs.

50 :

 50 Figure 50: Preliminary functional study of the FGF12-B variant V127M (A) Current density-voltage and (B) activation-Vm relationships in Na v 1.5-stable HEK cells not transfected as a control (n = 6), or transfected with either WT (N = 8) or the V127M FGF12-B variant (n = 9). Sodium current density and activation-Vm relationship are not affected by the coexpression of the WT or the V127M FGF12-B variant. (C) Steady-state inactivation-Vm relationships were shifted towards negative potential with both the WT and the V127M FGF12-B variant compared to nontransfected condition. n = number of cells

  three truncated mutants display both gain-and loss-of-function effects. They induced a drastic reduction in I Na with a positive shift in the activation curve. The decrease in I Na is most probably due to a decreased surface expression of the mutants, since all these truncated Na v 1.5 abolished the sites of interaction with many partner proteins involved in membrane channel stabilization355 . Moreover, the decrease in I Na is consistent with the results from Cormier et al demonstrating that truncation of Na v 1.5 distal C-terminus (L1921X truncated construct) reduced current density 491 . In addition, L1821fs*10 and R1860Gfs*12 mutants induced a negative shift of the steady-state inactivation decreasing channel availability, while the mutant D1816Vfs*7 induced a positive shift of the steady-state inactivation and enhanced the recovery from inactivation, effects that would increase channel availability. Moreover, L1821fs*10 and R1860Gfs*12 increased the fast and slow time constants of inactivation and increased I NaL , parameters that were unaffected by the mutant D1816Vfs*7. The observed alteration in the inactivation by the two mutants L1821fs*10 and R1860Gfs*12 is consistent with the involvement of the proximal region of the C-terminus in channel inactivation. It has been demonstrated that truncation of the sixth helix in the proximal region and the distal region of Na v 1.5 (S1885X truncated construct) decreased I Na , induced a negative shift of the steady-state inactivation, and increased I NaL 491 . However, it remains unknown why the mutant D1816Vfs*7 had a different effect on the steady-state inactivation with no I NaL . One possibility is that the mutant D1816Vfs*7 could affect the interaction with unknown partner proteins that modulate channel inactivation. In the heterozygous state, neither of the three truncated mutants had a dominantnegative effect on WT channels. Nevertheless, alteration of the biophysical properties were observed for both D1816Vfs*7 and R1860Gfs*12: positive shift of activation and steady-state inactivation, with enhanced recovery from inactivation for D1816Vfs*7; and significant negative shift of steady-state inactivation, prolonged time constants of inactivation and I NaL for R1860Gfs*12. The complex functional effects caused by the C-terminal truncating mutations could be responsible for the mixed clinical picture observed, but they do not explain the prominence of atrial arrhythmias in patients carrying the R1860Gfs*12 mutation compared to the other two mutations which caused atrial and ventricular arrhythmias. This led us to hypothesize that the significant negative shift of steady-state inactivation and the presence of I NaL combined with the constitutive difference in the resting membrane potential between atria and ventricles could be responsible for the prominence of atrial arrhythmia in this family. Mimicking this difference in HEK293 cells or using computer-model simulation evidenced more pronounced effects of the mutant R1860Gfs*12 in the atria compared to the ventricles. The R1860Gfs*12 biophysical defects associated with the constitutive difference in resting membrane potentials between atrium and ventricle might induce a prominence of atrial arrhythmias It has been shown in both dogs 470 and rabbits 471 that the resting membrane potential of atrial cells is more depolarized than ventricular cells (-83 vs -86 mV). In addition, I Na density is more important in atria than in ventricles, with an hyperpolarization of the steady-state inactivation of -16mV 470 . Moreover, the critical depolarization and current thresholds for action potential initiation are smaller in atrial cells than in ventricular cells indicating that atrial cells are more readily excitable than ventricular ones 471 . The impact of some SCN5A mutations might thus be greater in the atria than in the ventricles, and may predispose to the development of atrial arrhythmias more readily than to ventricular ones. By measuring I Na in HEK293 cells transfected with the WT and/or the R1860Gfs*12 mutant channels, and maintained at a holding potential mimicking the resting membrane potentials of atrial or ventricular cardiomyocytes, we demonstrated that the mutant induced a more important decrease in I Na at atrial potential compared to ventricular one. Moreover, the fact that the steady-state inactivation of Na v 1.5 in the atria is shifted by -16mV compared to the ventricles 470 could exacerbate this loss of current density, since a greater percentage of atrial Na v 1.5 channels would be inactivated at a given resting or take-off potential compared to the ventricular Na v 1.5 channels. We confirmed our results by including the biophysical properties of the WT and the heterozygous currents into computer-model simulations of single atrial and ventricular cell membrane action potentials 493 . A marked decrease in AP maximum upstroke velocity ([dV/dt] max ), representing the maximum speed of depolarization, and a lengthening of the AP duration were observed in the atrial compared to the ventricular myocytes. We hypothesized that the significant shift of steady-state inactivation leads to a severe reduction in channel availability in atrial cardiomyocytes, which are more depolarized compared to the ventricular ones. Compared to the mutant R1860Gfs*12, the D1816Vfs*7 mutation carriers have both atrial and ventricular arrhythmias. The mutant D1816Vfs*7 increased channel availability but it also caused a significant positive shift of activation, which is a major lossof-function feature, since channels are able to open at more depolarized potentials. We hypothesized that this positive shift of activation in the heterozygous state of the mutant D1816Vfs*7 might lead to a marked loss of I Na in both atrial and ventricular cells. This effect may account for the occurrence of atrial and ventricular arrhythmias compared to the mutant R1860Gfs*12 showing an unaffected channel activation in the heterozygous state. Computer modeling of the D1816Vfs*7 mutation might be needed in order to test our hypothesis. From a clinical point of view, regarding the important role of Na v 1.5 channels in the SAN function, the negative shift of steady-state inactivation in the heterozygous state and the persistent current could account for the sinus node dysfunction observed in the family carrying the R186Gfs*12 mutation. Indeed, the SAN AP model showed that the negative shift of steadystate inactivation decreases the sinus rate, by decreasing the diastolic depolarization rate, and the persistent current decreases the sinus rate by AP prolongation despite the simultaneous slight increase in diastolic depolarization rate 211 . However, the combination of a persistent current and a shift of inactivation lead to a decrease in the sinus rate and abolish the slight increase in the depolarization rate seen with persistent current alone. Moreover, an increase in the persistent current above a critical level leads to a plateau oscillation and repolarization failure resulting in sinus pause or arrest 211 . In addition, the association of the prolonged time constant of fast and slow inactivation and the I NaL, might cause repolarization failure, early

  Nevertheless, characterization of ion channel mutations in heterologous expression systems provides information about the biophysical phenotypes possibly leading to the clinical symptoms. Further studies are needed in a physiological environment like a mouse model, or human cardiomyocytes, in order to better understand the pathogenic pathway underlying the disease.A specific biophysical defect of a given SCN5A mutation might be able to explain the variability in the clinical expression between different mutations, but it cannot explain the variability in the clinical phenotypes and in the severity of the disease among family members carrying the same mutation. This has drawn our attention to the participation of the patient's genetic background in the modulation of the disease phenotype.The genetic background of the SCN5A mutation carriers could modulate the clinical phenotypeBy screening 17 SNPs linked to AF, and 3 SNPs linked to BrS in the family carrying the R1860Gfs*12 mutation, we showed that the proband, who had severe SSS and early onset AF, was the only mutation carrier to have one at-risk allele within the locus 4q25 upstream of the PITX2 gene. The 4q25 variants have been widely associated with AF development,

  inducing early atrial morphological changes and further loss-of-function of Na v 1.5 channels in the proband's atrium, accounting for the occurrence of severe SSS with AF at a young age. A recent study by Ritchie et al 498 has demonstrated that the 4q25 variants, especially rs2200733 and rs10033464, modified the clinical expression of signaling molecule and ion channel gene mutations associated with familial AF. In this study, seventy subjects in eleven families, in which AF was present in two or more family members, were included. Family members shared an AF causing mutation, and the probands had lone AF. Genotyping of the rs2200733 and rs10033464 SNPs was performed in the seventy subjects. Statistical analysis demonstrated a significant association between the presence of both 4q25 variants and AF mutations in the development of AF. All these data support the hypothesis that 4q25 variants could act as genetic modifiers of the clinical phenotype by modulating the expression of cardiac ion channels. However, there are several questions, which remain to be answered. The first is how these reported variants could alter the expression and/or the function of PITX2.Secondly, whether the arrhythmogenic substrate formed by the alteration of PITX2 expression is established during the embryonic stage496,499 , or during later stages of heart development497,475 . Moreover, how the 4q25 variants interact with the other AF-associated variants to modulate the phenotypes, and which of these variants has the most important effect in AF development remain to be elucidated. Quantitative PCR of PITX2 mRNA using human atrial biopsies from patients carrying the 4q25 variants could be useful to test if these variants alter PITX2 expression. Modulation of the phenotypic expression of ion channel gene mutations by common genetic variants is a major focus of recent research on the genetics of arrhythmia. Remme et al in 2009 have demonstrated that the genetic background can modulate disease severity 500 .By comparing the phenotypic expression of the SCN5A mutation 1798insD in two different mouse genetic backgrounds (129P2 and FVB/N), they demonstrated that the 129P2 mice had a more severe phenotype than the FVB/N ones. Recently, GWAS for different diseases and traits have identified common variants (SNPs) that could modulate disease susceptibility.

Table 1 : Summary of AF-susceptibility loci identified by linkage analysis

 1

......................

Table 2 : Different ion channel genes involved in AF

 2

..........................................................

Table 3 : SNPs associated with AF

 3

........................................................................................

Table 4 : Na + channel mutations associated with sinus node dysfunction

 4

.........................

Table 5 : HCN4 mutations associated with SSS syndrome

 5

..................................................

Table 6 : Mutations in genes associated with Brugada syndrome

 6

......................................

Table 7 : Forward (-F) and reverse (-R) primers used for site-directed mutagenesis ....Table 8 : Primary antibodies used in western blot

 78

.............................................................

Table 9 : qPCR primer pairs used for detecting FGF12 and FGF13 transcripts in adult human and mouse hearts

 9

.............................................................................

Table 10 : Primers used for FGF12

 10

	HPRT: Hypoxanthine guanine phosphoribosyl transferase
	HV: His-ventricular
	IB2: Islet-brain-2
	I Ca,L , I Ca,T : Calcium currents
	ICC: Interstitial cells of Cajal
	ICD: Implantable cardioverter defibrillator
	I f : Funny current
	IFM: Isoleucine, Phenylalanine, Methionine
	I Na : Inward sodium current
	I NaL : Late sodium current
	I to : Transient outward potassium current
	IV: Intravenous
	IVF: Idiopathic ventricular fibrillation
	JLNS: Jervell and Lange-Nielsen syndrome
	K + : Potassium ion
	KChIP2: K + channel-interacting protein
	LB: Lysogeny broth medium
	LGMD2G: Limb girdle muscular dystrophy
	LQTS: Long QT syndrome
	MAGUK: Membrane associated guanylate kinase
	MAP: Mitogen activated protein
	MDP: Maximum diastolic potential
	MOG1: Multicopy of suppressor gsp1
	Na + : Sodium ion
	Na v 1.5: Sodium channel 1.5 NCX: Na + /Ca 2+ exchanger
	Nedd4-2: Ubiquitin-protein ligases
	NHS: N-hydroxysuccinimide
	NPPA: Atrial natriuretic peptide
	PAC: Premature atrial contraction
	PBS: Phosphate buffered saline
	PCCD: Progressive cardiac conduction disease
	PKA: Protein kinase A
	PKC: Protein kinase C
	PMCA: Plasma membrane Ca 2+ ATPase
	PTPH1: Protein tyrosine phosphatase 1
	PVC: Premature ventricular contraction
	PY: Proline, Tyrosine
	qPCR: Quantitative reverse-transcriptase polymerase chain reaction
	RBBB: Right bundle branch block
	RFP: Red fluorescent protein
	RPL32: Ribosomal protein L32
	RVOT: Right ventricular outflow tract
	RyR2: Ryanodine receptors
	SAN: Sinoatrial node
	SAP97: Synapse associated protein 97
	SCA27: Spinocerebellar ataxia 27
	SCD: Sudden cardiac death
	SERCA: Sarco/endoplasmic reticulum Ca 2+ ATPase
	SIDS: Sudden infant death syndrome

-B sequencing ............................................................. Table11: BrS associated SNPs in the family with the R1860Gfs*12 SCN5A mutation .

php II.2.3.1 Different types of AF

		terminates spontaneously, usually within 48 hours.
		o Persistent AF: AF lasts more than seven days or does not terminate
		spontaneously requiring electrical or pharmalogical cardioversion.
		o Long-standing persistent AF: AF that has lasted one or more years
		before rhythm control is decided.
		o Permanent AF: The presence of AF is accepted by the patient and
		the physician, and as such, no further rhythm control interventions
		are carried out.
	II.2.3.2	Symptoms of AF
	Atrial fibrillation has a wide variety of clinical presentations, ranging from
	asymptomatic to patients presenting with symptoms of AF complications such as brain stroke,
	heart failure, or cardiovascular collapse. However, the most common symptoms are
	palpitations, dyspnea, fatigue, lightheadedness, and chest pain. Since these symptoms are not
	specific, diagnosis of AF cannot only be made on the clinical presentation.
	Based on etiology, AF can be divided into secondary and lone AF. Secondary AF, the
	most common form, is caused by coexisting medical conditions, cardiac or non-cardiac, such
	as ischemic heart disease, hypertension, electrolyte depletion, pneumonia, lung cancer and
	thyrotoxicosis. AF is also common after cardiothoracic operation. In addition, alcohol and
	caffeine consumption, as well as both physical and emotional stresses can increase AF risk.
	Lone AF occurs in younger patients with structurally normal hearts and in the absence
	of other secondary medical conditions, accounting for 10-30% of AF 30 . It is always a
	diagnosis of exclusion.
	Genetic risk factors, especially mutations in genes encoding ion channels, can also be
	involved.
	The European Society of Cardiology (ESC) issued an AF classification according to
	episode timing and termination, thus AF can be divided into five types 31 :
		o First diagnosed AF: A patient presents for the first time with AF
		regardless of its duration or presence and severity of symptoms.
		o Paroxysmal AF: AF episode that lasts less than seven days and

http://www.emedu.org/ecg/oni.

Table 2 : Different ion channel genes involved in AF

 2

	OMIM	Genes	Chromosome	Protein	Current	Functional consequences
	ATFB	KCNQ1	11p15	K v LQT1	I Ks	Gain-of-function effect on I Ks current 57-61
		KCNE1	21q22.12	minK	I Ks	Gain-of-function effect on I Ks current 68
	ATFB	KCNE2	21q22	MiRP1	I Ks	Gain-of-function effect on I Ks current 69
		KCNE5	Xq22.3	MiRP4	I Ks	Gain-of-function effect on I Ks current 70
	ATFB	NPPA	1p36-p35	ANP	I Ks	Gain-of-function effect on I Ks current 54,60
		KCNH2	7q36.1	K v 11.1	I Kr	Gain-of-function effect on I Kr current 71,72
		KCNE3	11q13.4	MiRP2	I Kr	Gain-of-function effect on I Kr and I to currents 73
	ATFB	KCNA5	12p13	K v 1.5	I Kur	Gain-of-function effect on I Kur crrent 74,75 Loss-of-function effect on I Kur current 75-78
		KCND3	1p13.2	K v 4.3	I to	Gain-of-function effect on I to current 81
	ATFB	KCNJ2	17q23-q24	Kir2.1	I K1	Gain-of-function effect on I K1 current 50,82-84
		KCNJ8	12p11.23	Kir6.1	I KATP	Gain-of-function effect on I KATP current 85,86
	ATFB 12	ABCC9	12p12.1	SUR2A	I KATP	Loss-of-function effect on I KATP current 87
	ATFB 10	SCN5A	3p21	Na v 1.5	I Na

Table 3 : SNPs associated with AF

 3

	rs11708996 130

* Identified by candidate gene associations studies

Table 4

 4

, some of these mutations are associated with mixed clinical pictures. Studies of several mutations (T220I, P1298L, delF1617, and E161K) in a two-dimensional model of intact SAN-atrium tissue showed that these mutations slowed down pacemaking and compromised AP conduction across the SAN-atrium, leading to possible sinus arrest or SAN exit block. These abnormalities are likely to be exacerbated by vagal nerve activity, and this may account for a high risk of cardiac arrest in SSS patients at night, when vagal activity is prominent

198

.

Recently, other mutations in SCN5A gene have been identified in patients with SSS without functional studies. The compound heterozygous mutations A735V/D1792N 199 , D349N/D1790N 200 , and the frame shift mutation F1775Lfs*15 201 , all are new mutations, except A735V which was previously reported in BrS 193 .

A mouse model with a null mutation in the SCN5A gene further confirmed the role of the sodium current in pacemaker function and the contribution of SCN5A mutations in the pathophysiology of SSS. The homozygous SCN5A -/-mice died in utero and showed severe defects in ventricular morphogenesis 202 . However, heterozygous mice had a normal survival rate but presented with a number of electrophysiological abnormalities, including impaired atrio-ventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of re-entrant excitation 202 .

Table 4 ,

 4 most LQT3 mutations associated with sinus node abnormalities induce both a persistent sodium current and a negative shift of inactivation. In order to determine if these alterations may account for the sinus bradycardia and the sinus pause seen in LQT3 patients, Veldkamp et al 211 studied the 1795inD mutation identified in a family with a mixed clinical picture of sinus bradycardia, CCD, LQTS and BrS, in a SAN AP model. This model shows that the 10-mv negative shift of inactivation decreased the sinus rate by decreasing the diastolic depolarization rate, and the persistent current decreased sinus rate by AP prolongation despite the simultaneous slight increase in diastolic depolarization rate. However, the combination of persistent current (1% to 2%) and the shift of inactivation led to a 10% decrease in sinus rate and abolished the slight increase in the depolarization rate seen with persistent current alone.

	Mutations	Channel characteristics	Current characteristics	Phenotype	References
	E161K	Loss-of-function	Reduced	cur	rent	density,	SSS, BrS, LQT	Smits et al., 2005 190
			positive shift in activation
	T187I	Non-functional	No current				SSS, BrS	Makiyama et al., 2005 191
	T220I	Loss-of-function	Reduced	current	density,	SSS	Benson et al.,
			negative shift in inactivation	DCM, AF, heart block	Olson et al., 2005 91
	R367H	Non-functional	No current				Atrial standstill, BrS	Takehara et al., 2004 192
							SUNDS	Vatta et al., 2002 193
	G514C	Loss-of-function / gain-of-function Positive shift in activation	CCD, bradycardia	Tan et al., 2001 194
			reducing I Na . Destabilized
			inactivation tending to
			increase I Na
	R878C	Non-functional	No current				SSS	Zhang et al., 2008 195
	D1275N	Loss-of-function	Positive shift in activation, fast	SSS, DCM, PCCD	Olson et al., 2005 91
			recovery				Atrial stand still with	Groenewegen et al., 2003 196
							connexin SNP
	P1298L	Loss-of-function	Reduced	current	density,	SSS	Benson et al.,
			negative shift in inactivation
	G1408R	Non-functional	No current				SSS	Benson et al.,
							BrS or CCD	Kyndt et al., 2001 188
	W1421X	Non-functional	No current (predicted)		SSS	Niu et al., 2006 197
	K1578fs/52	Non-functional	No current				SSS, BrS on provocation	Makiyama et al., 2005 191
	delF1617	Loss-of-function	Reduced	current	density,	SSS	Benson et al.,
			negative in shift inactivation	LQT	Splawski et al., 2000 189

Table 4 : Na + channel mutations associated with sinus node dysfunction

 4

Modified from

Lei M. et al. Prog Biophys Mol Biol 2008 219

  221,222 , syncope with QT prolongation and torsade de pointes225 , symptomatic sinus bradycardia with normal chronotropic and exercise capacity223 , or syncope with intermittent AF and chronotropic incompetence 220 . One mutation was reported in sporadic cases 220 , while the others were familial and transmitted in an autosomal dominant way[221][222][223] .

	Mutation	Channel characteristics	Current characteristics		Phenotype
	P544fsX30	Loss-of-function	Negative		shift	of	Patient with symptomatic sinus
	(HCN4 573X) 220		activation activation		at	long protocol,	bradycardia, intermittent AF, chronotropic incompetence
			insensitive to increased
			cAMP,		dominant-
			negative on WT
	D553N 225	Loss-of-function	Reduced I f current caused	Patient	with	sinus	node
			by decreased surface	dysfunction, QT prolongation,
			expression	of	the	PVT, torsade de pointes, AD
			channel,		dominant-
			negative on WT
	S672R 221	Loss-of-function	Negative		shift	of	Familial	with	asymptomatic
			activation,				faster	bradycardia, AD
			deactivation, no change
			of	cAMP-induced
			channel activation
	G480R 222	Loss-of-function	Reduced I f current, slow	Familial	with	asymptomatic
			and negative shift of	bradycardia, normal exercise
			activation					capacity, AD
	A485V 223	Loss-of-function	Drastic decreased in I f ,	Familial	with	symptomatic
			negative		shift	of	bradycardia, AD
			activation
	K530N 224	Loss-of-function	Negative		shift	of	Familial	with	tachycardia-
			inactivation	at	the	bradycardia	syndrome	and
			heterozygous state		persistent AF in an age-dependent
								fashion, AD

Recently, the mutation K530N was identified in a family which developed tachycardia-bradycardia syndrome and persistent AF in an age-dependent manner, indicating that the dysfunction in the f-channel may contribute to the development of atrial arrhythmias 224 .

Table 5 : HCN4 mutations associated with SSS syndrome AD

 5

= autosomal dominant.

Table 6 : Mutations in genes associated with Brugada syndrome

 6 * May act as modifier factors.Modified from Nielsen M. W. et al. Front Physiol 2013 303 The presence of conduction abnormalities, specifically right bundle branch block (RBBB), were involved in the early definition of BrS, in association with the persistent ST segment elevation in the right precordial leads 249 . However, in the consensus reports of 2002 340 , 2005 250 , and recently 2013 287 , ST elevation is the only maintained diagnostic ECG criterion of BrS. Nevertheless, several studies have shown that patients with BrS have various conduction abnormalities at different levels of the conduction system 261-264 . A recent multicentric study by Maury et al 264 including 325 BrS patients showed that only 21% of patients did not have any conduction abnormality on the ECG. The majority of patients had various conduction abnormalities including increased P-wave duration, first degree AV block, right bundle branch block, fascicular block, and prolongation of Hisventricular (HV) interval, occurring separately or in various combinations in the same patient.

	OMIM	Gene	Locus	Protein	Current	Functional effects	Reference
	BrS1	SCN5A	3p21	Na v 1.5	I Na	Loss-of-function	Chen et al., 1998 308
	BrS2	GPD1-L	3p22.3	GPD1L	I Na	Loss-of-function	London et al., 2007
	BrS3	CACNA1C	12p13.3	Ca v 1.2	I Ca-L	Loss-of-function	Antzelevitch et al.,
	BrS4	CACNB2	10p12	Ca v ß2	I Ca	Loss-of-function	Antzelevitch et al.,
	BrS5	SCN1B	19q13.1	Na v ß1	I Na	Loss-of-function	Watanabe et al., 2008 327
	BrS6	KCNE3	11q13-14	MiRP2	I to	Gain-of-function	Delpón et al., 2008
	BrS7	SCN3B	11q23.3	Na v ß3	I Na	Loss-of-function	Hu et al., 2009 329
	BrS8	HCN4	15q24.1	HCN4	I f	Loss-of-function	Ueda et al., 2009 *330
		KCNH2	7q36.1	hERG1	I Kr	Gain-of-function	Itoh et al., 2009 331 ; Verkerk et al., 2005 *332
		KCNJ8	12p11.23	Kir6.1	I KATP	Gain-of-function	Medeiros-Domingo et al., 2010 86
		CACNA2D1	7q21-q22	Ca v α2δ-1	I Ca-L	Not available	Burashnikov et al.,
		RANGRF	17p13.1	MOG1	I Na	Loss-of-function	Kattygnarath et al.,
		KCNE5	Xq22.3	MiRP4	I to	Gain-of-function	Ohno et al., 2011 *335
		KCND3	1p13.3	K v 4.3	I to	Gain-of-function	Giudicessi et al., 2011 336
		SLMAP	3p14.3	SLMAP	I Na	Loss-of-function	Ishikawa et al., 2012 337
		TRMP4	19q13.3	TRMP4	NSC Ca	Both	Liu et al., 2013 338
		SCN2B	11q23	Na v ß2	I Na	Loss-of-function	Riuró et al., 2013 339
		FGF12	3q28	FHF1	I Na	Loss-of-function	Hennessey et al., 2013 304
		PKP2	12p11	PKP2	I Na	Loss-of-function	Cerrone et al., 2013

  . However, the results concerning CaMKII effects on Na v 1.5 are conflicting. It has been shown that adenovirus-mediated over-expression of CaMKIIδc, the prominent cardiac isoform, in rabbit ventricular cardiomyocytes led to a

	negative shift of steady-state inactivation, enhanced intermediate inactivation and slow
	recovery from inactivation, and increased late I Na	411 . Another study showed that the inhibition
	of CaMKII in rat ventricular myocytes by KN93, a selective antagonist of CaMKII, has the
	same effects, with a decreased late current 412 . This discrepancy may be due to differences in
	expression systems, procedures or protein isoforms used in different studies. It has been
	recently shown that CaMKIIδc interacts with the linker I-II of Na v 1.5, and phosphorylates the
	serine S516 and threonine T594 residues 413 .

  . Replacement of FGF13 by WT or mutant FGF12-B restored the calcium current density and the localization of Ca v 1.2. However, only the WT FGF12-B was able to restore I Na and the negative shift of steady-state inactivation of Na v 1.5 304 . These results suggest that FFG12-B, the highly expressed FHF isoform in the human heart, is a new candidate gene in BrS. FGF12-B mutations could lead to BrS by affecting the sodium channel without perturbing the calcium channel, and vice versa.

Table 8 : Primary antibodies used in western blot

 8

Table 10 : Primers used for FGF12-B sequencing III.2 DNA concentration measurement

 10

Table 1 . Biophysical and kinetic properties of I Na from WT and mutant channels

 1

	Activation

Table 1 : Genotypes of SNPs associated with atrial fibrillation in the patient's family

 1 Risk alleles are in red and protective alleles in blue. MAF = minor allele frequency in HapMap-CEU.

	MG132 MG132	WT R1860G WT ---+ + + WT R1860G WT WT R1860G WT WT R1860G WT ---+ + +	WT R1860G WT ---WT R1860G WT ---	WT R1860G WT + + + WT R1860G WT + + +
	Na v 1.5 Na v 1.5			Blot Ab: GFP Blot Ab: GFP
	FHF1B FHF1B			Blot Ab: His 6 Blot Ab: His 6
		Total cell lysates Total cell lysates	IP with His 6 Ab IP with His 6 Ab

This work was supported by Institut National de la Santé et de la and Marie Curie, the Agence Nationale de la Recherche (ANR-09-GENO-003-CaRNaC, the French Ministry of Health (P.H.R.C. AOR04070, P040411) and NIH R01 (HL094450) (ID).

THESIS OBJECTIVES

Several mutations in the N-terminal domain of Na v 1.5 have been reported, however the functional role of this region remains unknown. In this study, we aimed to investigate the role of the N-terminal domain of Na v 1.5 on channel function, subcellular localization, and protein expression, by the characterization of the functional effects of the mutant R104W, as well as another N-terminal mutation, R121W, previously published 214,306 . Moreover, we created two constructs: ∆Nter, where the N-terminal region was deleted, and Nter, where only the N-terminal region was present.

Methods and Results

Patch-clamp analysis in HEK293 cells transfected with the WT, the mutant channels, R104W, R121W, or the construct ∆Nter showed that the two N-terminal mutants and the ∆Nter abolished the sodium current. Moreover, co-expression of the N-terminal mutants with the WT channels led to a dominant-negative effect on the WT channels with a positive shift in the activation curve, but not the ∆Nter. Immunocytochemical analysis in rat neonatal cardiomyocytes co-transfected with the N-terminal mutants and the CD4 construct carrying the KKXX endoplasmic reticulum (ER) retention motif showed that the N-terminal mutants were retained in the ER, and their expression with the WT led to WT channel retention. In

Solutions and drugs

Thirty-six hours after transfection, HEK293 cells were trypsinized and seeded to a density that enabled single cells to be identified. Some HEK293 cells transfected with R104W or R121W mutants were cultured with the class I anti-arrhythmic agent mexiletine at several concentrations (30, 60, 140, 250 or 500 µM) for 24h after transfection. Cells were then returned to their normal buffer 30 min before patch-clamp recordings.

RNA extraction and reverse transcriptase-PCR (RT-PCR)

Forty-eight hours after transfection with WT or mutant channels, selected cells were treated with DTT 500 µM for one hour to induce UPR (Unfolded Protein Response) as a positive control. Total RNA was extracted from DTT-treated and non-treated cells using TRIzol reagent with the PureLink mRNA Mini Kit (Invitrogen, CA, USA). One µg of total mRNA was reverse-transcribed with the SuperScriptIII RT-PCR kit (Invitrogen, CA, USA). PCR was then performed as previously reported using primers XBP1-forward CCTTGTAGTTGAGAACCAGG and XBP1-reverse GGGGCTTGGTATATATGTGG 1 .

Supplemental Figure 3: RT-PCR of XBP-1 mRNA from HEK293 cells transfected with N-terminal Na v 1.5 mutants. Accumulation of unfolded proteins in the ER can cause reticular stress, and activate signaling pathways including the unfolded protein response (UPR) 2 . UPR activation leads to the XBP-1 mRNA splicing of 26 nucleotides (S-XBP-1) causing a frameshift and the generation of a longer active protein. Analysis of these spliced transcripts is used as a marker of reticular stress 1 . MW indicates molecular weight and NT non-transfected cells. Only cells incubated with DTT, known to induce reticular stress and used as a positive control, contained S-XBP-1 mRNA, suggesting that the mutant Nav1.5 channel transfection does not cause reticular stress by UPR activation. Objective: To characterize a distal truncating SCN5A mutation, R1860Gfs*12, identified in a family with different phenotypes including sick sinus syndrome (SSS), atrial fibrillation (AF), atrial flutter and atrioventricular-block.

Supplemental references

Methods: Patch-clamp and biochemical analysis were performed in HEK293 cells transfected with wild-type (WT) and/or mutant channels.

Results:

The mutant channel expressed alone caused a 70% reduction in I Na density compared to WT currents, consistent with its partial proteasomal degradation. It led also to a negative shift of steady-state inactivation and to a persistent current. When mimicking the heterozygous state of the patients by co-expressing WT and R1860Gfs*12 channels, the biophysical properties of I Na were still altered, and the mutant channel α-subunits still interacted with the WT ones. Since the proband developed paroxysmal AF at young age, we screened 17 polymorphisms associated with AF risk in this family, and showed that the proband carries at-risk polymorphisms upstream of PITX2, a gene widely associated with AF development. In addition, when mimicking the difference in resting membrane potentials between cardiac atria and ventricles in HEK293 cells, or using computer-model simulation, R1860Gfs*12 induced a more drastic decrease in I Na at the atrial potential.

Conclusion:

We have identified a distal truncated SCN5A mutant associated with gain-and loss-of-function effects, leading to SSS and atrial arrhythmias. A constitutively higher susceptibility to arrhythmias of atrial tissues and genetic variability could explain the complex phenotype observed in this family.

Keywords

Methods

Patient

Blood samples were obtained after signed written informed consent for genetic analyses and after approval by the local ethics committee. The study was conducted according to the principles of the Helsinki Declaration.

Mutation and SNP analysis

Genomic DNA was isolated from leukocytes according to standard procedures. Screening for mutations was performed by genomic DNA amplification of all exons and splice junctions of several genes responsible for arrhythmias (Supplemental methods). We also genotyped single nucleotide polymorphisms (SNP) associated with AF 6,7 (Supplemental Table 1).

PCR products were directly sequenced with the Big Dye Terminator v. Current-voltage relationships (I/V curves) and the steady-state inactivation-V m protocols were as previously reported 8 . Data for the activation-V m and steady-state inactivation-V m relationships of I Na were fitted to the Boltzmann equation as previously reported 8 .

The putative involvement of a persistent Na + current was assessed by using 100 µM tetrodotoxine (TTX). Currents were elicited by a 500-ms step to -20 mV from a holding potential of -120 mV. The percentage of persistent current was calculated by dividing the TTX-sensitive I Na amplitude determined at the end of the pulse by the peak I Na amplitude obtained before TTX application.

Computer simulations of atrial and ventricular membrane action potentials were performed using Oxsoft Heart Model V. 4.8 as described in the Supplemental methods.

Protein extraction and Western blot

They were performed as previously reported 8 . Primary antibodies used were: rabbit anti-GFP (1:2000, Torrey Pines Biolabs, USA), mouse anti-Flag (1:500, Sigma, USA), and rabbit anti-GAPDH (1:2000, Abcam, UK). Total protein signals were firstly normalized to GAPDH levels and then to WT levels.

Co-immunoprecipitation

Co-immunoprecipitation experiments were performed as described in the Supplemental methods. Briefly, total cell lysates were incubated with Dynabeads (Dynal, Norway) crosslinked to anti-GFP antibody (Torrey Pines Biolabs, USA) for 2 h at room temperature. Western blots were revealed using either rabbit anti-GFP (Torrey Pines Biolabs, USA), or mouse anti-Flag (Sigma, USA) antibodies. located upstream of the PITX2 gene, while the proband carried only one copy (Figure 1A and Supplemental Table 1). The multimarker risk score for AF based on combined rs2200733, rs17570669 and rs3853445 genotypes 9 was higher in the proband (1.74) compared to her father (<1) and uncle (<1).

The R1860Gfs*12 mutation produced a loss-and gain-of-function of Na v 1.5

Na + currents were recorded in HEK293 cells 36 h after transfection with WT or mutant channels. I Na traces and I/V relationships are shown in Figure 2. Peak current densities and Pvalues are given in Table 1. Alone, R1860Gfs*12 showed a drastic reduction of I Na density compared to WT channels (Figure 2A andB). Furthermore, the biophysical characteristics of the C-terminal mutant channels were impaired (Table 1, Figure 2C andD). Indeed, the V 1/2 of activation for R1860Gfs*12 was shifted by +7.1 mV, and the V 1/2 of inactivation was shifted by -24.5 mV compared to WT. No change in recovery from inactivation was observed (Supplemental Figure 3). Fast and slow time constants of inactivation (τ fast and τ slow ) were significantly increased compared to WT channels (Table 1, Figure 3A-C).

The presence of a TTX-sensitive persistent Na + current was assessed using 100 µM TTX (Figure 3D and Supplemental Figure 4). The mutant channel generated a persistent current of 4% of the peak current.

Co-expression of WT and R1860Gfs*12 channels also led to a loss-and gain-of-function of Na v 1.5

To mimic the heterozygous state of the patient, cells were co-transfected with the WT channel (no tag) and the R1860Gfs*12 mutant channel (GFP tagged) in a 1:1 ratio. In cotransfected cells, peak current densities were slightly reduced (Figure 2A andB), but not significantly different from the WT alone, as for the V 1/2 of activation. In contrast, the V 1/2 of steady-state inactivation remained significantly shifted by -16.6 mV compared to WT (Table 1, Figure 2C andD). Moreover, τ fast and τ slow were significantly increased at the heterozygous state compared to WT alone (Table 1, Figure 3A andB), and the persistent current was still present (Table 1, Figure 3D and Supplemental Figure 4).

Since the steady-state inactivation of the mutant channels alone or co-expressed with WT channels were drastically shifted, we measured I Na successively from a holding potential (HP) of -86 and -83 mV mimicking the ventricular vs atrial resting membrane potentials 10,11 .

Interestingly, we showed that the 3-mV difference in HPs was sufficient to enhance the reduction of I Na due to the mutation in the atrium compared to the ventricle (Figure 3E).

Supplemental material

Methods

Genes screened in the proband

Mutational analyses were performed for the following genes: KCNQ1 (NM_000218),

KCNA5 (NM_002234), KCNJ2 (NM_00891), GJA5 (NM_005266 and 181703), SCN1B

(NM_001037 and 199037), SCN2B (NM_004588) and SCN3B (NM_018400).

Solutions and drugs

Thirty-six hours after transfection, HEK293 cells were trypsinized and seeded to a density

III. Fibroblast Growth Factor Homologous Factor 1B III.1 The proximal and distal regions of the C-terminal domain of Na v 1.5 interacted with FHF1B

The C-terminal domain of Na v 1.5 is the site of interaction of several partner proteins regulating channel function. Among these partners is the Fibroblast Growth Factor Homologous Factor 1B (FHF1B), which regulates channel inactivation and surface expression 304,454,456,458 . Recently, the FGF12 gene encoding FHF1B has been reported as a new candidate in BrS 304 . The site of interaction of FHF1B on Na L1821fs*10 are partially degraded by the ubiquitin-proteasome system, we checked whether increasing total protein expression with MG132, a 26S-subunit ubiquitin-proteasome inhibitor, could restore the interaction with FHF1B. Immunoprecipitation using anti-His 6 antibody was done after treating the cells with MG132 (Figure 47A). Although we increased total protein expression of the two mutants with MG132, this did not restore the interaction with FHF1B (Figure 47A). Reciprocal co-immunoprecipitation was also performed (Figure 47B). FHF1B protein was successfully precipitated with the WT channels but not with the two mutants.

The loss of interaction of the two mutants with FHF1B confirms the involvement of both the proximal and distal parts of the Na v 1.5 C-terminus in the interaction with FHF1B as reported by Wang et al 456 .

DISCUSSION

characterized in this study, no functional defects were observed, which suggests that they may be rare polymorphisms and not disease-causing mutations, and that we should be cautious when interpreting the results of genetic testing. Nevertheless, in this study none of these mutations was studied in the heterozygous state, and other unknown factors may contribute to the disease phenotype.