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The multi-faceted aspects of the complex cardiac Nav1.5 in mem- brane function and pathophysiology

Nicola Detta, Giulia Frisso, Francesco Salvatore

PII: S1570-9639(15)00202-2 DOI: doi: 10.1016/j.bbapap.2015.07.009 Reference: BBAPAP 39635

To appear in: BBA - Proteins and Proteomics

Received date: 16 April 2015 Revised date: 9 July 2015 Accepted date: 19 July 2015

Please cite this article as: Nicola Detta, Giulia Frisso, Francesco Salvatore, The multi- faceted aspects of the complex cardiac Nav1.5 proteins in membrane function and patho- physiology, BBA - Proteins and Proteomics (2015), doi: 10.1016/j.bbapap.2015.07.009

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The multi-faceted aspects of the complex cardiac Nav1.5 proteins in membrane function and pathophysiology

Nicola Detta a, Giulia Frisso a,b, Francesco Salvatore a,c,*

a CEINGE‐Biotecnologie Avanzate s.c.ar.l., Naples, Italy; b Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy; c IRCCS-Fondazione SDN, Naples, Italy.

*Corresponding author: Francesco Salvatore, M.D., Ph.D., CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486 - Naples, Italy. Tel.: +39 081 7463133; Fax: +39 081 7463650; E-mail: [email protected]

KEYWORDS: Cardiac , , Nav1.5, SCN5A, sodium channel kinetics, Na 1.5 biologyACCEPTED MANUSCRIPT v

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Index

ABSTRACT ...... 3

1. INTRODUCTION ...... 4

2. STRUCTURE OF THE VOLTAGE-GATED SODIUM CHANNEL ...... 4

3. FUNCTION AND VOLTAGE-DEPENDENT ACTIVATION OF NAV1.5 ...... 6

4. FAST INACTIVATION OF NaV1.5 ...... 7

5. SLOW INACTIVATION OF Nav1.5 ...... 8

6. POST-TRANSLATION MODIFICATIONS ...... 8

6.1 Glycosylation...... 8

6.2 Phosphorylation ...... 9

6.3 Ubiquitination ...... 10

6.4 Other post-transcriptional modifications ...... 10

7. INTERACTING PROTEINS ...... 11

8. CHANNELOPATHIES ...... 12

8.1 Long QT syndrome ...... 13

8.2 ...... 14

8.3 Sick sinus syndrome ...... 16 8.4 Progressive CardiacACCEPTED Conduction Disease ...... MANUSCRIPT...... 17 8.5 Inherited Dilated Cardiomyopathy ...... 18

9. CONCLUSIONS AND PERSPECTIVES ...... 19

ACKNOWLEDGEMENTS ...... 20

ABBREVIATIONS ...... 21

REFERENCES ...... 22

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ABSTRACT

The aim of this mini-review is to draw together the main concepts and findings that have emerged from recent studies of the cardiac channel Nav1.5. This complex protein is encoded by the

SCN5A that, in its mutated form, is implicated in various diseases, particularly channelopathies, specifically at cardiac tissue level. Here we describe the structural, and functional aspects of Nav1.5 including post-translational modifications in normal conditions, and the main human channelopathies in which this protein may be the cause or trigger. Lastly, we also briefly discuss interacting proteins that are relevant for these channel functions in normal and disease conditions.

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1. INTRODUCTION

The voltage-gated sodium channels are large transmembrane proteins responsible for

the initial and rapid phase of the action potential in most electrically excitable cells [1]. All

sodium channels consist of 2 different subunits: the α-subunit that constitutes the voltage-

gated sodium-selective aqueous pore, and the β-subunits (one or two) that are responsible

for channel regulation and localization [2]. Nav1.5 is the voltage-gated sodium channel

isoform expressed specifically in cardiac tissue [1]. It is distributed in atrial and ventricular

myocytes and Purkinje fibers. This channel is an essential player in cardiac action

potential initiation and subsequent propagation through the heart [3]. Given the key role

played by Nav1.5 in cardiac electrical excitability and stability, mutations that alter channel

function are involved in the pathogenesis of arrhythmic, and also in structural, inherited

genetic disorders. This mini-review provides an overview and update of the human cardiac

sodium channel protein Nav1.5 and related diseases.

2. STRUCTURE OF THE VOLTAGE-GATED SODIUM CHANNEL

The human cardiac sodium channel (Nav1.5) is a member of the voltage-dependent

family of Na channels. This family consists of 9 members (Nav1.1-1.9), encoded by 9

(SCN1-5A, ACCEPTED and SCN8-11A); the amino MANUSCRIPT acid sequences of the proteins are more

than 50% homologous [4]. These channels consist of the heteromeric assembly of an α-

subunit (the pore-forming component) which is functionally modulated by its association

with two ancillary β-subunits. Currently 5 β-subunits, encoded by 4 genes (SCN1B-

SCN4B), have been identified in mammals (β1-β4 and β1B) [5]. The α-subunit is sufficient

to allow sodium ions to flow through the cell membrane and thus to generate the sodium

currents (INa). β-subunits are unable to generate sodium currents independently of the α-

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subunit, and they probably serve to facilitate sodium channel migration towards the cell membrane by directly interacting with the α-subunit [4].

Nav1.5 is encoded by the SCN5A gene that is located on 3p21 and consists of 28 exons [6]. The Nav1.5 α-subunit is a large integral membrane protein (227 kDa) consisting of 2,016 amino acids organized in four homologous transmembrane domains, designated D1-D4 (D1, aa 127-416; D2, aa 712-939; D3, aa 1201-1470; D4, aa

1524-1772) (Fig. 1A). These 4 domains assemble in a specific 3D-structure to create an aqueous pore, with alternation of positive charges, which connects the cytoplasmic compartment of the cell to the extracellular medium (Fig. 1B) [4]. This 3D organization exposes specific binding sites and facilitates ion movement. Each of the 4 domains contains 6 α-helical trans-membrane segments (S1-S6) and is connected to the adjacent domain by interdomain linker loops (ID1-2, aa 416-711; ID2-3, aa 940-1200; ID3-4, 1471-

1523) (Fig.1A). The S4 segment (voltage sensor) of each domain contains positively charged amino acids that alternate every 3 or 4 positions. The movement of S4, following voltage variation (depolarization), causes channel opening [4].

The P-loop is a highly conserved pore-forming region between S5 and S6 of each domain. Being inserted in the phospholipid bilayer, the P-loop creates the hole through which sodium ionsACCEPTED cross the cell membrane MANUSCRIPT (Fig. 1A). This region is important in determining sodium ion selectivity and for the conductance/permeation properties especially thanks to the presence of a selectivity filter constituted by aspartic and glutamic acid, lysine and alanine residues (the DEKA ring) [7]. In fact, a single amino acid variation in the P-loop can alter ion selectivity [8].

The region of fast inactivation regulation is located between ID3 and ID4 (see Figure

1A), whereas the intracellular C-terminal tail (aa 1772-2016) is a protein-interacting region.

The correct location of Nav1.5 at the cell membrane is mediated by several cell-trafficking

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proteins including the β-subunits (hβ1) that also play a role in the regulation of the activation, inactivation and recovery from inactivation of the cardiac sodium channel [5].

3. FUNCTION AND VOLTAGE-DEPENDENT ACTIVATION OF NAV1.5

Within cardiac tissue, the Nav1.5 protein generates and propagates the cardiac action potential (CAP) that is responsible for normal heart contraction. The CAP consists of 5 phases lasting about 200-300 milliseconds overall:, rapid depolarization, which is mediated by a rapid influx of sodium ions (induced by Nav1.5 opening, INa) (phase 0); early repolarization (phase 1), which is caused by the transient outward potassium current (IKto) that induces a rapid and brief repolarization of cell membrane; plateau (phase 2), which is maintained by the simultaneous exit of potassium ions (IKur) and the entry of calcium ions

(ICaL); final repolarization (phase 3), which restores the resting potential. Final repolarization results from outward potassium currents: rapid (IKr) and slow (IKs); and phase 4 the resting phase (about -85 mV). In the ventricules, this phase is due to IK1 current (Kir2.1).

The Nav1.5 channel generates phase 0 - that triggers the cardiac CAP. Once a cell is electrically stimulatedACCEPTED by a current from adjacent MANUSCRIPT cells, the cardiac sodium channels open thereby causing a rapid influx of sodium ions into the cell. This movement of ions induces rapid depolarization of the cell membrane from the resting potential (about -85 mV, phase

4) to a positive voltage (about +20 to +45 mV, phase 0). The flow of sodium ions through the cell membrane, responsible for the phase 0 trigger, is induced by the transition of the channel from a closed state (at the resting potential, -85 mV) to an open state. Specifically, the S4 segment moves outward with respect the S1-S2 and S3 segments; it rotates around its own axis and finally tilts with respect to the S4-S5 linker loop. These changes drive the movement of the S4-S5 linker loop that causes the entire S1-S4 block (called 6 ACCEPTED MANUSCRIPT

voltage sensing domain, VSD, Fig. 1A) rotation and enables sodium ions to enter the cell

[9] .

4. FAST INACTIVATION OF NaV1.5

The ion flow across the cell membrane stops when the channel closes. In fact, when depolarization is maintained, the channel enters into a closed non-conducting state [10].

When the membrane is depolarized, the cardiac sodium channel closes thanks to the inactivation process. Nav1.5 is inactivated by two physically distinct inactivation processes: fast inactivation and slow inactivation [11]. The fast inactivation process is coupled to the activation process. In fact, it starts as activation proceeds [12]. This rapid (milliseconds) mechanism of closure of Nav1.5 is driven by the IDIII-IV. Specifically, a highly conserved amino acid triad, consisting in isoleucine-phenylalanine-methionine (IFM; residues 1485-

1487, Fig. 1A), is directly and critically involved in this rapid inactivation. In fact, mutation of the IFM triad with three glutamine residues (IFM→QQQ) abolishes channel inactivation

[13]. The 54 amino acid residues of the ID3-4 linker-loop region are probably involved in modulation of inactivation properties. In fact, mutations/deletions in this loop generally negatively affect Nav1.5 inactivation thereby leading to cardiac channelopathies (i.e.,

Brugada syndromeACCEPTED [BrS] and Long QT syndrome MANUSCRIPT [LQTS]) [14]. Fast inactivation of Nav1.5 occurs via a “ball & chain” mechanism where the “ball”, which occludes the pore, is represented by the interdomain linker loop ID3-4. Thus, ID3-4 interrupts the flow of ions through the channel.

Mutations in the cytosolic interdomain linker loop ID1−2 also accelerate the fast inactivation process, although it is not known if the control of the fast inactivation kinetics mediated by ID1−2 is induced by direct interaction of ID1−2 with the “standard” fast inactivation apparatus (namely ID3−4) or by allosteric modulation of the gate kinetics [15].

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Also the C-terminal tail of Nav1.5, by interacting directly or indirectly with the ID3−4, may be involved in modulation of channel inactivation (Fig. 1A) [16].

5. SLOW INACTIVATION OF Nav1.5

Slow inactivation of Nav1.5 differs greatly from fast inactivation. In fact, unlike fast inactivation, this process is not coupled to activation, it does not start during activation progression, and can be sustained for as long as a few seconds [17]. The sodium channel enters this stable non-conducting state when channel activation (depolarization of membrane) is prolonged. The slow inactivation mechanism is induced by conformational modifications that directly involve the pore structure. The rearrangement of the pore, which blocks sodium ion transport through the membrane, is driven by 3 extracellular domains: the trans-membrane segment S4/D4, the loop between S5-S6/D2 (P-loop/D2) and segment S6/D2. The conformational changes mediated by these regions of the protein induce channel inactivation, whereas the entrance of the pore is unaffected [11]. Mutations in the P-loop regions of Nav1.5 (C373F in P-loop/D1 and H880Q in P-loop/D2) destabilize the slow inactivation states – a finding that implicates also P-loop/D1 in slow inactivation modulation, and confirms the role of P-loop/D2 in slow inactivation of the channel [18]. ACCEPTED MANUSCRIPT

6. POST-TRANSLATION MODIFICATIONS

6.1 Glycosylation

Nav1.5 undergoes several post-translational modifications that modulate its functional behavior. These modifications can modulate both the channel kinetic and targeting of the protein toward the cell membrane.

The sodium channel is a heavily glycosylated protein. Both channel activation and channel inactivation are regulated by this post-translational modification. Studies of the 8 ACCEPTED MANUSCRIPT

role of sialylation in ST3Gal4 (sialyltransferase)-deficient mice demonstrated that the lack of this post-translational modification significantly shifts activation/inactivation to more positive potentials (depolarizing shift) and, in addition, the recovery from fast inactivation is accelerated, whereas targeting of Nav1.5 toward the cell membrane is not affected [19].

6.2 Phosphorylation

Phosphorylation can alter both trafficking and gating of the cardiac sodium channel. In fact, direct phosphorylation of Nav1.5 by protein kinase A (PKA) increases channel density on the cell surface thereby facilitating targeting of the protein probably by masking the endothelial reticulum retention signals [20]. Mutations affecting the PKA phosphorylation consensus site of Nav1.5 induce the loss of PKA-mediated phosphorylation and results in dysregulation of trafficking and signaling (R526H associated to Brugada syndrome) [21].

On the contrary, the phosphorylation of Nav1.5 mediated by protein kinase C (PKC) induces a reduction of INa amplitude. This effect is due to distribution of the sodium channel away from the plasma membrane and to modification of the intracellular trafficking rate [22]. Moreover, Nav1.5 can be phosphorylated by CaMKII. Particularly, S571 phosphorylation reduces channel availability and enhances persistent current thereby increasing the susceptibilityACCEPTED to after depolarizations MANUSCRIPT [23]. S571 and the proximate residues are a critical site for regulation of channel function. In fact, increased levels of S571 phosphorylation have been found in acquired cardiac diseases and, moreover, mutations in S571 neighboring residues (i.e. A572D and Q573E) alter the CaMKII-induced modulation of Nav1.5, and results in altered susceptibility to pro-arrhythmic phenotypes

[23, 24]. In addition, CaMKII-induced phosphorylation of pS516 and pS594 alter channel availability. Indeed, it shifts Na channel availability to a more negative potential and increases the accumulation of the channel in an intermediate inactivated state [25]. A mass spectrometry study of the cardiac sodium channel of ventricular cardiomyocytes 9 ACCEPTED MANUSCRIPT

from adult mice, showed that all the phosphorylated sites were located in the intracellular interdomain linkers ID1−2 (pS457, pS460, pS483, pS484, pS497, pS510, pS524 and/or pS525, pS571, pS664, pS667); except some sites that were located in the N-terminal tail

(pS36, pT38, S39 and/or pS42) [26].

6.3 Ubiquitination

Ubiquitination of Nav1.5 also alters the trafficking of the protein to the cell membrane.

This post-translational modification is implicated in the degradation and/or internalization of membrane proteins, as well as in such non-proteolytic effects as the control of DNA- damage and/or cell signaling [27]. The cardiac sodium channel has a conserved PY-motif in the C-terminal tail that is a binding site for ubiquitin-protein ligases (Nedd4/Nedd4-like).

Nedd4-2-mediated ubiquitination of the protein (mono- or oligo-ubiquitination) reduces the density of Nav1.5 on the cell surface probably by accelerating channel internalization. This modification does not affect channel kinetics (voltage-dependent activation and inactivation) [28]. There is evidence that Nav1.5 is ubiquitinated not only by the Nedd4-2 protein, but also by other ubiquitin-protein ligases, albeit to a lesser extent (Nedd4-1 and

WWP2). WWP2-mediated but not Nedd4-1-mediated ubiquitination decreases channel density at the cell membraneACCEPTED (<30%) [29]. MANUSCRIPT

6.4 Other post-transcriptional modifications

The Nav1.5 protein can also undergo such post-translational modifications as S- nitrosylation. This modification of the channel protein causes the persistence of the late sodium current in cardiomyocytes [30]. A novel Nav1.5 post-transcriptional modification, i.e. arginine methylation (ArgMe), has been identified in arginine residues in the intracellular interdomain linker ID1−2 [31]. This Nav1.5 modification results from protein arginine methyltransferases (PRMT-1,-3,-5) and leads to a mono- or dimethylated arginine

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[32]. The ArgMe modification improves the cell surface migration of Nav1.5. In fact, current density was increased in cells expressing Nav1.5 together with PRMT-3 or PRMT-5, whereas channel kinetics (activation, inactivation, and recovery from inactivation) were unchanged. These findings suggest that the increased current density is due to enhanced channel expression in the plasma membrane [33].

7. INTERACTING PROTEINS

The Nav1.5 protein is involved in a number of electrical (long QT syndrome [LQTS],

Brugada syndrome [BrS], sick sinus syndrome [SSS], and progressive cardiac conduction disease [PCCD]) and structural (dilated cardiomyopathy [DCM]) cardiac diseases. The extensive pathologic implications of Nav1.5 suggest that its function is linked to a complex network of proteins. In fact, Nav1.5 interacts with several proteins implicated in sodium current regulation, cellular trafficking, and post-transductional modifications (Table 3). The principal interacting region is the cytosolic C-terminal tail that binds various proteins

(syntrophin, dystrophin, calmodulin, SAP97, -like, FGF13, and PTP-H1); the intracellular interdomain linker loops are also implicated in these protein interactions [34].

An interesting interaction was that with α-actinin-2 at the level of the ID3-4 that is implicated in the ACCEPTEDtrafficking of Nav1.5 toMANUSCRIPT the plasma membrane, while biophysical properties of the channel were not altered [35].

Other proteins are thought to be Nav1.5 interactors, but their specific interaction location has not yet been identified (table 3). Two distinct pools of Nav1.5 expression have been found at two different subcellular regions of cardiac cells: at the lateral membrane and at the intercalated disk; a third pool at T-tubule level is hypothesized [34]. Each pool of expression differs in their protein-protein interactions with Nav1.5. At the lateral membrane pool, Nav1.5 interacts with the dystrophin/syndtrophin multicomplex, (at the C- terminal tail) that is responsible for sodium channel anchoring to the plasma membrane, 11 ACCEPTED MANUSCRIPT

which, in turn, is required for correct channel function and expression at this membrane, although protein SAP97 protein has been recently implicated in the expression sodium channel at the lateral membrane [36]. Instead, the pool of Nav1.5 at the intercalated disks interacts, in vivo, with ankirin-G that is also linked with the desmosomal protein plakophilin-

2 [37]. Importantly, also the kinetic properties of the sodium channel differed between the two Nav1.5 pools: compared with the pool of Nav at the intercalated disk, the Nav1.5 at the lateral membrane had a smaller current amplitude, a negative shift in the inactivation kinetic and a slower recovery from inactivation [38]. The functions and implications of these distinct pools of expression in cardiac disorders are not yet clear and remain an area of active investigation. A recent study demonstrated interaction between two α-subunits in the endoplasmic reticulum that is important for channel protein migration. In fact, this interaction, involving N-terminus segments, directs the complex (two α-subunits) to the plasma membrane. Mutations in the N-terminal tail of an α-subunit causes a dominant negative effect that leads to BrS [39].

8. CHANNELOPATHIES

The cardiac sodium channel is involved in several inherited diseases collectively known as cardiac channelopathiesACCEPTED (Table 1). These MANUSCRIPT cardiac disorders manifest with different electrocardiographic patterns and sometimes with different clinical features, although they are often characterized by similar arrhythmic events. All can occur in subjects who do not manifest clinical symptoms or a pathological phenotype but they carry the genetic defect present in clinically affected relatives. Specifically, mutations in the cardiac sodium channel-encoding gene (SCN5A), which trigger biophysical alterations in the Nav1.5 protein, are strictly related to the inherited cardiac channelopathies LQTS, BrS, sick sinus syndrome (SSS) and progressive cardiac conduction disease (PCCD). Genetic variations

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in SCN5A are also linked to other inherited disorders characterized by structural alterations of the heart, namely, DCM [40].

8.1 Long QT syndrome

Long QT syndrome is an inherited arrhythmic disorder characterized by abnormal prolongation of the QT interval on the surface 12-lead ECG in patients with a structurally normal heart [41]. Prolongation of the QT interval is associated with delayed ventricular repolarization. The disease features polymorphic ventricular tachycardia (known as torsade de pointes), syncope and sudden cardiac death [42]. Diagnosis is based on a standard 12-lead ECG where the QT is measured and normalized by heart rate (Bazett’s formula: QTc= QT interval/ √ RR, where RR is the interval from the onset of one QRS complex to the onset of the next QRS complex. LQTS is diagnosed in case of an LQTS

Schwartz diagnostic score ≥ 3.5 in the absence of a secondary cause of QT interval prolongation; when genetic screening reveals an unequivocally pathogenic mutation in an

LQTS-related gene; and in case of QTc ≥500 ms in repeated 12-lead ECG and without a secondary cause of QT interval prolongation. LQTS can be diagnosed also with a QTc ranging from 480 ms to 499 ms (in repeated 12-lead ECG) in patients with unexplained syncope in the absenceACCEPTED of secondary cause MANUSCRIPT for prolongation of the QT interval and of pathogenic mutations [43]. Heritable LQTS is classified in 13 subtypes (LQT1-LQT13) each of which is linked to specific gene alteration. LQT1, LQT2 and LQT3 are the most frequent genetic forms, and are related to mutations of cardiac -encoding genes. LQT3 is associated with

SCN5A mutations. LQT3 patients show arrhythmia, and often, also bradycardia.

Arrhythmic events commonly occur during rest or sleep, unlike LQT1 and LQT2 where they manifest during exercise (especially swimming) or emotional stress (sudden noises like the ringing of an alarm clock), respectively [44]. Thousands of LQTS-related mutations 13 ACCEPTED MANUSCRIPT

have been identified in the 13 LQTS-associated genes, and many have been functionally characterized. About 15% of all LQTS-causing mutations are in the SCN5A gene. They are spread throughout the entire protein, and cause an increased in sodium channel activity. LQT3 is triggered by gain-of-function mutations in Nav1.5 that cause increased levels of the depolarizing sodium current (INa). In fact, this surplus of inward depolarizing sodium current, during phase 0 slows the repolarization phase, thus prolonging the CAP.

The biophysical hallmark of LQT3 is increased levels of persistent sodium current that are evoked by a disruption of the fast inactivation gate. LQT3-linked mutations can also accelerate recovery from inactivation, disturb the voltage-dependence of activation, and increase channel availability and sodium current density [3].

Treatment of LQT3 with β-blockers (suggested with Class I consensus recommendation in LQTS patients) may not be very effective, thus a class Ib sodium channel blocker (mexiletine) is usually administered as add-on therapy in these patients

[45]. Recently, ranolazine, which inhibits several ion currents, is now being administered as an antiarrhythmic drug for LQT3. This compound has two advantages. First, in atrial cells it inhibits the peak INa , and second it inhibits the late INa [46]. Thus, ranolazine can prevent and LQT3. Ranolazine inhibits also the IK and ICa,L currents, although it has beenACCEPTED associated with mild sideMANUSCRIPT effects [46]. Consequently, long-term safety monitoring is necessary before this treatment can be routinely applied in the clinical setting

[47]. Despite the advances made in the pharmacological treatment of LQT3, implantation of a cardioverter defibrillator (ICD) is currently the treatment of choice for high-risk patients

[43].

8.2 Brugada syndrome

Brugada syndrome is an autosomal inherited clinically characterized by episodes of syncope, ventricular arrhythmia that can lead to sudden cardiac death, often in 14 ACCEPTED MANUSCRIPT

apparently healthy young subjects (≤ 40 years old) [48]. Symptoms usually appear in the third decade of life and occur more often in men than women. These clinical alterations appear in a structurally normal heart and usually occur during rest or sleep [48]. In some

BrS patients, fever unmasks the Brugada ECG and also triggers fatal arrhythmias [49].

Diagnosis of BrS can be difficult because the ECG changes are dynamic and variable.

Three ECG subtypes have been recognised: Type 1 BrS features a coved-shaped ST elevation in right precordial leads (V1-V3) with a J wave or ST elevation of ≥ 0.2 mV at its peak followed by a negative T wave, with little or no isoelectric separation. Type 2 also has a high take-off ST segment elevation, but the J wave amplitude (≥ 0.2 mV) gives rise to a gradually descending ST-segment elevation that remains ≥1 mV above baseline, followed by a positive or biphasic T-wave that results in a saddleback configuration. Type 3 features right precordial ST elevation of < 0.1 mV of the saddleback type or coved type [50, 51].

Based on these ECG features, BrS is diagnosed in patients with a type I morphology in ≥1 lead among the right precordial leads V1, V2, occurring either spontaneously or challenge test with intravenous administration of Class I antiarrhythmic drugs, [43].

Loss-of-function mutations in the SCN5A gene, which reduce the INa peak, or alter trafficking, and result in the absence of the sodium channel in the plasma membrane, have been related to BrSACCEPTED in approximately 10% toMANUSCRIPT 30% of cases [52]. Patch-clamp analysis has demonstrated that some SCN5A mutations impair cardiac sodium channel gating more severely at higher temperatures, leading to further reduction in the INa peak during fever and increasing arrhythmogenicity in BrS patients [53]

However, it remains to be established whether arrhythmias arise consequent to altered repolarization, abnormal depolarization, or both. The “repolarization hypothesis” is based on transmural dispersion of repolarization between the endocardium and epicardium at the level of the right ventricular (RV) outflow tract [54]. In contrast, the

“depolarization hypothesis” is based on RV conduction slowing, caused by a consistent 15 ACCEPTED MANUSCRIPT

reduction of the depolarizing current that is unable to initiate the action potential, and on mild structural changes that may not become evident with conventional cardiac imaging approaches [51].

Although SCN5A is the gene most often associated with the BrS phenotype, other ion channels (CACNA1C and HERG) and/or accessory subunit-encoding genes have been implicated in BrS [55]. The genes associated with BrS are listed in Table 2.

Treatment of BrS patients is either pharmacological (e.g. isoprotenerol), radiofrequency catheter ablation or ICD implantation, which is the only strategy able to prevent sudden cardiac death [43].

8.3 Sick sinus syndrome

Sick sinus syndrome is a cardiac disorder triggered by dysfunction of the sinus atrial node (SAN) [56]. In SSS, the pacemaking function of SAN is disrupted, which results in alteration of the normal cardiac impulse conduction. Patients manifest a variety of ECG conditions: inappropriate sinus bradycardia, sinus arrest or exit block, a combination of sinoatrial and atrioventricular nodal conduction disturbances, and atrial tachyarrhythmias

[57]. It can also manifest as the coexistence of atrial tachycardia and bradycardia, in which cases is called tachycardiaACCEPTED-bradycardia syndrome. MANUSCRIPT SSS occurs mainly in subjects older than 50 years, although children can also be affected [56]. The symptoms of SSS can include central nervous system alterations (e.g., syncope or pre-syncope) or cardiovascular alterations (e.g. angina pectoris) [57]. Other symptoms are dizziness, errors in judgment, digestive disturbance. During exercise, many affected individuals experience chest pain, difficulty in breathing, or excessive tiredness (fatigue). Once symptoms of SSS appear, they usually worsen with time. However, some people with the condition never experience any related health problems [57].

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SSS can result from genetic or environmental factors. In many cases, the cause is unknown. More commonly, SSS is caused by factors that, inherently (e.g. idiopathic degenerative fibrotic infiltration, amyloidosis and cardiomyopathies) or extrinsically (e.g. hyperkalemia, hypoxia, and pharmacologic agents), alter the structure or function of the

SAN [57]. In children SSS is attributed to congenital abnormalities or sinoatrial nodal artery deficiency [57].

Genetic changes are an uncommon cause of SSS. The condition is usually inherited as an autosomal recessive trait associated, primarily, with compound heterozygous mutations in the SCN5A gene. The recessive inherited form of SSS includes double heterozygous mutations in the SCN5A, LMNA A/C, EMD and GJA5 genes. The disorder may also be associated to mutations in the HCN4 gene; this SSS form is inherited as an autosomal dominant trait. Autosomal dominant inheritance of SSS due to an SCN5A mutation has recently been reported [58].

The most typical biophysical feature of SSS-causative SCN5A mutations is the loss- of-function property that reduces electric coupling between the sinus node and surrounding atria that, in turn, results in conduction block (exit block) [59]. However, some

SCN5A gain-of-function mutations cause SSS by prolonging the sinus node action potential and disruptingACCEPTED its complete repolarization MANUSCRIPT, which in turn, leads to a reduction in sinus rate [60]. In case of severe SSS symptoms can only be treated with permanent pacemaker implantation. In fact, pharmacological treatment is not reliable [61].

8.4 Progressive Cardiac Conduction Disease

Progressive cardiac conduction disease (PCCD) is an electric disorder characterized dysfunctions of the conduction system. It is also called Lev-Lenègre syndrome. The disorder affects the impulse propagation through the His-Purkinje network and causes right/left bundle branch block and enlargement of the ECG QRS complex. These electric 17 ACCEPTED MANUSCRIPT

alterations can lead to syncope and sudden death. Deposition of fibrous tissue has been described in patients with conduction abnormality in ventricles [62]. Pacemaker implantation is required to prevent sudden death. Loss-of-function mutations, due to point mutations or haploinsufficiency in the SCN5A gene, cause a reduction in the availability of channels and are associated to PCCD [63, 64]. In some cases, the same mutation in the

SCN5A gene can lead either to Brugada syndrome or to an isolated cardiac conduction defect or to overlapping phenotypes (e.g. LQTS and conduction defects) [65, 66].

8.5 Inherited Dilated Cardiomyopathy

Dilated cardiomyopathy is a cardiac disorder characterized by dilatation of the cardiac chambers and alteration of ventricle contractility with a consequent reduction in systolic function. Symptomatic DCM patients can manifest heart failure, arrhythmias, and conduction defects [67]. The causes of DCM are various and include coronary artery disease, cardiotoxic drugs, and endocrine diseases; whereas, it is defined idiopathic in

50% of cases. About 30% of idiopathic DCM patients have a family history of the condition, which suggests a familial form of the disorder (FDCM). The condition is inherited as an autosomal dominant/recessive, maternal, or X-linked trait [68], although autosomal dominant transmissionACCEPTED is the most frequent. MANUSCRIPT More than 40 genes have been linked to FDCM, most of which were identified in the cardiac z-disc or in sarcomeric protein- encoding genes [69]. A recent study shows that 1.7% of FDCM cases are associated with

SCN5A mutations (point or truncation mutations [70]) that are implicated in electrical excitability dysfunction. Probably, altered excitability can lead to dilatation of the cardiac chambers [71]. In fact, recent findings suggest that point mutations in VSD (R222Q and

R225W) generate gating pore currents leading to dilatation of cardiac chambers [72]. An alternative DCM-causing mechanism is cardiomyocyte acidification induced by a mutation in SCN5A that causes an H+ leak through the mutated channel [73]. 18 ACCEPTED MANUSCRIPT

9. CONCLUSIONS AND PERSPECTIVES

The Nav1.5 protein is located in the cell membrane and is responsible for the rapid upstroke of the CAP. It is implicated in several electrical and structural cardiac diseases

(LQTS, BrS, SSS, PCCD and DCM); in fact, alteration of its biophysical behavior and its regulation are critical for the onset of these diseases. Moreover, cardiac sodium channelopathies manifest in different clinical phenotypes, and consequently require different pharmacological therapies. Given the important role of this protein in action potential generation and the complex network of interacting proteins, Nav1.5 is probably involved in other cardiac and non-cardiac diseases. Thus, the identification of sodium channel modulators and other genetic natural variants could shed light on Nav1.5-related diseases and help to understand how alterations of this protein can induce diverse clinical phenotypes. Considering that many cardiac sodium channel disorders lead to sudden cardiac death, the study of the biological and electrophysiological properties of Nav1.5, coupled with genetic analysis of SCN5A and the modulator encoding genes could enable risk stratification in patients. In addition. the demonstration of the modulator effect induced by sodium channel-interacting proteins coupled with the electrophysiological characterization of natural variants (in Nav1.5 and its interacting proteins) may be helpful for genetic diagnosisACCEPTED and, probably, in the MANUSCRIPTidentification of pharmacological targets. Lastly, the recent unexpected finding that Nav1.5 is implicated in the invasiveness of breast, colon, prostate, and lung cancer cells [74, 75] indicates that Nav1.5 plays various complex roles, some of which yet to be discovered.

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ACKNOWLEDGEMENTS

We thank Jean Ann Gilder (Scientific Communication srl., Naples, Italy) for writing assistance and Vittorio Lucignano (CEINGE-Biotecnologie Avanzate) for assistance in graphic design. This work was supported by grant PON01_02589 (MICROMAP) and grant

PON02_00677 (BIOGENE) potenziamento lab.8 - 2012 from the Italian Ministry of

University and Research (both to F.S.) and Grant POR Campania FSE 2007/2013

(CAMPUS-Bioframe) from the Regione Campania, Italy (to F.S.).

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ABBREVIATIONS aa, amino acids

BrS, Brugada syndrome

CAP, cardiac action potential

DCM, dilated cardiomyopathy

ECG, electrocardiogram

FDCM, familial dilated cardiomyopathy

HGMD, human genetic mutation database

ICaL, L-type calcium current

ICD, implantable cardioverter defibrillator

IKr, rapid rectifier potassium current

IKs, slow rectifier potassium current

IKto, transient outward potassium current

IKur, ultra-rapid potassium current

INa, sodium current

LQTS, long QT syndrome mV, millivolts

Na, sodium ACCEPTED MANUSCRIPT

PCCD, progressive cardiac conduction diseases

RV, right ventricular

SAN, sinus atrial node

SSS, sick sinus syndrome

VSD, voltage sensing domain

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Figure legend

Figure 1. (A) Schematic representation of the Nav1.5 cardiac sodium channel. IFM is the fast inactivation region represented by isoleucine, phenylalanine and methionine, the grey circle represents the β-subunit interaction region; VSD is the Voltage Sensing Domain; ID1-2, ID2-3, ID3-4 are the interdomain linkers. On the extreme right is a schematic representation of the β-subunit. (B) Schematic tridimensional representation of Nav1.5. The blue, red, yellow and green blocks represent domains 1, 2, 3 and 4, respectively. The grey circles represent the β-subunits. The light blue circle represents the aqueous pore.

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Fig. 1

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Graphical abstract

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Table 1. Cardiac disorders associated to Nav1.5 alterations Nav1.5-related ECG disease Clinical features Gating alterations alterations

Ventricular ↑ persistent Na current tachyarrhythmias (TdP) ↑ channel availability Long QT syndrome QT-interval ventricular fibrillation ↑peak sodium current (LQTS) prolongation syncope density cardiac arrest slowed inactivation

ST-segment elevation in (V1- Brugada syndrome Ventricular arrhythmias V3) ↓ inward Na current (BrS) sudden cardaic death ↑ PQ- and QRS-duration (with normal QT interval)

slow heart rate sinus bradycardia irregular heart sinus arrest/exit block rhythm or Sick sinus syndrome SA and AV nodal ↓ inward Na current pauses>2 sec (SSS) conduction disturbances slow-fast atrial tachyarrhythmias alternation of heart rhythm

right and/or left bundle Progressive cardiac branch block complete conduction defects atrioventricular block ↓ inward Na current QRS -widening (PCCD) syncope sudden death

No unique ECG features Bifid P wave ST segment ACCEPTED MANUSCRIPT depression and T wave inversion Left (or right) Structural defects bundle branch Dilated cardiomyopathy arrhythmias and/or mixed block (DCM) fibrillation* Left axis deviation Premature ventricular complex Ventricular bigeminy

↑ increasing ; ↓ decreasing; TdP, torsade des pointes; SA, sinoatrial; AV, atrioventricular * in presence of SCN5A mutations [71]

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Table 2. Proteins implicated in Brugada syndrome and number of mutations identified

N° mutation described Gene Protein (HGMD)

SCN5A Sodium channel, voltage gated, type V, 366 alpha polypeptide CACNA1C , voltage-dependent, l type, 10 alpha 1c subunit TRPM4 Transient receptor potential cation channel, 9 subfamily M, member 4 CACNB2 Calcium channel, voltage-dependent, beta 2 7 subunit KCNH2 Potassium voltage-gated channel, subfamily 6 H, member 2 (HERG) CACNA2D1 Calcium channel, voltage-dependent, alpha 4 2/delta subunit 1 HCN4 Hyperpolarization activated cyclic 3 nucleotide-gated 4 SCN1bb Sodium channel, voltage-gated, type 1, beta, 3 isoform b ABCC9 ATP-binding cassette, sub-family C 3 (CFTR/MRP), member 9 KCND3 Potassium voltage-gated channel, Shal- 2 related subfamily, member 3 SCN3B Sodium channel, voltage-gated, type III, beta 2 SLMAP Sarcolemma associated protein 2 SCNN1A Sodium channel, non-voltage gated 1, alpha 2 SEMA3A Semaphorin-3A 2 CACNA1ct23 Calcium channel, voltage-dependent, l type, 1 alphaACCEPTED 1c subunit, transcript MANUSCRIPT variant 23 CACNB2i5 Calcium channel, voltage-dependent, beta 2 1 subunit, isoform 5 GPD1L Glycerol-3-phosphate dehydrogenase 1-like 1 KCNE3 Potassium voltage-gated channel, Isk- 1 related family, member 3 KCNJ8 Potassium inwardly-rectifying channel, 1 subfamily J, member 8 PKP2 Plakophilin 2 1 RANGRF RAN guanine nucleotide release factor 1 SCN2B Sodium channel, voltage-gated, type II, beta 1 KCNT1 Potassium channel, subfamily T, member 1 1 KCNJ16 Potassium inwardly-rectifying channel, 1 subfamily J, member 16 KCNB1 Potassium voltage-gated channel, Shab- 1 related subfamily, member 2

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HGMD, Human Gene Mutation Database

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Table 3. Nav1.5-interacting proteins, relative binding domains and functions affected [34]

Protein Mass (kDa) Nav1.5 interacting region Channel regulation

SAP97 100 C-terminus (PDZ domain) Ankyrin-G 480 interdomain D2-3 linker MOG1 20 interdomain D2-3 linker Nav1.5 227 N-terminus Trafficking α-actinin-2 104 interdomain D3-4 linker Calmodulin 56 C-terminus (IQ motif) and interdomain D3-4

Desmoglein -2 122 Not determined Plakophilin-2 97 Not determined FGF proteins 28 C-terminus (aa 1773-1832) 28 14-3-3η interdomain D1-2 linker Gating regulation Caveolin-3 17 Not determined GPD1-L 38 Not determined Telethonin 19 Not determined ZASP 77 Not determined 0

PTPH1 104 C-terminus (PDZ domain) Nedd4-like family 112 C-terminus (PY domain) Post-translational modifications CAMKIIδc 56 interdomain D1-2 linker and via spectrina

Syntrophin 54 C-terminus (PDZ domain) ACCEPTED MANUSCRIPTAdaptation and stabilization

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HIGHLIGHTS

 Nav1.5 is responsible for the initiation of the cardiac action potential

 Post-translational modifications of Nav1.5 regulate its activity and/or trafficking

 Cross-talk with a vocabulary of proteins regulates Nav1.5 functions

 Mutations in Nav1.5 are causes of arrhythmic and structural cardiac diseases

ACCEPTED MANUSCRIPT

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