Cardiac Sodium Channel Nav1.5 Mutations and Cardiac Arrhythmia

Abstract

As a major cardiac voltage-gated sodium channel isoform in the heart, Nav1.5 channel is essential for the cardiac action potential initiation and the subsequent propagation throughout the heart. Mutations of Nav1.5 have been linked to a variety of cardiac disease such as long QT syndrome (LQTs), Brugada syndrome, cardiac conduction defect, atrial fibrillation and dilated cardiomyopathy. Mutagenesis approach and heterologous expression systems are most frequently used to study the function of this channel. This review is primarily focused on recent findings on Nav1.5 mutations that are associated with type 3 long QT syndrome (LQT3) in particular. Understanding the functional changes of the Nav1.5 mutation may offer critical insight into the mechanism of long QT3 syndrome. In addition, this review will provide the updated information on the current progress of using various experimental model systems to study primarily the long QT3 syndrome.

Structure and physiological function of voltage-gated sodium channels

Voltage-gated sodium (Nav) channels initiate action potential depolarization and are responsible for propagating the action potential throughout the heart [43]. These channels are composed of a single pore forming α subunit with molecular weight 33–36 kD and may interacts with ancillary β subunits [7]. The α subunit consists of four structurally homologous transmembrane domains designated DI–DIV [46]. Each domain is composed of six putative transmembrane segments, which is named as S1–S6. The extracellular linker region between segment 5 and 6 within each domain joins together forming the channel pore referred to as “P loop” or “P segment”, which controls ion selectivity and permeation (Figure 1). Site-directed mutagenesis approach helped to identify the functional regions on S4 of the sodium channel [49]. Positively charged residues such as arginine or lysine at every third position (with mostly nonpolar residues intervening between these basic residues) on S4 segments are voltage sensors, which can lead to the fast activation of the sodium channel when the membrane potential is depolarized. The α subunit itself can form a functional channel, which is able to conduct ion and performs voltage-dependent gating process.

Figure 1.

Schematic linear representation of the structure of voltage-gated sodium channel. This figure is modified from the review article by Yu FH et al. [60]. The putative inactivation motif on the intracellular linker between DIII and DIV are indicated. The voltage sensor on segment 4 of each domain is marked with “+” and highlighted in grey. Extracellular loops between segments 5 and 6 within each domain forms the channel pore region. TTX sensitivity site is located in the extracellular loop of DI.

Upon activation, the channel is unable to open again due to the channel gating process called “inactivation”. This process varies from milliseconds (steady-state fast inactivation) to a sustained time up to a few seconds (slow inactivation). Inactivation is the process that causes the termination of the current flow [19]. Molecular biology and advanced biochemistry techniques allow us to know certain regions of the sodium channel that are responsible for channel fast inactivation. These regions are: the IFM motif with three hydrophobic amino acids Ile-Phe-Met [58] in the segment between transmembrane domain III and IV (DIII, DIV), the intracellular linker between S4 and S5 of DIII and DIV, the P loop, and the C-terminal domain of the channel [15].

As mentioned above, sodium channel also consists of auxiliary β subunits. So far, there are four sodium channel β subunits (β₁ to β₄) that have been identified. These four subunits can be divided into two groups. One group contains β₁ (encoded by SCN1B, localized in brain neuronal tissue, skeletal muscle and cardiac tissue [31]), and β₃ (encoded by SCN3B, localized primarily in neuronal tissue [36]; while the other group contains β₂ and β₄. β₁ and β₃ are very similar in amino acid sequence and they are noncovalently linked with α subunits [21, 36]. Studies indicates that the β subunit interacts with DI and DIV of the α subunit [32]. Co-expression of β₁ subunit with α subunit modulates channel gating kinetics [32]. It has been shown that co-expression of the β₁ subunit with the neuronal or skeletal muscle α subunit in Xenopus oocytes increases cell surface channel expression. Further, channel inactivation and activation rates were both altered. Channel inactivation curve was shifted to more negative potentials when β₁ subunit was co-expressed with the cardiac sodium channel α subunit [31, 38]. Lori Isom’s group studied the expression of α and β subunits in heart tissue, and identified Nav1.1 α subunit, Nav1.5 α subunit, β₁, and β₂. Yet other groups have indentified the existence of Nav1.1, Nav1.3, Nav1.6 and Nav1.5 in cardiomyocytes [29, 30]. β₃ has also been detected in the heart [12, 14, 36]. The developmental time course of β₂ subunit expression suggests that it is at detectable levels from postnatal day 15. Both Nav1.5 and Nav1.1 were shown to be co-expressed with β₁ and β₂ subunits [12]. In cardiomyocytes, it was shown that, although the α subunit associates with both β₁ and β₂ subunits, only the β₁ subunit has a modulatory effect on the electrophysiological properties of Nav1.5. The β₂ subunit seems to have no detectable effects on electrophysiological properties of cardiac sodium channels, suggesting that effects of β₂ in the heart in vivo may involve cell adhesion and cytoskeletal communication instead of modulating channel gating process [12].

So far, there are nine subtypes of voltage-gated sodium channels namely Nav1.1 to Nav1.9, and they are encoded by different genes [16]. Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are found in neurons. Nav1.1, Nav1.2 and Nav1.6 are found in the brain. Nav1.4 is found highly expressed in the skeletal muscle. In cardiac tissue, the predominant form of sodium channel is Nav1.5, which is encoded by SCN5A gene. Mutations of this gene have been linked to many types of cardiac arrhythmia such as long QT3 syndrome, atrial fibrillation, sick sinus node syndrome, Brugada syndrome, conduction defect, and dilated cardiomyopathy [5, 6, 11, 34, 47, 53, 54].

Cardiac sodium channel Nav1.5 tissue distribution

As mentioned above, the major cardiac sodium channel α subunit in the heart is Nav1.5 [60]. The α subunit is encoded by the SCN5A gene. Most mutations that affect the cardiac sodium channel are located on this gene. Cardiomyocytes also express neuronal Nav channel isoforms, such as Nav1.1, Nav1.3, and 1.6, but the location of these channels are mainly located on T-tubules [30]. The Nav1.5 isoform was found at high levels and is the major sodium channel isoform in adult rat heart [43]. Nav1.5 isoform is detectable in embryonic, neonatal skeletal muscle and denervated skeletal muscle [25]. However, it is not detectable in adult skeletal muscle. Like other types of voltage-gated sodium channels, these channels contain a principle α subunit and one or more auxiliary β subunits. The α subunit is the pore forming subunit, which can form a functional channel when it is expressed alone in the heterologous expression system. When expressed, this large α subunit accounts for the defining properties of voltage-gated sodium channels. It contains characteristic toxin-binding sites, channel pore, gates, and voltage sensors.

The tissue distribution of the cardiac sodium channel was demonstrated by both immunohistochemistry and conventional RT-PCR experiments. It was found that these channels are localized not only on the cardiomyocyte surface, but also on T-tubules. Later on, it was also demonstrated that cardiac sodium channels are concentrated at the terminal of the intercalated discs [9, 30], which separate adjacent cells and contain gap junctions that are responsible for propagating the cardiac action potential. The specific distribution of Nav1.5 as compared to the neuronal forms of sodium channel in the heart suggest that it may be crucial for action potential initiation while the action potential propagates from one cell to the other within the heart. However, the specialized distribution pattern of neuronal forms of sodium channels within the heart suggests that they are responsible for conducting action potentials inside of the myocyte to activate the contractile machinery [10].

Function of Nav1.5 on long QT3 syndromes

In the patient population that has been identified to carry LQTs, about 50% was associated with mutations on channels or associated proteins. Since the first identification of genetic mutations that cause LQTs in 1991, mutations have been discovered in 12 different genes. The classification of the inherited LQTs was based on the chronological order in which the location of the mutation was identified. The first SCN5A mutation was identified to be associated with LQT3 in 1995 by Wang et al [54]. Sodium channel mutations account for about 7% of the total identified LQTs-causing mutations. The typical ECG study for LQT3 indicates the long ST-segment interval before the onset of a late prominent T wave, which is the characteristic of LQT3 phenotype. Mutations in the alpha subunit of cardiac sodium channel have been identified and were linked to a series of other cardiac diseases such as Brugada syndrome, sick sinus node disease, cardiac conduction defect, dilated cardiomyopathy, and atrial fibrillation [40, 47, 48, 50].

LQT3 disease-causing gene was mapped to 3p21–24 by Mark Keating’s group using “candidate gene approach” [24]. Three amino acid KPQ deletions at position 1505–1507 of the α subunit in affected members of two distinct families with LQTs history were found by this group and this triple amino acid deletion is the most extensively characterized. This three amino acid in-frame deletion Lys-1505, Pro-1506, Gln-1507 was predicted to be in the cytoplasmic linker between DIII and DIV. The presence of exactly identical deletions in two unrelated families with LQT3 history provide strong evidence that SCN5A mutation is the most likely cause of LQT3. In 1995, Bennett et al. reported biophysical properties of this mutant channel. They showed that fast inactivation of the mutant channel is delayed since time constants for current decay were decreased in the ΔKPQ mutant (WT: τ _fast = 1.47 ± 0.11 ms, τ _slow = 8.59 ± 0.71 ms; ΔKPQ: τ _fast = 0.98 ± 0.07 ms, τ _slow = 5.40 ± 0.55 ms, p < 0.01; data are presented as means ± s.e.m.) not increased as was predicted. However, unlike WT channels, there are abnormal sustained sodium current (I_sus) in ΔKPQ expressing cells. The I_sus (~ 5% of the peak inward current at −20 mV) was shown not to decay within a 200 ms depolarization pulse [4, 52]. This sustained current, also called late I_Na was also found in a large number of other LQT3 related sodium channel mutations [2]. Single channel recordings indicated that ΔKPQ channels present multiple intermittent reopenings, which induces an I_sus or delayed inactivation. Beside phenotypical sodium channel changes which are indicated by this first identified ΔKPQ sodium channel mutation, other gain-of-function electrophysiological changes including left-ward shift of the activation curve, increase of window current, faster recovery from inactivation and ramp current are also identified to be correlated with LQT3 syndrome [3, 20].

Voltage-gated sodium channels underline the rapid depolarization phase in ventricular cardiomyocytes and also conduct a small portion of current during the plateau phase of the action potential [1]. Therefore, a subtle abnormality of sodium channel function such as delayed sodium channel inactivation, or altered voltage dependence of channel inactivation could delay the cardiac repolarization phase. As a consequence, sodium channel functional defects can cause small increase in net inward sodium current and lead to QT interval prolongation and arrhythmia. Typically, the SCN5A mutations that are correlated with LQT3 are gain-of-function mutations (Table 1). Studies have demonstrated that gain-of-function mutations disturb the delicate balance between outward and inward currents which are involved in the plateau phase of the action potential repolarization process. In the presence of APD prolongation, there is a propensity for cardiomyocytes to develop early afterdepolarizations, which can trigger TdP. Under normal conditions, during the plateau phase nearly 99% of the channels become inactivated and transitioned into a non-conducting state while the remaining channels stay in the open activated state, sustaining a small window-current that contributes to the plateau phase. Therefore, mutations that disrupt the inactivation process will dramatically enhance the window-current and lead to the action potential prolongation [4].

Table 1.

List of representative SCN5A mutations that are associated with LQT3 syndrome.

Mutation	Locus in Nav1.5 protein	Biophysical consequences	References
ΔKPQ1505–1507	in the DIII–IV linker	↑late INa, defect in inactivation, ↑rate of recovery from inactivation	Ref [8, 54]
F1473C	in the DIII–DIV linker	↑late INa, depolarizing shift of the steady state inactivation, speeds the recovery from fast inactivation, uarr; ramp current	Ref [3]
F1473S	in the DIII–DIV linker	↑late INa, depolarizing shift of the steady state inactivation	Ref [44]
ΔQKP1507–1509	in the DIII–DIV linker	↑late INa, depolarizing shift of the steady state activation	Ref [26]
I1768V	in DIVS6 near the C-terminal end	↑speed of recovery from inactivation, and less slow inactivation	Ref [17]
V411M	in DIS6	↑late INa, depolarizing shift of the steady state activation	Ref [20]
L619F	in the DI-DII linker	↑late INa, depolarizing shift of the steady state inactivation	Ref [56]

Approaches that have been and are currently used to study mutation-induced LQTs including LQT3 syndrome

Heterologous expression system

Early studies on voltage-gated sodium channel were performed in vitro using Xenopus oocytes as a heterologous expression system [57, 58]. These studies offered detailed fundamental characterizations of the voltage-gated sodium channel. Human embryonic kidney (HEK) cells such as HEK293 cells are most frequently used to study functional changes following SCN5A mutations [3]. Other HEK cells such tSA cells and Chinese hamster ovary (CHO) cells are also used [33, 41] as heterologous expression systems. Together, these varieties of expression systems offer a platform to study the physiological function of the sodium channel and pathophysiological changes following gene mutations. However, the advantage of these systems is limited due to the fact that the cellular signaling component might be different from the cardiomyocyte.

Mouse model of LQT3

With the development of transgenic animal technique, experimental mouse models are emerging as a useful tool to study the LQT3 syndrome in vivo (Table 2). Transgenic mouse models carrying ΔKPQ and N1325S mutants were made [18, 39, 59]. In vivo studies from these mice indicated the presence of prolonged QT interval, spontaneous ventricular tachycardia and ventricular fibrillation. In vitro electrophysiology studies indicated that cardiomyocytes from these mice have prolonged APD, increased I_sus, and early afterdepolarizations [13, 42, 51]. These animal models provide an opportunity to study the underlying mechanism of LQT3. However, limitation of the mouse model must be taken into account since there are significant electrophysiological differences between mouse hearts and human hearts [37]. Based on this, one must be very cautious while interpreting data generated using mouse model.

Table 2.

Representative mouse models of SCN5A related LQT3 syndrome.

Model	Locus of the mutation	Genetic approach	Biophysical consequences	References
WT/Δ1505–1507KPQ	intracellular loop between DIII and DIV of Nav1.5	knock-in	↑late INa,	Ref [13, 39, 40]
WT/N1325S	intracellular region between S4–S5 of DIII of the Nav1.5	transgenic	↑late INa	Ref [51]
WT/1798insD	C-terminal domain	knock-in	↑late INa, ↓INa	Ref [42]

Computational approach

So far, a number of studies have used computational approach to simulate the action potential profile changes subsequent to Nav1.5 channel mutations and drug application [23, 55].The predicted computational analysis method allow to explore the consequences of disease-causing mutations on cellular electrical activity. One is beneficial not only from modeling the action potential profile itself but also from pacing the cell with different frequency and getting to know cellular activity changes as a consequence of gene mutation or drug application based on computational approach. Therefore, mechanistic insights on mutations can be obtained. Although this approach is widely used, yet ultimate changes of action potential profile are still hypothetical. Other approaches such as using primary myocytes are indeed needed to be explored.

Neonatal cardiomyocyte expression model

Recently, neonatal mouse cardiomyocytes have been used as a mammalian expression system to study ion channel mutations found in patients with LQTs [28]. In this study, fresh isolated cardiomyocytes from neonatal mouses were transiently transfected with the cDNA encoding the channel of interest using Amaxa Nucleotranfection kit (Lonza Inc.). The advantage of using neonatal cardiomyocytes as an expression system is that these cells have essential signaling components, which may play very crucial role in regulating ion channels. Although this approach has not been applied to study the sodium channel mutation that causes LQT3, this could be one of the future experimental approaches.

Recently, we have extended our study on SCN5A mutations using neonatal rat cardiomyocytes which express a mutant SCN5A cDNA. The mutation was previously identified in human patients. Ex vivo gene transfection method has been used by transfecting the gene using Nucleofection transfection kit. Using this approach, we have studied the functional consequence of a mutation on intracellular loop III and found that this mutation not only causes a series of gain-of-function changes in electrophysiological properties and gating process, but also leads to a significant prolongation of the action potential duration (unpublished data). Spontaneous early after depolarizations were also found (unpublished data). Although the exact copy number of the plasmids that were introduced into myocytes cannot be determined, this approach offers us a new strategy to study electrophysiological properties of the cardiac sodium channel using primary cells.

Future direction of modeling cardiac LQT3 syndrome with pluripotent stem cells

Itzhaki et al and Moretti et al established the most recent new paradigm for in vitro disease modeling of LQT1 and LQT2 syndromes [22, 35]. In their studies, induced pluoripotent stem cells (iPS cells) from patients suffering from the disease with characteristic of LQTs were differentiated into cardiomyocytes. This new advancement offers a new platform and strategy for mechanistically studying the channel mutation. In addition, it is useful for testing therapeutic compounds. In the future, iPS cell lines from the LQT3 patients can be used to differentiate into cardiomyocytes, and the pathophysiological changes that are caused by a specific mutation can be potentially explored. Therapeutic compounds can be tested on LQT3-based cardiomyocytes. Therefore, mutation-specific and individualized therapy can be potentially established.

Conclusions

This review summarizes the current understanding of sodium channel and focus particularly on their physiological and pathophysiological functions in cardiac arrhythmia. Various approaches to study the LQTs-related channel mutations have been discussed in this review. We have used a strategy to characterize the LQT3 syndrome disease-causing SCN5A mutations in neonatal rat cardiomyocytes. Functional changes of the action potential profile such as action potential duration prolongation and the onset of spontaneous early afterdepolarizations as a consequence of certain sodium channel mutations were identified. Therefore, our approach using neonatal rat cardiomyocytes to study the LQT3-related sodium channel mutations is a useful and effective strategy for analyzing the physiological phenotypes of various sodium channel mutations.