Cardiac sodium channelopathies
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- Amin, A.S., Asghari-Roodsari, A. & Tan, H.L. Pflugers Arch - Eur J Physiol (2010) 460: 223. doi:10.1007/s00424-009-0761-0
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Cardiac sodium channel are protein complexes that are expressed in the sarcolemma of cardiomyocytes to carry a large inward depolarizing current (INa) during phase 0 of the cardiac action potential. The importance of INa for normal cardiac electrical activity is reflected by the high incidence of arrhythmias in cardiac sodium channelopathies, i.e., arrhythmogenic diseases in patients with mutations in SCN5A, the gene responsible for the pore-forming ion-conducting α-subunit, or in genes that encode the ancillary β-subunits or regulatory proteins of the cardiac sodium channel. While clinical and genetic studies have laid the foundation for our understanding of cardiac sodium channelopathies by establishing links between arrhythmogenic diseases and mutations in genes that encode various subunits of the cardiac sodium channel, biophysical studies (particularly in heterologous expression systems and transgenic mouse models) have provided insights into the mechanisms by which INa dysfunction causes disease in such channelopathies. It is now recognized that mutations that increase INa delay cardiac repolarization, prolong action potential duration, and cause long QT syndrome, while mutations that reduce INa decrease cardiac excitability, reduce electrical conduction velocity, and induce Brugada syndrome, progressive cardiac conduction disease, sick sinus syndrome, or combinations thereof. Recently, mutation-induced INa dysfunction was also linked to dilated cardiomyopathy, atrial fibrillation, and sudden infant death syndrome. This review describes the structure and function of the cardiac sodium channel and its various subunits, summarizes major cardiac sodium channelopathies and the current knowledge concerning their genetic background and underlying molecular mechanisms, and discusses recent advances in the discovery of mutation-specific therapies in the management of these channelopathies.
KeywordsArrhythmia Action potential Cardiac electrophysiology Cardiomyocyte Ion channels
The effects of SCN5A mutations on the normal structure, expression, and function of cardiac sodium channels are routinely investigated by cloning and expression of channel proteins in heterologous systems (e.g., human embryonic kidney cells, Chinese hamster ovary cells, Xenopus oocytes). The effects of mutations in genes encoding the regulatory proteins are studied by co-expression of the mutant protein with the normal sodium channel protein. Additionally, genetically engineered mice carrying mutations in a specific gene of interest are increasingly used to study the effect of mutations in the native environment of the myocyte . These expression models, in conjunction with various experimental techniques (e.g., electron crystallography to study channel structure, polymerase chain reaction to study mRNA expression, Western blot and immunohistochemistry to study protein expression, co-immunoprecipitation to study protein–protein associations, and patch-clamp technique to measure currents) have greatly enhanced our understanding of the role of sodium channel dysfunction in the pathophysiology of cardiac sodium channelopathies. Furthermore, they have provided basic rationale for gene-specific and even mutation-specific approaches in the clinical management of subject with such channelopathies. This review briefly describes the structure and function of the cardiac sodium channel and its modulation by regulatory proteins. Moreover, it discusses the well-recognized cardiac sodium channelopathies and the nature and role of sodium channel dysfunction in the underlying mechanisms of these diseases.
The cardiac sodium channel
Although Nav1.5 is sufficient to generate sodium current in heterologous expression systems, the obtained current is quite different from INa present in isolated cardiomyocytes. This may be due to the absence of ancillary β-subunits and regulatory proteins in heterologous systems (Fig. 2b). So far, four β-subunits are known in the heart (β1 to β4), which are encoded by four genes (SCN1B to SCN4B) [1, 29, 33, 48, 89]. The β-subunits are proteins with an extracellular N terminus, one transmembrane segment, and a cytoplasmic C terminus. They increase the expression of Nav1.5 in the sarcolemma, augment the amplitude of INa, modulate its gating properties, and play crucial roles in the interaction of Nav1.5 proteins to extracellular matrix molecules, cytoplasmic cytoskeleton apparatus, and components of cardiac intercellular junctions (e.g., cadherins, connexins). The β-subunits may also contribute to the preferential localization of Nav1.5 proteins in the intercalated disks. Nav1.5 proteins may also directly interact with several regulatory proteins, including enzymes involved in their glycosylation and phosphorylation (e.g., Ca2+/calmodulin-dependent protein kinase II) [1, 77], adaptor proteins that connect them to the cytoskeleton (e.g., ankyrins) , and proteins that mediate their trafficking from the endoplasmic reticulum (ER) to the sarcolemma (e.g., glycerol-3-phosphate dehydrogenase 1-like protein) . Of note, mutations in genes encoding β-subunits and regulatory proteins are occasionally found in patients with clinical phenotypes similar to arrhythmogenic diseases caused by SCN5A mutations. This reflects the importance of these proteins for the normal functioning of the cardiac sodium channel .
Long QT syndrome type 3
Arrhythmic events in LQT-3 usually occur at rest or during sleep when the heart rate is slow. Accordingly, Isus is larger at slower stimulus frequencies, suggesting that the degree of Isus may be a strong determinant for arrhythmias to occur . This is confirmed by a report of homozygous carriers of an SCN5A mutation who displayed longer QT intervals and more frequent arrhythmias than heterozygous carriers of the same mutation. Consistently, homozygous expression of the mutation in heterologous system caused larger Isus densities than heterozygous expression of the mutation . The definite role of Isus in LQT-3 is further reflected by the effectiveness of drugs that inhibit Isus in the treatment of patients with LQT-3. Such drugs (e.g., ranolazine, mexiletine, flecainide) shorten QT intervals in patients with LQT-3 by preferentially blocking Isus over peak INa [11, 51, 69]. However, this effect may be mutation specific. Mexiletine has been shown to be especially effective in patients with SCN5A mutations that shift inactivation toward more negative potentials (i.e., earlier inactivation) . Moreover, in high concentrations these drugs may also block peak INa, and exert pro-arrhythmic effects by decreasing cardiac excitability and slowing electrical conduction velocity. Finally, beta-blockers (the cornerstone therapy in most long QT syndrome patients, particularly those with LQTS type 1 and, somewhat less so, in LQTS type 2) seem to be less effective in LQT-3 . This may be due to their pro-arrhythmic effect by slowing the heart rate and consequently increasing Isus, which opposes their anti-arrhythmic effect (i.e., preferential block of Isus by direct binding to Nav1.5). The anti-arrhythmic effect of beta-blockers in LQT-3 may also be mutation-specific (e.g., when inactivation occurs at more negative potentials), and limited to some agents (propranolol, carvedilol, but not metoprolol) .
LQTS and mutations in sodium channel regulatory proteins
Three less common types of LQTS are caused by mutations in genes encoding proteins that regulate the expression or function of Nav1.5 proteins. A mutation in SCN4B, encoding the β4-subunit, has been linked to LQTS type 10. When co-expressed heterologously with SCN5A, the mutation shifted the inactivation toward more positive potentials, but did not change the activation. This resulted in increased window currents at membrane potentials corresponding to the phase 3 of the action potential . Mutations in CAV3, encoding caveolin-3, are linked to LQTS type 9 (LQT-9). Caveolin-3 co-localizes and interacts with Nav1.5 proteins at the sarcolemma of cardiomyocytes (Fig. 2b). When co-expressed with SCN5A, the mutant caveolin-3 proteins induce Isus through a yet unknown mechanism . Finally, a mutation in SNTA1 has been linked to LQTS type 12. SNTA1 encodes α1-syntrophin, a cytoplasmic adaptor protein that enables the interaction between Nav1.5, nitric oxide synthase (NOS), and sarcolemmal calcium ATPase (PMCA). PMCA inhibits nitric oxide synthesis by NOS. By inducing nitrosylation of Nav1.5 proteins, nitric oxide decreases channel inactivation and increases Isus densities. The mutation in SNTA1 disrupted the interaction between Nav1.5 and PMCA, and consequently caused increased Nav1.5 nitrosylation and Isus densities .
INa reduction decreases the upstroke velocity of action potential phase 0, and, as a result, slows atrial and ventricular electrical conduction (Fig. 6c). This is often reflected by prolonged atrioventricular and ventricular conduction intervals (PR and QRS intervals, respectively) on the ECGs of BrS patients with an SCN5A mutation (Fig. 6a) . During electrophysiological studies in such patients, electrical conduction is particularly delayed between the His bundle and the ventricles (HV interval prolongation), indicating the importance of INa for the initiation and propagation of action potentials in Purkinje fiber myocytes and the ventricular conduction system . The right-precordial ST segment elevation is less well understood, and explained by two hypotheses. The first hypothesis focuses on the presence of transmural voltage gradients due to heterogeneity in action potential duration between the right ventricular epicardium and endocardium. Indeed, action potential durations are shorter in the epicardium, where the repolarizing transient outward potassium current (Ito) is more prominently expressed. INa reduction would further shorten epicardial action potential durations, and facilitate reentrant excitation waves between depolarized endocardium and prematurely repolarized epicardium . The second hypothesis involves preferential conduction slowing in the right ventricular outflow tract. Regional differences in conduction velocity in the right ventricular epicardium would be aggravated by INa reduction, and trigger the occurrence of epicardial reentrant excitation waves . This hypothesis is supported by the increased prevalence of mild (subclinical) structural abnormalities in the right ventricles of BrS patients (e.g., inflammation, fatty tissue replacement, and fibrosis) . Regardless of the mechanism, since ST segment elevation is equally seen in SCN5A mutation carriers and non-carriers, other genetic variants (e.g., in genes encoding ion channels or regulatory proteins) and exogenous factors (e.g., electrolyte imbalances, hormones, body temperature) may contribute to the pathophysiology of BrS [4, 30, 46, 74].
Arrhythmic events in BrS occur more frequently in men, and at rest or during sleep. Experimental studies suggest that the effects of gender on the BrS phenotype may be due to intrinsic differences in ion channel expression between men and women (e.g., higher Ito densities in men) or due to differences in hormone levels (e.g., higher testosterone levels in men) [7, 74]. The latter is confirmed by the disappearance of typical BrS ECG changes after castration . The occurrence of arrhythmias during sleep may be due to an increased vagal activity and/or decreased sympathetic activity. This is confirmed in BrS patients by right-precordial ST segment elevation following intracoronary injection of acetylcholine, and decreased levels of norepinephrine in the synaptic cleft on positron emission tomography [32, 54]. Moreover, the typical ECG changes and arrhythmias in BrS may also be triggered by fever . Although the mechanism is not fully understood, some SCN5A mutations have been shown to alter the gating properties of cardiac sodium channels in a temperature-dependent manner, e.g., more slow inactivation at higher temperatures [6, 21]. Finally, the BrS ECG changes are reported to worsen during exercise. This may be partially attributed to an enhanced slow inactivation in mutant channels, leading to an accumulation of the mutant channels in the slow inactivation state at fast heart rates. However, other factors (e.g., autonomic nervous system, ion current imbalances) may also play a role .
So far, no mutation-specific therapy is available for BrS. Decreased sarcolemmal expression of mutant Nav1.5 proteins can be restored with cardiac sodium channel blocking drugs (e.g., mexiletine). Such drugs bind to mutant proteins, and act as molecular chaperones to rescue their trafficking from the ER to the sarcolemma . However, it is questionable whether these drugs can be used as therapy, because (once expressed on the sarcolemma) the mutant proteins display arrhythmia-causing gating defects (e.g., Isus) . Moreover, since sodium channel blocking drugs reduce INa, they may aggravate ECG changes or trigger arrhythmias in BrS, and should therefore be avoided [30, 59]. An implantable cardioverter defibrillator and adequate treatment of fever are currently the only effective therapies to prevent sudden death in BrS.
BrS and mutations in sodium channel regulatory proteins
INa reduction in BrS may also be due to mutations in genes encoding β-subunits or regulatory proteins of the cardiac sodium channel. A mutation in SCN1B was found in one family with BrS. When heterologously expressed, the mutation resulted in formation of truncated β1-subunits, which failed to interact with Nav1.5 proteins and to increase INa densities as normal β1-subunits did . A missense mutation in SCN3B was found in one subject with BrS. The mutation reduced INa by disrupting the trafficking of Nav1.5 proteins from the ER to the sarcolemma, and by altering the gating properties (e.g., earlier and faster inactivation) . Finally, a mutation in GPD1-L, encoding glycerol-3-phosphate dehydrogenase 1-like protein (GPD1-L), was found in a family with BrS . Recently, GPD1-L was shown to inhibit the phosphorylation of Nav1.5 proteins at residues where phosphorylation would lead to INa reduction (probably by decreasing the sarcolemmal expression of Nav1.5 proteins). The mutant GPD1-L failed to inhibit the phosphorylation of Nav1.5 proteins, and resulted in reduced INa densities .
Progressive cardiac conduction disease
Whether the age-dependent fibrosis of the conduction system is a primary degenerative process in PCCD, or a physiologic process that is accelerated by INa reduction remains to be investigated. However, the latter is suggested by histological studies, in which fibrosis was found in cardiac biopsies of BrS patients , and animal studies in heterozygous SCN5A knockout mice (50% INa reduction), in which conduction defects and myocardial fibrosis that deteriorated progressively with age were observed . So far, no molecular therapy for PCCD exists and the implantation of a pacemaker is the most effective treatment.
PCCD and mutations in sodium channel regulatory proteins
Two loss-of-function mutations in SCN1B have been identified in patients with PCCD who carried no mutation in SCN5A. One mutation resulted in truncated β1-subunits that failed to interact with Nav1.5 proteins, to increase INa densities and to alter the gating properties as normal β1-subunits did. The other mutation decreased the ability of normal β1-subunits to increase INa densities and to facilitate the activation of the co-expressed sodium channels . Interestingly, normal and mutant β1-subunits were expressed to a higher degree in the Purkinje fibers than in the ventricles. This indicates that INa reduction in Purkinje fiber myocytes may underlie prolongation of PR and QRS intervals, and right or left bundle branch block in PCCD.
DCM is characterized by ventricular dilatation and impaired systolic function, which may proceed into congestive heart failure. Although DCM is a final common pathway of various acquired diseases, up to 50% of cases are reported to be idiopathic (i.e., without any obvious aetiological trigger). Approximately 20% of idiopathic DCM cases display familial prevalence, and have been linked to mutations in various genes that encode proteins involved in the contractile apparatus and the cytoskeleton. Initially, linkage analysis in a large family with DCM mapped the disease locus to a region on the short arm of chromosome 3 (3p22-p25), which harbors the SCN5A gene . Afterwards, a missense mutation in SCN5A was associated with the disease phenotype in this family . Since then, several other missense and truncation mutations in SCN5A have been linked to DCM [25, 53, 56]. Remarkably, most of these mutations are found in patients who display multiple phenotypes, including DCM, sinoatrial node dysfunction, atrial flutter, atrial fibrillation, atrioventricular block, bundle branch block, ventricular tachycardia, and/or ventricular fibrillation. When expressed heterologously, DCM-linked SCN5A mutations usually do not disrupt the sarcolemmal expression of channel proteins, but cause diverse loss-of-function and gain-of-function changes in their gating properties (e.g., earlier or delayed activation, earlier inactivation, disrupted fast inactivation, and increased INaL or window current) [25, 53]. Although it remains unclear how such divergent gating changes lead to DCM, it is speculated that SCN5A mutations in DCM disrupt the interactions between cardiac sodium channels and intracellular (or extracellular) proteins that are essential for normal cardiomyocyte structure and architecture. However, it must be noted that mutations linked to DCM cases with conduction defects often cause INa reduction. This is supported by reduced SCN5A gene transcription and INa densities in a mouse model with DCM that also displayed prolonged conduction parameters . No molecular therapy for DCM exists, and the standard therapy for congestive heart failure is applied.
Sick sinus syndrome
Pacemaker cells in the sinoatrial node reach the voltage range for the sodium window current during action potential phase 4. The small inward window current contributes to the gradual depolarization of the sarcolemma of these cells, and allows spontaneous action potential generation, which underlies cardiac automaticity. In addition to their contribution to cardiac pacemaker activity, sodium channels also play an essential role in the propagation of action potentials from the central area of the sinoatrial node through its peripheral regions to the surrounding atrial muscle [37, 84]. Experimental studies in heterozygous SCN5A knockout mice suggest that the main mechanism of INa reduction to cause SSS indeed involves reduced automaticity, and conduction slowing or blocking of action potentials from the sinoatrial node to the surrounding atrial muscle . Interestingly, LQT-3 patients with SCN5A gain-of-function mutations may also suffer from sinus bradycardia and sinus arrest. Computer simulation models showed that Isus in pacemaker cells may decrease sinus rate and induce sinus arrest by delaying the repolarization and prolonging action potential durations .
AFib is the most prevalent clinically relevant cardiac arrhythmia, and is characterized by a disorganized electrical activation of the atria. It usually affects elderly patients with structural heart disease. However, AFib may also occur in young patients with structurally normal hearts (i.e., lone AFib), and display a familial occurrence. SCN5A mutations were first linked to lone AFib in patients with DCM . Next, a common polymorphism (H558R) in SCN5A was found more often in patients with lone AF than matched controls. Of note, H558R is known to reduce INa in heterologous systems . Subsequently, an SCN5A loss-of-function mutation was identified in a family with lone AFib . It was speculated that INa reduction may predispose to AFib by slowing the electrical conduction velocity. Conduction slowing is an essential determinant for maintaining reentrant excitation waves in the atria. This mechanism is supported by an increased prevalence of lone AFib, accompanied with prolonged atrial conduction intervals (e.g., P wave duration), in BrS patients . Nevertheless, two SCN5A gain-of-function mutations have also been linked to lone AFib. Interestingly, both mutations were found to induce gain-of-function by delaying channel inactivation [38, 43]. In one mutation, larger INa densities corresponded with increased atrial excitability in mutation carriers . The other mutation induced spontaneous action potential formation when expressed in atrial cardiomyocytes . Thus, loss-of-function mutations in SCN5A predispose to AFib by facilitating the maintenance of reentrant excitation waves, while gain-of-function mutations may initiate AFib by increasing atrial excitability.
Although both gain-of-function mutations discussed above did not prolong QT intervals, another SCN5A gain-of-function mutation has recently been described in a family with LQT-3 and lone AFib. The mutation was shown to induce gain-of-function by causing Isus, suggesting that Isus may promote AFib by prolonging action potential duration and triggering EADs. Importantly, through inhibition of Isus, flecainide not only shortened the QT intervals in the affected family members, but also effectively restored the normal sinus rhythm . In contrast, flecainide therapy in patients with AFib due to SCN5A loss-of-function mutations may be contraindicated, because flecainide also blocks the peak INa, and therefore may aggravate INa reduction in these patients.
AFib and mutations in sodium channel regulatory proteins
Recently, mutations in SCN1B and SCN2B (encoding the β1- and β2-subunits of the cardiac sodium channel, respectively) have been identified in patients with lone AFib. Interestingly, the reported patients with AFib and mutations in SCN1B or SCN2B often displayed an ECG pattern suspect for BrS. When heterologously co-expressed with SCN5A, the mutant β1-subunits failed to increase INa as normal β1-subunits did, and caused delayed channel activation. Mutant β2-subunits also induced loss-of-function, but only by delaying channel activation .
Sudden infant death syndrome
SIDS is diagnosed when an infant under the age of 1 year suddenly and unexpectedly dies, and when a detailed review of the clinical history, extensive examination of the death scene, and a complete medical autopsy fail to provide an explanation for the death. Although various exogenous factors are recognized to increase the risk for SIDS (e.g., tobacco and alcohol use by the mother, low socioeconomic status, prone sleeping), higher SIDS rates in some ethnicities suggest a role for genetic factors in the development of SIDS. Initially, this suggestion was reinforced when SIDS was linked to prolonged repolarization intervals as seen in LQTS . This was followed by anecdotal reports linking SIDS to gain-of-function mutations in SCN5A and to mutations that were previously associated with LQT-3 [68, 90]. Subsequently, postmortem genetic testing in a population-based cohort indicated that gain-of-function mutations in SCN5A may be the most prevalent genetic cause of SIDS . SCN5A mutations in SIDS commonly increase Isus, mostly in combination with altered gating properties that result in INa gain-of-function (e.g., earlier activation, delayed inactivation, or increased window current). Interestingly, some SIDS-linked SCN5A mutations display only Isus under acidic conditions, supporting the role of exogenous factors (here acidosis) in the development of SIDS . Less frequently, SCN5A loss-of-function mutations have also been found in infants with SIDS . However, it may be possible that in these patients SIDS represents a severe form of BrS that manifests during early childhood .
SIDS and mutations in sodium channel regulatory proteins
Recently, postmortem genetic testing in population-based cohorts identified mutations in CAV3 and GPD1-L as possible causative genetic factors in the development of SIDS [20, 80]. As mentioned earlier, mutations in CAV3 are linked to LQT-9, and mutations in GDPL-1 are found in patients with BrS [39, 81]. When co-expressed heterologously with SCN5A, SIDS-linked mutations in CAV3 or GDPL-1 exerted similar effects on the cardiac sodium channel as their equivalents in LQT-9 or BrS, respectively (i.e., increased Isus in the presence of mutant CAV3 proteins, and decreased peak INa in the presence of mutant GDPL-1 proteins). These data support the hypothesis that SIDS may be a severe form of LQTS or BrS during infancy.
Overlap syndromes involve overlapping clinical symptoms or arrhythmias of various arrhythmogenic diseases (i.e., sodium channelopathies). The designation is also used when a mutation causes various arrhythmogenic phenotypes in different families or members of one family. Not surprisingly, SCN5A loss-of-function mutations have often been associated with overlapping phenotypes of BrS and PCCD or BrS and SSS [15, 35, 42, 44, 73, 83]. Since the underlying molecular mechanisms of these diseases implicate INa reduction, it is plausible that the clinical phenotype is determined by the degree of INa reduction, the nature of altered gating properties, the presence of exogenous factors (e.g., electrolyte imbalance, drugs, hormones, body temperature, subclinical cardiac structural changes) and co-inherited genetic variants (e.g., polymorphisms in SCN5A or genes encoding regulatory proteins).
More surprisingly, some SCN5A mutations cause symptoms of both LQT-3 (INa gain-of-function) and BrS (INa loss-of-function) in members of one family, or LQT-3 in one family and BrS in another [15, 26, 42]. Although exogenous and genetic factors discussed before may play a role, such mutations are believed to alter gating properties in a manner that results in both INa gain-of-function and loss-of-function. For example, the insertion of an aspartic acid residue at position 1795 of the Nav1.5 protein (1795insD) was the first mutation found in a multigenerational family with ECG signs of both LQT-3 and BrS . While QT interval prolongation was found to be caused by an increased Isus, BrS was shown to be the result of delayed activation, earlier inactivation, and slower recovery from inactivation of the mutant channels . Interestingly, while SCN5A mutations that are linked to LQT-3 often lead to increased Isus levels, mutations linked to overlap syndromes (LQT-3 and BrS with or without SSS) usually also induce earlier inactivation (i.e., inactivation at more negative membrane potentials) and enhanced tonic block by flecainide . Finally, administration of sodium channel blocking drugs may induce typical ECG signs of BrS in patients with LQT-3, limiting the use of such drugs to inhibit Isus and restore delayed repolarization in these patients .
Cardiac sodium channelopathies that are described in this review emphasize the importance of INa for normal cardiac electrical activity. The association of most channelopathies to mutations not only in SCN5A but also in genes encoding the β-subunits or regulatory proteins indicates that, for normal functioning of the cardiac sodium channel, the contribution of various channel subunits is required. Most of our understanding of the molecular mechanisms of cardiac sodium channelopathies originates from experimental studies in heterologous expression system and transgenic mouse models. Although these models have greatly increased our knowledge of the structure and function of the cardiac sodium channel in normal hearts, and the nature and role of INa dysfunction in diseased hearts, they have by and large failed to explain the mechanism of different phenotypes or diseases caused by one single mutation in SCN5A. Since the native environment of cardiomyocytes is absent, the effects of intracellular and extracellular molecules on the function of cardiac sodium channels are greatly lost in heterologous expression models. Although this shortcoming is remedied in transgenic mouse models, the absence of environmental factors and co-inherited genetic variants (e.g., polymorphisms, mutations in non-coding genetic regions) in mouse models remains a limitation. These limitations compel careful interpretation of the experimental data and their translation into the patient phenotypes, and make it clear that future research is needed to design more appropriate expression systems (e.g., cardiomyocytes derived from induced pluripotent stem cells). Future research is also needed to discover novel gene-specific and mutation-specific pharmacological therapies in the management of cardiac sodium channelopathies. Currently, molecular therapy for cardiac sodium channelopathies due to INa loss-of-function is lacking, and the efficiency of sodium channel blocking drugs to restore QT intervals in patients with LQT-3 seems to be limited to mutations that induce earlier inactivation of the sodium channels.
Conflict of interest
The authors have declared that no conflict of interest exists.
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