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Inherited Sodium Channelopathies A Continuum of Channel Dysfunction
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Inherited Sodium Channelopathies A Continuum of Channel Dysfunction Prakash C. Viswanathan*, and Jeffrey R. Balser Voltage-gated sodium channels are transmembrane proteins that produce the ionic current responsible for the rising phase of the cardiac action potential and play a fundamental role in the initiation, propagation, and maintenance of normal cardiac rhythm. Inherited mutations in SCN5A, the gene encoding the pore-forming subunit of the cardiac Na+ channel, have been associated with distinct cardiac rhythm syndromes: the congenital long QT syndrome, Brugada syndrome, and isolated conduction disease. Electrophysiologic characterization of heterologously expressed mutant Na+ channels have revealed gating defects that, in many cases, can explain the distinct phenotype associated with the rhythm disorder. However, recent studies have revealed significant overlap between aberrant rhythm phenotypes, and single mutations have been identified that evoke multiple rhythm disorders with common gating lesions. These new insights enhance understanding of the structure-function relationships of voltage-gated Na+ channels, and also highlight the complexities involved in linking single mutations, ion-channel behavior, and cardiac rhythm. (Trends Cardiovasc Med 2004;14:28–35) n 2004, Elsevier Inc.
Voltage-gated sodium (Na+) channels are transmembrane proteins that are responsible for the rapid upstroke of the action potential (AP) in nerve and muscle fibers (Balser 2001, Catterall 2000). In the heart, Na+ channels play a major role in the
Prakash C. Viswanathan is at the Department of Anesthesiology, Vanderbilt University, Nashville, Tennessee, USA. Jeffrey R. Balser is at the Departments of Anesthesiology and Pharmacology, Vanderbilt University, Nashville, Tennessee, USA. *Address correspondence to: Prakash C. Viswanathan, PhD, Room 560, Preston Research Building, Vanderbilt University School of Medicine, 2220 Pierce Avenue, Nashville, TN 37232-6602, USA. Tel.: (+1) 615-936-2407; fax: (+1) 615-936-0456; e-mail:[email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter
initiation, propagation, and maintenance of normal cardiac rhythm. Inherited mutations in the family of genes that encode voltage-gated Na+ channels poreforming subunits (SCNXA) underlie a wide spectrum of neurologic, musculoskeletal, and cardiovascular disorders. Mutations in SCN5A, the cardiac Na+ channel gene, are known to evoke multiple life-threatening disorders of cardiac rhythm that range from tachyarrhythmias to bradyarrhythmias that often require pacemaker implantation (Tan et al. 2003). Although patients with SCN5A mutations linked to either the long QT or Brugada syndromes may experience sudden, life-threatening arrhythmias, patients with isolated conduction disease exhibit heart rate slowing (bradycardia) that manifests clinically as syncope, or perhaps only as ‘‘lightheadedness.’’
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Na+ channels are dynamic molecules that undergo rapid structural rearrangements in response to changes in the transmembrane electrical field, a process known as gating (Figure 1) (Hodgkin and Huxley 1952). Upon membrane depolarization, Na+ channels rapidly undergo conformational changes that lead to opening of the channel pore to allow for Na+ influx, a process termed ‘‘activation.’’ Simultaneously, depolarization triggers initiation of ‘‘fast inactivation’’ (Ifast) that terminates Na+ influx. Inactivation differs qualitatively from channel closure in that inactivated channels do not normally open unless the membrane potential is hyperpolarized, often for a sustained period. In addition, Na+ channels may inactivate without ever opening (so-called ‘‘closed-state’’ inactivation) (Horn et al. 1981). With prolonged depolarizations, Na+ channels progressively enter ‘‘slow inactivated’’ states (Islow) with diverse lifetimes ranging from hundreds of milliseconds to many seconds (Cummins and Sigworth 1996, Rudy 1978). Slow inactivation reduces cellular excitability, particularly in pathophysiologic conditions associated with prolonged membrane depolarization, such as epilepsy (Spampanato et al. 2001), neuromuscular diseases (Cannon 1996), or cardiac arrhythmias (Veldkamp et al. 2000). It is increasingly clear that a great many single amino acid substitutions within the SCN5A coding region can evoke a broad spectrum of cardiac rhythm behavior by modulating these gating processes. At the same time, common sequence variants (‘‘polymorphisms’’) in the Na+ channel gene have also been implicated as risk factors in cardiac diseases (Viswanathan et al. 2003), as well as determinants of drug sensitivity (Splawski et al. 2002). Recent
Figure 1. Simplified scheme of the Na+ channel gating conformational changes in response to membrane depolarization. Activation and inactivation occur nearly simultaneously, so that channels sometimes reach the fast/-inactivated state (Ifast) without opening (closed-state inactivation). Sustained depolarization induces channels to occupy stable, slow, inactivated states (Islow).
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studies have shown that polymorphisms in the Na+ channel gene can confer enhanced drug sensitivity, promoting arrhythmias (Splawski et al. 2002), or even modulate the biophysical effects of disease-causing mutations (Viswanathan et al. 2003). Functional studies of mutations associated with cardiac diseases have provided a wealth of information that highlights the exquisite sensitivity of cardiac rhythm to Na+ channel function. This article reviews some of the recent findings that link multiple gating lesions to distinct cardiac disorders, to demonstrate the complex relationship between Na+ channel function and cardiac rhythm.
Long QT Syndrome: Gain of Function Mutations
The long QT syndrome, as the name suggests, manifests as prolongation of the QT interval as reflected in the bodysurface electrocardiogram (ECG) (Wang et al. 1995). Patients with prolonged QT intervals are predisposed to ventricular tachyarrhythmias caused by unstable repolarization, often leading to sudden cardiac death. Multiple genes have been associated with the inherited form of the long QT syndrome. Mutations in the cardiac Na+ channel linked to autosomal-dominant inheritance (LQT3) disrupt fast inactivation and thereby allow for sustained channel opening (Tan et al. 2003). This evokes a small but persistent Na+ current (a gain of channel function) during the AP plateau that delays myocyte repolarization, evokes ECG QT interval prolongation, and predisposes patients to polymorphic ventricular tachycardia (‘‘torsade de pointes’’). Figure 2A shows Na+ currents recorded from normal and mutant Na+ channels expressed in tSA-201 cells during depolarization. The mutation induces a sustained component of inward current (f1.5% of the peak inward current) throughout the depolarization period. In many cases, the development of arrhythmogenic episodes correlates with bradycardia during sleep or relaxation. Surprisingly, the size of the sustained inward current during the AP plateau is extremely small (f0.5% – 2%) compared with the peak inward Na+ current. Nonetheless, the role of this miniscule current in prolonging repolarization and generating cardiac arrhythmias has been val-
idated in quantitative models of the AP (Clancy and Rudy 1999). The first reported LQT3 mutation was a deletion of three residues in the III-IV linker of Na+ channel (1505 – 1507 DKPQ), a cytoplasmic channel domain that has been implicated in fast inactivation of the Na+ channel (Wang et al. 1995). Although expression of DKPQ mutant channels in heterologous expression systems enabled the initial characterization of altered channel kinetics, more recent studies (Nuyens et al. 2001) using genetically altered mice lacking the same triplet of residues revealed ECG features of LQT3 syndrome, and a predisposition to ventricular tachyarrhythmias in response to sudden rate accelerations. Mutations linked to LQT3 have been primarily localized to domains III and IV, the cytoplasmic linker connecting these two domains, and the C terminus of the Na+ channel. The highly conserved acidic domain in the C terminus appears to be a ‘‘hot spot’’ for LQT3 mutations (Wei et al. 1999). Although two chargealtering LQT3 mutations in this region (E1784K, 1795insD) evoke a small, persistent current similar to DKPQ and other LQT3 mutants, a third charge deletion (D1790G) produces both a small plateau and alters the voltage dependence of inactivation in a proarrhythmic manner (Alings and Wilde 1999). Although most of the mutations associated with LQT3 produce a sustained inward plateau current, some of the mutations that are causally linked to LQT3 have exhibited other gating disorders that also led to prolongation of the QT interval. These include faster recovery from inactivation and accompanying shifts in the voltage dependence of activation and inactivation (Abriel et al. 2001, Rivolta et al. 2002). Because a very delicate balance of inward and outward currents maintains the plateau of the AP, these subtle shifts in the balance of currents during repolarization may provoke QT prolongation and torsade de pointes.
Brugada Syndrome: Loss of Function Mutations
Another group of SCN5A mutations produces exceptional risk for idiopathic ventricular fibrillation with a high mortality rate in the absence of QT interval prolongation or structural heart disease (Bru-
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Figure 2. A single amino-acid insertion in the C terminus of the Na+ channel modifies two distinct gating processes. (A) The 1795insD mutation induces a sustained component of inward current, consistent with LQT3 phenotype. The inset shows tetrodotoxin (TTX)-sensitive difference current, revealing (arrows) a sustained plateau current for the mutant (f1.5% of the peak inward current). (B) Intermediate kinetic component of inactivation (slow inactivation) as evaluated using the protocol shown in the inset. 1795insD enhanced the magnitude of the slow component, consistent with loss of Na+ channel function and Brugada phenotype. WT, wild type. (Reprinted with permission from Veldkamp MW, Viswanathan PC, Bezzina C, et al.: 2000. Two distinct congenital arrhythmias evoked by a multidysfunctional Na(+) channel. Circ Res 86:E91 – E97. Copyright 2000, Lippincott, Williams and Wilkins.)
gada and Brugada 1992). The clinical disorder, known as ‘‘Brugada syndrome,’’ is usually characterized by ST segment elevation in the right precordial leads (V1 – V3), right bundle branch block, left axis deviation, and an increased HV interval (indicative of conduction disturbances). Multiple mutations have been identified and linked to Brugada syndrome; however, unlike LQT3, Brugada syndrome mutations are seen in virtually all regions of the Na+ channel (Tan et al. 2003). Although some of the Brugada syndrome mutations render the Na + channel entirely nonfunctional, many of the mutations alter Na+ channel gating function in a manner that reduces the Na+ current, in contrast to LQT3 mutants
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that invariably cause a gain of Na+ channel function. The cellular basis for the Brugada syndrome is thought to involve an outward shift of net transmembrane current at the end of phase 1 of the ventricular epicardial AP (Alings and Wilde 1999). The transient outward potassium current, Ito—which plays a role in determining the spike and dome morphology of the cardiac AP (during phase 1)—is more prominent in the epicardium than in the endocardium, particularly in the right ventricular epicardium. An increase in outward current at the end of phase 1 could accentuate this AP ‘‘notch’’ and lead to an all-or-none repolarization at the end of phase 1, causing a loss of AP
dome and a marked reduction in AP duration (Figure 3C). Hence, mutations of the Na+ channel that reduce the inward Na+ current could lead to an outward shift of the net transmembrane current, leading to an all-or-none repolarization and premature shortening of the epicardial AP. Because Ito is small in the endocardium, early repolarization and AP shortening due to reduced Na+ current is probably not observed in this region. As such, heterogeneous loss of the AP dome between the epicardium and endocardium creates a transmural voltage gradient responsible for elevation of the ST segment characteristic of Brugada syndrome. Computational studies (Tan et al. 2001) using a mathematical
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Figure 3. Balanced gating effects resulting from G514C mutation. (A) The voltage dependence of channel inactivation assessed using the protocol shown in the inset. G514C mutation shifts the voltage dependence of inactivation by +7 mV. This would increase the number of channels available to open at any given potential (a gain of function). (B) The voltage dependence of channel activation as evaluated using the protocol shown in the inset. G514C caused a +10 mV shift in the activation curve, suggesting that channels are less likely to open at any given membrane potential (a loss of function). (C) Simulated endocardial and epicardial action potentials obtained with use of the Luo-Rudy cable model. Wildtype (WT) action potentials, with arrow indicating the epicardial ‘‘notch’’ due to prominent ITo potassium current in epicardial cell (left panel). Simulated effect of the T1620M Brugada syndrome channels, with gating lesions that cause an unopposed reduction in Na+ channel function (Dumaine et al. 1999) leading to early epicardial repolarization, consistent with Brugada syndrome ECG ST segment elevation (middle panel). G514C, with activation and inactivation modified as observed in (A) and (B) (right panel). The defect does not elicit early epicardial repolarization, but reduces the action potential upstroke (as shown in the inset), which determines the rate of impulse propagation through the myocardium and explains the clinical phenotype of conduction slowing. (Reprinted with permission from Tan HL, Bink-Boelkens MT, Bezzina CR, et al.: 2001. A sodium-channel mutation causes isolated cardiac conduction disease. Nature 409:1043 – 1047. Copyright 2001, Nature Publishing Group.)
model of the AP that incorporated the gating defects associated with T1620M— a Brugada syndrome mutation—showed that heterogeneous loss of the AP dome could occur in a one-dimensional model of AP propagation. Further studies (Gima and Rudy 2002) have also shown that this loss of the AP dome in epicardial cells can increase the voltage gradient in the transmural ventricular wall, leading to ST segment elevation in a mathematically computed ‘‘ECG.’’
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The opposing nature of the functional defects of LQT3 and Brugada syndrome mutations (gain vs. loss of function) might suggest that these two disorders should not coexist in the same patient. Surprisingly, a mutation in the C terminus of the Na+ channel, 1795insD, was described in which affected individuals exhibited the ECG manifestations of both syndromes: QT-interval prolongation and distinctive ST-segment elevations (Veldkamp et al. 2000). The molecular
mechanisms whereby a single amino acid insertion evokes both arrhythmia syndromes was related to disruption of the rapid component of inactivation (fast inactivation), causing a plateau of persistent INa during sustained depolarization (Figure 2A), prolonging QT interval at slow heart rates. Simultaneously, the mutation also induces channels to undergo excessive slow inactivation during the sustained depolarization period intrinsic to the cardiac AP (Figure 2B).
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Figure 4. A common SCN5A polymorphism, H558R, attenuates the gating defects of a neighboring mutation. (A) Voltage dependence of activation and inactivation of wild type (WT), T512I, and H558R/T512I obtained using the protocols shown in the insert. Note the hyperpolarizing shifts in activation and inactivation curves as a result of the T512I mutation as well as their restoration by H558R. (B) Slow kinetic component of inactivation as evaluated using the protocol shown in the inset (and in Figure 2B). Although T512I dramatically enhanced slow inactivation, similar to 1795insD (Figure 2B), H558R attenuated the extent of slow inactivation caused by T512I. (Reprinted with permission from Viswanathan PC, Benson DW, Balser JR: 2003. A common SCN5A polymorphism modulates the biophysical effects of an SCN5A mutation. J Clin Invest 111:341 – 346. Copyright 2003, Journal of Clinical Investigation.)
This abnormal behavior reduces Na+ channel availability primarily at rapid heart rates, and contributes to the heterogeneous repolarization of AP that is thought to underlie ST segment elevation in the Brugada syndrome. By conferring this dual defect on Na+ channel gating function, this single mutation provokes two distinct cardiac arrhythmia syndromes at the opposing extremes of heart rate.
Conduction Disease: Loss of Function Mutations
Cardiac conduction defects are among the most common cardiac rhythm disturbances and are characterized by progressive alteration of cardiac conduction through the His-Purkinje system with right or left bundle branch block and
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widening of the QRS complex. The disorder may progress to complete atrioventricular (AV) block, with syncope and, in some cases, sudden death. Although inherited mutations in SCN5A were first associated with rapid heart rhythms (Wang et al. 1995, Chen et al. 1998), more recent studies (Schott et al. 1999, Tan et al. 2001) have also associated SCN5A mutations to isolated conduction diseases. Considering that Na+ channels play a fundamental role in the initiation, maintenance, and conduction of normal cardiac rhythm, association of inherited mutations in the Na+ channel to isolated conduction diseases is not surprising. As with long QT syndrome and Brugada syndrome, multiple SCN5A mutations have been identified in patients with isolated conduction diseases (Tan et al. 2003). Functional studies of
SCN5A mutations associated with conduction disease have also consistently revealed a loss of Na+ channel function (Wang et al. 2002, Herfst et al. 2003, Probst et al. 2003, Viswanathan et al. 2003). A loss of Na+ current would make it more difficult for the membrane potential to reach the threshold for AP activation, thereby slowing conduction. Schott et al. (1999) first associated isolated conduction disease with mutations in SCN5A. Subsequently, five members of a Dutch family carrying a mutation in the Na + channel I-II linker (G514C) exhibited isolated cardiac conduction disease requiring pacemaker therapy (Tan et al. 2001). The biophysical characterization of this mutation revealed balanced changes in Na+ channel gating function, with both activation and inactivation gating requiring stronger depolarizing membrane potentials (Figures 3A and B). A parallel depolarizing shift in activation (loss of function) and inactivation (gain of function) might evoke little overall net change in Na+ channel activity. However, in this case, the activation shift predominated (by f3 mV), yielding a slight net decrease in Na+ channel function. In a computational model of cardiac conduction (Tan et al. 2001), this loss of function was not sufficient to induce premature epicardial AP repolarization and Brugada syndrome, but did reduce AP upstroke velocity by 20%, an effect that is predicted to slow conduction and explain the observed phenotype (Figure 3C). Excessive slow inactivation of Na + channels has previously been linked to Brugada syndrome (Veldkamp et al. 2000, Wang et al. 2000). However, recent studies (Viswanathan et al. 2003) have also linked excessive slow inactivation to isolated conduction disease. A 2-year-old with second-degree AV block, carries a mutation, T512I, in the DI-DII cytoplasmic linker, and is also homozygous for a common polymorphism (H558R) present in the Na+ channel DI-DII linker with a frequency of 20% (Yang et al. 2002). Studies showed that the polymorphism alone had no effect on the channel, but had a modulatory effect on the gating lesions caused by the T512I mutation (Viswanathan et al. 2003). Functional studies of the T512I mutation alone revealed shifts in the voltage dependence of activation and inactivation (Figure 4A) and, more importantly, a significant en-
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Figure 5. Diagram depicting overlap between long QT syndrome (LQT3), Brugada syndrome (BS), and cardiac conduction defects (PCCD). Figure highlights the complexities involved in linking single mutations, ion channel behavior, and cardiac rhythm. (Reprinted from Cardiovascular Research, 49, Bezzina CR, Rook MB, Wilde AAM, Cardiac sodium channel and inherited arrhythmia syndromes, 257 – 271, 2001, with permission from European Society of Cardiology.)
hancement of slow inactivation (Figure 4B) (Viswanathan et al. 2003). However, the polymorphism reversed the shifts in activation and inactivation (Figure 4A) and also mitigated the extent of slow inactivation, while still maintaining a significant difference compared with normal channels (Figure 4B). In this case, it would seem that a slight increase in slow inactivation could delay the recovery of Na+ channels between successive stimuli, causing a cumulative loss of function, leading to conduction slowing.
gating defects as well as other ‘‘unseen’’ regulatory factors. Single nucleotide polymorphisms— DNA sequence variations that are common in the population—have been implicated in phenotypic variability in physiology, pharmacology, and pathophysiology by altering gene function and susceptibility to disease. Studies have linked gene polymorphisms to ele-
vated risk for cystic fibrosis (Hull and Thomson 1998), Alzheimer’s disease (Roses 1998), or even heart disease (Wilkins et al. 2000). In addition to their role in disease, polymorphisms are also thought to confer sensitivity to drug therapy, as well as proarrhythmic risk from drug therapy. Recently, a polymorphism in SCN5A (S1102Y) was identified in individuals of African descent and implicated in an elevated risk for arrhythmia (Splawski et al. 2002). Electrophysiologic and computational analyses predicted negligible effects on AP properties as a result of the polymorphism. Surprisingly, however, the polymorphism increased AP duration and the susceptibility to the development of arrhythmogenic early afterdepolarizations in the background of reduced outward potassium current as a result of drug block (Splawski et al. 2002). Other studies (Yang et al. 2002) have identified a more common polymorphism, H558R, in the I-II linker of the Na+ channel (20% allelic frequency). As discussed above (Figure 4), the H558R polymorphism had no effect on wild-type Na+ channel current, but attenuated the gating defects caused by an intrageneic
Loss of Na+ Channel Function: Brugada Syndrome or Conduction Disease?
Electrophysiologic analysis of mutations linked to Brugada syndrome or isolated conduction disease has revealed defects that consistently lead to loss of channel function (Wang et al. 2000, Herfst et al. 2003, Probst et al. 2003, Viswanathan et al. 2003). Whereas even a single Na+ channel mutation may cause multiple changes in gating function that, each considered alone, could drastically increase or decrease the Na+ current, computational models of cardiac excitability equipped to consider the ensemble of these mutational effects may predict only a mild ‘‘net’’ increase or decrease in Na+ current. Furthermore, with the identification of several Na+ channel mutations that evoke multiple rhythm disturbances, such as 1795insD (Veldkamp et al. 2000), or DK1500 (Grant et al. 2002) (Figure 5), it is becoming clear that the manifestation of a particular phenotype is the result of the complex interplay between
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Figure 6. Simulated action potentials obtained using the Luo-Rudy mathematical model of the AP (Luo and Rudy 1994). Panel (A) shows action potentials (APs) computed from a ventricular cell (in this case, an epicardial cell) during three conditions: control, enhanced slow inactivation, and positive shift of voltage dependence of activation. Panel (B) shows APs computed from a Purkinje cell during the three different conditions. APs are superimposed to enable comparison. To simulate the longer AP characteristic of the Purkinje cell, the expression of the outward delayed rectifier potassium currents and inward calcium current were reduced (Schram et al. 2002). None of the interventions had a major effect on the ventricular cell AP, whereas Purkinje cell APs became irregular during conditions of enhanced slow inactivation, occasionally exhibiting complete loss of the AP dome (indicated by the arrow).
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mutation identified in a patient with isolated conduction defect (Viswanathan et al. 2003). Another study (Ye et al. 2003) also reported a similar modulatory role of this polymorphism when present along with a mutation linked to the long QT syndrome. Functional studies of disease-causing mutations have identified gating defects common to Brugada syndrome and isolated conduction disease. In particular, studies have shown enhanced slow inactivation as a common mechanism underlying these two syndromes (Veldkamp et al. 2000, Wang et al. 2000, Viswanathan et al. 2003). Reduction in Na+ current, irrespective of the underlying mechanism, would be expected to evoke both Brugada syndrome and conduction disease. Widening of the QRS and an increased PR interval, indicative of conduction slowing, was indeed observed in the original report describing Brugada syndrome (Brugada and Brugada 1992), and in other Brugada kindreds (Kyndt et al. 2001, Potet et al. 2003). However, such manifestations may be more the exception than the rule. For example, a 50% reduction in Na+ current in an SCN5AF mouse leads to only minor conduction abnormalities (Papadatos et al. 2002), whereas certain mutations evoke complete loss of Na+ channel function, yet the phenotype is only mild (Herfst et al. 2003, Probst et al. 2003, Smits et al. 2002). It therefore is difficult to consistently reconcile the phenotypic predominance of Brugada syndrome or conduction disease based on the reduction in channel current alone. It is important to note that the electrophysiologic characterization of SCN5A mutations associated with Brugada syndrome or conduction disease have been carried out in heterologous expression systems that do not necessarily recapitulate in vivo conditions. As such, although the severity of the Na+ channel functional defect may parallel the ECG phenotype in some cases, other unrecognized factors are certain to play a role. Such factors may include humoral regulation, auxiliary subunits, chaper one proteins, anchoring proteins, and transcriptional regulation. A recent study (Weiss et al. 2002) identified a new Brugada syndrome locus, distinct from SCN5A and associated with progressive conduction disease. Although this locus has not yet been associated with any ion channel or protein, the finding supports
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the notion that non-SCN5A gene products likely play a role in the manifestation of the conduction phenotype.
Loss of Na+ Channel Function: AV Conduction Slowing or PanConduction Slowing?
Inherited mutations in the Na+ channel have been associated with AV conduction slowing as well as ‘‘pan-conduction’’ slowing throughout the atria and ventricles. Although functional studies (Wang et al. 2002, Herfst et al. 2003, Probst et al. 2003, Viswanathan et al. 2003) have consistently shown that these mutations reduce Na+ current, it is still unclear as to how reduction in channel current can lead to these distinct phenotypes. Although additional studies are required to firmly establish causality between conduction block and channel gating, here we provide a framework for linking the biophysical observations and the observed phenotypes. Mutations identified in patients with conduction defects have revealed three distinct gating lesions: shifts in voltage dependence of activation and inactivation (Bezzina et al. 2003, Lupoglazoff et al. 2001, Tan et al. 2001), enhanced slow inactivation (Viswanathan et al. 2003, Wang et al. 2002), and entirely nonfunctional channels (Herfst et al. 2003, Schott et al. 1999). Mutations causing enhanced slow inactivation have generally been associated with AV conduction slowing (prolonged PR intervals), but no intraventricular or intraatrial conduction defect (normal P wave and QRS duration) (Viswanathan et al. 2003, Wang et al. 2002). In contrast, mutations leading to shifts in voltage dependence of activation and inactivation have often been associated with slow AV conduction, as well as delayed conduction throughout the atria and ventricles, including broad P waves, PR interval prolongation, and widening of the QRS complex (Bezzina et al. 2003, Lupoglazoff et al. 2001, Tan et al. 2001). Mutations that preferentially delay recovery from inactivation (via enhancing slow inactivation) could disproportionately affect cells with longer inherent AP duration (Purkinje cells). Hence, it is possible that excess slow inactivation, which would cause Na+ channels to recover from inactivation more slowly during diastole than do wild-type channels,
provides a mechanism whereby AV conduction is slowed in preference to atrial or ventricular conduction. In contrast, as in the case of G514C, mutations targeting the channel activation process could affect the myocardium more uniformly, as was observed. Consistent with this idea, it is noteworthy that H558R entirely eliminated the T512I effect on activation gating, but only partly corrected the slow inactivation defect (Viswanathan et al. 2003). As such, greater accumulation of Na+ channel slow inactivation upon successive stimuli in Purkinje cells— with their longer AP duration and smaller consequent diastolic interval— could lead to greater loss of Na+ channel function in these cells at rapid pacing rates, and thereby produce isolated AV conduction delay. Moreover, a premature stimulus could also further compromise the Purkinje diastolic interval and lead to a dramatic loss of Na+ current and result in all-or-none repolarization and conduction block (Figure 6).
Summary
It is becoming increasingly clear that even subtle changes in Na+ channel gating can dramatically affect cardiac rhythm. From functional studies of inherited mutations, it is evident that although in some cases the link between mutation-specific gating lesions and the clinical phenotype is direct, in other cases, the correlation is not straightforward. Yet-unrecognized factors such as humoral regulation, auxiliary subunits, and transcriptional regulation will surely play a role. Interestingly, heterogeneities in ion channel expression have been implicated as the major determinant underlying the functional properties of the varied cell types found in the myocardium (Schram et al. 2002). If mutationinduced gating defects are influenced by heterogeneities in Na+ channel expression, expression-related factors may prove to be critical in ultimately determining arrhythmia phenotype.
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Grant AO, Carboni MP, Neplioueva V, et al.: 2001. Long QT syndrome, Brugada syndrome, and conduction disease are linked to a single sodium channel mutation. J Clin Invest 110:1201 – 1209. Herfst LJ, Potet F, Bezzina CR, et al.: 2002. Na+ channel mutation leading to loss of function and non-progressive cardiac conduction defects. J Mol Cell Cardiol 35:549 – 557. Hodgkin AL, Huxley AF: 1978. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117:500 – 544. Horn R, Patlak JB, Stevens CF: 1999. Sodium channels need not open before they inactivate. Nature 291:426 – 427. Hull J, Thomson AH: 2003. Contribution of genetic factors other than CFTR to disease severity in cystic fibrosis. Thorax 53:1018 – 1021.
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generalized epilepsy with febrile seizures plus type 2. J Neurosci 21:7481 – 7490. Splawski I, Timothy KW, Tateyama M, et al.: 2002. Variant of SCN5A sodium channel implicated in risk of cardiac arrhythmia. Science 297:1333 – 1336. Tan HL, Bink-Boelkens MT, Bezzina CR: 2001. A sodium-channel mutation causes isolated cardiac conduction disease. Nature 409:1043 – 1047. Tan HL, Bezzina CR, Smits JP, et al.: 2003. Genetic control of sodium channel function. Cardiovasc Res 57:961 – 973. Veldkamp MW, Viswanathan PC, Bezzina C, et al.: 2000. Two distinct congenital arrhythmias evoked by a multidysfunctional Na(+) channel. Circ Res 86:E91 – E97. Viswanathan PC, Benson DW, Balser JR: 2003. A common SCN5A polymorphism modulates the biophysical effects of an SCN5A mutation. J Clin Invest 111:341 – 346. Wang DW, Makita N, Kitabatake A, et al.: 2000. Enhanced Na(+) channel intermediate inactivation in Brugada syndrome. Circ Res 87:E37 – E43. Wang DW, Viswanathan PC, Balser JR, et al.: 2002. Clinical, genetic, and biophysical characterization of SCN5A mutations associated with atrioventricular conduction block. Circulation 105:341 – 346. Wang Q, Shen J, Splawski I, et al.: 1995. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 80:805 – 811. Wei J, Wang DW, Alings M, et al.: 1999. Congenital long-QT syndrome caused by a novel mutation in a conserved acidic domain of the cardiac Na + channel. Circulation 99: 3165 – 3171.
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Inherited Sodium Channelopathies A Continuum of Channel Dysfunction Prakash C. Viswanathan*, and Jeffrey R. Balser Voltage-gated sodium channels are transmembrane proteins that produce the ionic current responsible for the rising phase of the cardiac action potential and play a fundamental role in the initiation, propagation, and maintenance of normal cardiac rhythm. Inherited mutations in SCN5A, the gene encoding the pore-forming subunit of the cardiac Na+ channel, have been associated with distinct cardiac rhythm syndromes: the congenital long QT syndrome, Brugada syndrome, and isolated conduction disease. Electrophysiologic characterization of heterologously expressed mutant Na+ channels have revealed gating defects that, in many cases, can explain the distinct phenotype associated with the rhythm disorder. However, recent studies have revealed significant overlap between aberrant rhythm phenotypes, and single mutations have been identified that evoke multiple rhythm disorders with common gating lesions. These new insights enhance understanding of the structure-function relationships of voltage-gated Na+ channels, and also highlight the complexities involved in linking single mutations, ion-channel behavior, and cardiac rhythm. (Trends Cardiovasc Med 2004;14:28–35) n 2004, Elsevier Inc.
Voltage-gated sodium (Na+) channels are transmembrane proteins that are responsible for the rapid upstroke of the action potential (AP) in nerve and muscle fibers (Balser 2001, Catterall 2000). In the heart, Na+ channels play a major role in the
Prakash C. Viswanathan is at the Department of Anesthesiology, Vanderbilt University, Nashville, Tennessee, USA. Jeffrey R. Balser is at the Departments of Anesthesiology and Pharmacology, Vanderbilt University, Nashville, Tennessee, USA. *Address correspondence to: Prakash C. Viswanathan, PhD, Room 560, Preston Research Building, Vanderbilt University School of Medicine, 2220 Pierce Avenue, Nashville, TN 37232-6602, USA. Tel.: (+1) 615-936-2407; fax: (+1) 615-936-0456; e-mail:[email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter
initiation, propagation, and maintenance of normal cardiac rhythm. Inherited mutations in the family of genes that encode voltage-gated Na+ channels poreforming subunits (SCNXA) underlie a wide spectrum of neurologic, musculoskeletal, and cardiovascular disorders. Mutations in SCN5A, the cardiac Na+ channel gene, are known to evoke multiple life-threatening disorders of cardiac rhythm that range from tachyarrhythmias to bradyarrhythmias that often require pacemaker implantation (Tan et al. 2003). Although patients with SCN5A mutations linked to either the long QT or Brugada syndromes may experience sudden, life-threatening arrhythmias, patients with isolated conduction disease exhibit heart rate slowing (bradycardia) that manifests clinically as syncope, or perhaps only as ‘‘lightheadedness.’’
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Na+ channels are dynamic molecules that undergo rapid structural rearrangements in response to changes in the transmembrane electrical field, a process known as gating (Figure 1) (Hodgkin and Huxley 1952). Upon membrane depolarization, Na+ channels rapidly undergo conformational changes that lead to opening of the channel pore to allow for Na+ influx, a process termed ‘‘activation.’’ Simultaneously, depolarization triggers initiation of ‘‘fast inactivation’’ (Ifast) that terminates Na+ influx. Inactivation differs qualitatively from channel closure in that inactivated channels do not normally open unless the membrane potential is hyperpolarized, often for a sustained period. In addition, Na+ channels may inactivate without ever opening (so-called ‘‘closed-state’’ inactivation) (Horn et al. 1981). With prolonged depolarizations, Na+ channels progressively enter ‘‘slow inactivated’’ states (Islow) with diverse lifetimes ranging from hundreds of milliseconds to many seconds (Cummins and Sigworth 1996, Rudy 1978). Slow inactivation reduces cellular excitability, particularly in pathophysiologic conditions associated with prolonged membrane depolarization, such as epilepsy (Spampanato et al. 2001), neuromuscular diseases (Cannon 1996), or cardiac arrhythmias (Veldkamp et al. 2000). It is increasingly clear that a great many single amino acid substitutions within the SCN5A coding region can evoke a broad spectrum of cardiac rhythm behavior by modulating these gating processes. At the same time, common sequence variants (‘‘polymorphisms’’) in the Na+ channel gene have also been implicated as risk factors in cardiac diseases (Viswanathan et al. 2003), as well as determinants of drug sensitivity (Splawski et al. 2002). Recent
Figure 1. Simplified scheme of the Na+ channel gating conformational changes in response to membrane depolarization. Activation and inactivation occur nearly simultaneously, so that channels sometimes reach the fast/-inactivated state (Ifast) without opening (closed-state inactivation). Sustained depolarization induces channels to occupy stable, slow, inactivated states (Islow).
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studies have shown that polymorphisms in the Na+ channel gene can confer enhanced drug sensitivity, promoting arrhythmias (Splawski et al. 2002), or even modulate the biophysical effects of disease-causing mutations (Viswanathan et al. 2003). Functional studies of mutations associated with cardiac diseases have provided a wealth of information that highlights the exquisite sensitivity of cardiac rhythm to Na+ channel function. This article reviews some of the recent findings that link multiple gating lesions to distinct cardiac disorders, to demonstrate the complex relationship between Na+ channel function and cardiac rhythm.
Long QT Syndrome: Gain of Function Mutations
The long QT syndrome, as the name suggests, manifests as prolongation of the QT interval as reflected in the bodysurface electrocardiogram (ECG) (Wang et al. 1995). Patients with prolonged QT intervals are predisposed to ventricular tachyarrhythmias caused by unstable repolarization, often leading to sudden cardiac death. Multiple genes have been associated with the inherited form of the long QT syndrome. Mutations in the cardiac Na+ channel linked to autosomal-dominant inheritance (LQT3) disrupt fast inactivation and thereby allow for sustained channel opening (Tan et al. 2003). This evokes a small but persistent Na+ current (a gain of channel function) during the AP plateau that delays myocyte repolarization, evokes ECG QT interval prolongation, and predisposes patients to polymorphic ventricular tachycardia (‘‘torsade de pointes’’). Figure 2A shows Na+ currents recorded from normal and mutant Na+ channels expressed in tSA-201 cells during depolarization. The mutation induces a sustained component of inward current (f1.5% of the peak inward current) throughout the depolarization period. In many cases, the development of arrhythmogenic episodes correlates with bradycardia during sleep or relaxation. Surprisingly, the size of the sustained inward current during the AP plateau is extremely small (f0.5% – 2%) compared with the peak inward Na+ current. Nonetheless, the role of this miniscule current in prolonging repolarization and generating cardiac arrhythmias has been val-
idated in quantitative models of the AP (Clancy and Rudy 1999). The first reported LQT3 mutation was a deletion of three residues in the III-IV linker of Na+ channel (1505 – 1507 DKPQ), a cytoplasmic channel domain that has been implicated in fast inactivation of the Na+ channel (Wang et al. 1995). Although expression of DKPQ mutant channels in heterologous expression systems enabled the initial characterization of altered channel kinetics, more recent studies (Nuyens et al. 2001) using genetically altered mice lacking the same triplet of residues revealed ECG features of LQT3 syndrome, and a predisposition to ventricular tachyarrhythmias in response to sudden rate accelerations. Mutations linked to LQT3 have been primarily localized to domains III and IV, the cytoplasmic linker connecting these two domains, and the C terminus of the Na+ channel. The highly conserved acidic domain in the C terminus appears to be a ‘‘hot spot’’ for LQT3 mutations (Wei et al. 1999). Although two chargealtering LQT3 mutations in this region (E1784K, 1795insD) evoke a small, persistent current similar to DKPQ and other LQT3 mutants, a third charge deletion (D1790G) produces both a small plateau and alters the voltage dependence of inactivation in a proarrhythmic manner (Alings and Wilde 1999). Although most of the mutations associated with LQT3 produce a sustained inward plateau current, some of the mutations that are causally linked to LQT3 have exhibited other gating disorders that also led to prolongation of the QT interval. These include faster recovery from inactivation and accompanying shifts in the voltage dependence of activation and inactivation (Abriel et al. 2001, Rivolta et al. 2002). Because a very delicate balance of inward and outward currents maintains the plateau of the AP, these subtle shifts in the balance of currents during repolarization may provoke QT prolongation and torsade de pointes.
Brugada Syndrome: Loss of Function Mutations
Another group of SCN5A mutations produces exceptional risk for idiopathic ventricular fibrillation with a high mortality rate in the absence of QT interval prolongation or structural heart disease (Bru-
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Figure 2. A single amino-acid insertion in the C terminus of the Na+ channel modifies two distinct gating processes. (A) The 1795insD mutation induces a sustained component of inward current, consistent with LQT3 phenotype. The inset shows tetrodotoxin (TTX)-sensitive difference current, revealing (arrows) a sustained plateau current for the mutant (f1.5% of the peak inward current). (B) Intermediate kinetic component of inactivation (slow inactivation) as evaluated using the protocol shown in the inset. 1795insD enhanced the magnitude of the slow component, consistent with loss of Na+ channel function and Brugada phenotype. WT, wild type. (Reprinted with permission from Veldkamp MW, Viswanathan PC, Bezzina C, et al.: 2000. Two distinct congenital arrhythmias evoked by a multidysfunctional Na(+) channel. Circ Res 86:E91 – E97. Copyright 2000, Lippincott, Williams and Wilkins.)
gada and Brugada 1992). The clinical disorder, known as ‘‘Brugada syndrome,’’ is usually characterized by ST segment elevation in the right precordial leads (V1 – V3), right bundle branch block, left axis deviation, and an increased HV interval (indicative of conduction disturbances). Multiple mutations have been identified and linked to Brugada syndrome; however, unlike LQT3, Brugada syndrome mutations are seen in virtually all regions of the Na+ channel (Tan et al. 2003). Although some of the Brugada syndrome mutations render the Na + channel entirely nonfunctional, many of the mutations alter Na+ channel gating function in a manner that reduces the Na+ current, in contrast to LQT3 mutants
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that invariably cause a gain of Na+ channel function. The cellular basis for the Brugada syndrome is thought to involve an outward shift of net transmembrane current at the end of phase 1 of the ventricular epicardial AP (Alings and Wilde 1999). The transient outward potassium current, Ito—which plays a role in determining the spike and dome morphology of the cardiac AP (during phase 1)—is more prominent in the epicardium than in the endocardium, particularly in the right ventricular epicardium. An increase in outward current at the end of phase 1 could accentuate this AP ‘‘notch’’ and lead to an all-or-none repolarization at the end of phase 1, causing a loss of AP
dome and a marked reduction in AP duration (Figure 3C). Hence, mutations of the Na+ channel that reduce the inward Na+ current could lead to an outward shift of the net transmembrane current, leading to an all-or-none repolarization and premature shortening of the epicardial AP. Because Ito is small in the endocardium, early repolarization and AP shortening due to reduced Na+ current is probably not observed in this region. As such, heterogeneous loss of the AP dome between the epicardium and endocardium creates a transmural voltage gradient responsible for elevation of the ST segment characteristic of Brugada syndrome. Computational studies (Tan et al. 2001) using a mathematical
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Figure 3. Balanced gating effects resulting from G514C mutation. (A) The voltage dependence of channel inactivation assessed using the protocol shown in the inset. G514C mutation shifts the voltage dependence of inactivation by +7 mV. This would increase the number of channels available to open at any given potential (a gain of function). (B) The voltage dependence of channel activation as evaluated using the protocol shown in the inset. G514C caused a +10 mV shift in the activation curve, suggesting that channels are less likely to open at any given membrane potential (a loss of function). (C) Simulated endocardial and epicardial action potentials obtained with use of the Luo-Rudy cable model. Wildtype (WT) action potentials, with arrow indicating the epicardial ‘‘notch’’ due to prominent ITo potassium current in epicardial cell (left panel). Simulated effect of the T1620M Brugada syndrome channels, with gating lesions that cause an unopposed reduction in Na+ channel function (Dumaine et al. 1999) leading to early epicardial repolarization, consistent with Brugada syndrome ECG ST segment elevation (middle panel). G514C, with activation and inactivation modified as observed in (A) and (B) (right panel). The defect does not elicit early epicardial repolarization, but reduces the action potential upstroke (as shown in the inset), which determines the rate of impulse propagation through the myocardium and explains the clinical phenotype of conduction slowing. (Reprinted with permission from Tan HL, Bink-Boelkens MT, Bezzina CR, et al.: 2001. A sodium-channel mutation causes isolated cardiac conduction disease. Nature 409:1043 – 1047. Copyright 2001, Nature Publishing Group.)
model of the AP that incorporated the gating defects associated with T1620M— a Brugada syndrome mutation—showed that heterogeneous loss of the AP dome could occur in a one-dimensional model of AP propagation. Further studies (Gima and Rudy 2002) have also shown that this loss of the AP dome in epicardial cells can increase the voltage gradient in the transmural ventricular wall, leading to ST segment elevation in a mathematically computed ‘‘ECG.’’
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The opposing nature of the functional defects of LQT3 and Brugada syndrome mutations (gain vs. loss of function) might suggest that these two disorders should not coexist in the same patient. Surprisingly, a mutation in the C terminus of the Na+ channel, 1795insD, was described in which affected individuals exhibited the ECG manifestations of both syndromes: QT-interval prolongation and distinctive ST-segment elevations (Veldkamp et al. 2000). The molecular
mechanisms whereby a single amino acid insertion evokes both arrhythmia syndromes was related to disruption of the rapid component of inactivation (fast inactivation), causing a plateau of persistent INa during sustained depolarization (Figure 2A), prolonging QT interval at slow heart rates. Simultaneously, the mutation also induces channels to undergo excessive slow inactivation during the sustained depolarization period intrinsic to the cardiac AP (Figure 2B).
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Figure 4. A common SCN5A polymorphism, H558R, attenuates the gating defects of a neighboring mutation. (A) Voltage dependence of activation and inactivation of wild type (WT), T512I, and H558R/T512I obtained using the protocols shown in the insert. Note the hyperpolarizing shifts in activation and inactivation curves as a result of the T512I mutation as well as their restoration by H558R. (B) Slow kinetic component of inactivation as evaluated using the protocol shown in the inset (and in Figure 2B). Although T512I dramatically enhanced slow inactivation, similar to 1795insD (Figure 2B), H558R attenuated the extent of slow inactivation caused by T512I. (Reprinted with permission from Viswanathan PC, Benson DW, Balser JR: 2003. A common SCN5A polymorphism modulates the biophysical effects of an SCN5A mutation. J Clin Invest 111:341 – 346. Copyright 2003, Journal of Clinical Investigation.)
This abnormal behavior reduces Na+ channel availability primarily at rapid heart rates, and contributes to the heterogeneous repolarization of AP that is thought to underlie ST segment elevation in the Brugada syndrome. By conferring this dual defect on Na+ channel gating function, this single mutation provokes two distinct cardiac arrhythmia syndromes at the opposing extremes of heart rate.
Conduction Disease: Loss of Function Mutations
Cardiac conduction defects are among the most common cardiac rhythm disturbances and are characterized by progressive alteration of cardiac conduction through the His-Purkinje system with right or left bundle branch block and
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widening of the QRS complex. The disorder may progress to complete atrioventricular (AV) block, with syncope and, in some cases, sudden death. Although inherited mutations in SCN5A were first associated with rapid heart rhythms (Wang et al. 1995, Chen et al. 1998), more recent studies (Schott et al. 1999, Tan et al. 2001) have also associated SCN5A mutations to isolated conduction diseases. Considering that Na+ channels play a fundamental role in the initiation, maintenance, and conduction of normal cardiac rhythm, association of inherited mutations in the Na+ channel to isolated conduction diseases is not surprising. As with long QT syndrome and Brugada syndrome, multiple SCN5A mutations have been identified in patients with isolated conduction diseases (Tan et al. 2003). Functional studies of
SCN5A mutations associated with conduction disease have also consistently revealed a loss of Na+ channel function (Wang et al. 2002, Herfst et al. 2003, Probst et al. 2003, Viswanathan et al. 2003). A loss of Na+ current would make it more difficult for the membrane potential to reach the threshold for AP activation, thereby slowing conduction. Schott et al. (1999) first associated isolated conduction disease with mutations in SCN5A. Subsequently, five members of a Dutch family carrying a mutation in the Na + channel I-II linker (G514C) exhibited isolated cardiac conduction disease requiring pacemaker therapy (Tan et al. 2001). The biophysical characterization of this mutation revealed balanced changes in Na+ channel gating function, with both activation and inactivation gating requiring stronger depolarizing membrane potentials (Figures 3A and B). A parallel depolarizing shift in activation (loss of function) and inactivation (gain of function) might evoke little overall net change in Na+ channel activity. However, in this case, the activation shift predominated (by f3 mV), yielding a slight net decrease in Na+ channel function. In a computational model of cardiac conduction (Tan et al. 2001), this loss of function was not sufficient to induce premature epicardial AP repolarization and Brugada syndrome, but did reduce AP upstroke velocity by 20%, an effect that is predicted to slow conduction and explain the observed phenotype (Figure 3C). Excessive slow inactivation of Na + channels has previously been linked to Brugada syndrome (Veldkamp et al. 2000, Wang et al. 2000). However, recent studies (Viswanathan et al. 2003) have also linked excessive slow inactivation to isolated conduction disease. A 2-year-old with second-degree AV block, carries a mutation, T512I, in the DI-DII cytoplasmic linker, and is also homozygous for a common polymorphism (H558R) present in the Na+ channel DI-DII linker with a frequency of 20% (Yang et al. 2002). Studies showed that the polymorphism alone had no effect on the channel, but had a modulatory effect on the gating lesions caused by the T512I mutation (Viswanathan et al. 2003). Functional studies of the T512I mutation alone revealed shifts in the voltage dependence of activation and inactivation (Figure 4A) and, more importantly, a significant en-
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Figure 5. Diagram depicting overlap between long QT syndrome (LQT3), Brugada syndrome (BS), and cardiac conduction defects (PCCD). Figure highlights the complexities involved in linking single mutations, ion channel behavior, and cardiac rhythm. (Reprinted from Cardiovascular Research, 49, Bezzina CR, Rook MB, Wilde AAM, Cardiac sodium channel and inherited arrhythmia syndromes, 257 – 271, 2001, with permission from European Society of Cardiology.)
hancement of slow inactivation (Figure 4B) (Viswanathan et al. 2003). However, the polymorphism reversed the shifts in activation and inactivation (Figure 4A) and also mitigated the extent of slow inactivation, while still maintaining a significant difference compared with normal channels (Figure 4B). In this case, it would seem that a slight increase in slow inactivation could delay the recovery of Na+ channels between successive stimuli, causing a cumulative loss of function, leading to conduction slowing.
gating defects as well as other ‘‘unseen’’ regulatory factors. Single nucleotide polymorphisms— DNA sequence variations that are common in the population—have been implicated in phenotypic variability in physiology, pharmacology, and pathophysiology by altering gene function and susceptibility to disease. Studies have linked gene polymorphisms to ele-
vated risk for cystic fibrosis (Hull and Thomson 1998), Alzheimer’s disease (Roses 1998), or even heart disease (Wilkins et al. 2000). In addition to their role in disease, polymorphisms are also thought to confer sensitivity to drug therapy, as well as proarrhythmic risk from drug therapy. Recently, a polymorphism in SCN5A (S1102Y) was identified in individuals of African descent and implicated in an elevated risk for arrhythmia (Splawski et al. 2002). Electrophysiologic and computational analyses predicted negligible effects on AP properties as a result of the polymorphism. Surprisingly, however, the polymorphism increased AP duration and the susceptibility to the development of arrhythmogenic early afterdepolarizations in the background of reduced outward potassium current as a result of drug block (Splawski et al. 2002). Other studies (Yang et al. 2002) have identified a more common polymorphism, H558R, in the I-II linker of the Na+ channel (20% allelic frequency). As discussed above (Figure 4), the H558R polymorphism had no effect on wild-type Na+ channel current, but attenuated the gating defects caused by an intrageneic
Loss of Na+ Channel Function: Brugada Syndrome or Conduction Disease?
Electrophysiologic analysis of mutations linked to Brugada syndrome or isolated conduction disease has revealed defects that consistently lead to loss of channel function (Wang et al. 2000, Herfst et al. 2003, Probst et al. 2003, Viswanathan et al. 2003). Whereas even a single Na+ channel mutation may cause multiple changes in gating function that, each considered alone, could drastically increase or decrease the Na+ current, computational models of cardiac excitability equipped to consider the ensemble of these mutational effects may predict only a mild ‘‘net’’ increase or decrease in Na+ current. Furthermore, with the identification of several Na+ channel mutations that evoke multiple rhythm disturbances, such as 1795insD (Veldkamp et al. 2000), or DK1500 (Grant et al. 2002) (Figure 5), it is becoming clear that the manifestation of a particular phenotype is the result of the complex interplay between
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Figure 6. Simulated action potentials obtained using the Luo-Rudy mathematical model of the AP (Luo and Rudy 1994). Panel (A) shows action potentials (APs) computed from a ventricular cell (in this case, an epicardial cell) during three conditions: control, enhanced slow inactivation, and positive shift of voltage dependence of activation. Panel (B) shows APs computed from a Purkinje cell during the three different conditions. APs are superimposed to enable comparison. To simulate the longer AP characteristic of the Purkinje cell, the expression of the outward delayed rectifier potassium currents and inward calcium current were reduced (Schram et al. 2002). None of the interventions had a major effect on the ventricular cell AP, whereas Purkinje cell APs became irregular during conditions of enhanced slow inactivation, occasionally exhibiting complete loss of the AP dome (indicated by the arrow).
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mutation identified in a patient with isolated conduction defect (Viswanathan et al. 2003). Another study (Ye et al. 2003) also reported a similar modulatory role of this polymorphism when present along with a mutation linked to the long QT syndrome. Functional studies of disease-causing mutations have identified gating defects common to Brugada syndrome and isolated conduction disease. In particular, studies have shown enhanced slow inactivation as a common mechanism underlying these two syndromes (Veldkamp et al. 2000, Wang et al. 2000, Viswanathan et al. 2003). Reduction in Na+ current, irrespective of the underlying mechanism, would be expected to evoke both Brugada syndrome and conduction disease. Widening of the QRS and an increased PR interval, indicative of conduction slowing, was indeed observed in the original report describing Brugada syndrome (Brugada and Brugada 1992), and in other Brugada kindreds (Kyndt et al. 2001, Potet et al. 2003). However, such manifestations may be more the exception than the rule. For example, a 50% reduction in Na+ current in an SCN5AF mouse leads to only minor conduction abnormalities (Papadatos et al. 2002), whereas certain mutations evoke complete loss of Na+ channel function, yet the phenotype is only mild (Herfst et al. 2003, Probst et al. 2003, Smits et al. 2002). It therefore is difficult to consistently reconcile the phenotypic predominance of Brugada syndrome or conduction disease based on the reduction in channel current alone. It is important to note that the electrophysiologic characterization of SCN5A mutations associated with Brugada syndrome or conduction disease have been carried out in heterologous expression systems that do not necessarily recapitulate in vivo conditions. As such, although the severity of the Na+ channel functional defect may parallel the ECG phenotype in some cases, other unrecognized factors are certain to play a role. Such factors may include humoral regulation, auxiliary subunits, chaper one proteins, anchoring proteins, and transcriptional regulation. A recent study (Weiss et al. 2002) identified a new Brugada syndrome locus, distinct from SCN5A and associated with progressive conduction disease. Although this locus has not yet been associated with any ion channel or protein, the finding supports
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the notion that non-SCN5A gene products likely play a role in the manifestation of the conduction phenotype.
Loss of Na+ Channel Function: AV Conduction Slowing or PanConduction Slowing?
Inherited mutations in the Na+ channel have been associated with AV conduction slowing as well as ‘‘pan-conduction’’ slowing throughout the atria and ventricles. Although functional studies (Wang et al. 2002, Herfst et al. 2003, Probst et al. 2003, Viswanathan et al. 2003) have consistently shown that these mutations reduce Na+ current, it is still unclear as to how reduction in channel current can lead to these distinct phenotypes. Although additional studies are required to firmly establish causality between conduction block and channel gating, here we provide a framework for linking the biophysical observations and the observed phenotypes. Mutations identified in patients with conduction defects have revealed three distinct gating lesions: shifts in voltage dependence of activation and inactivation (Bezzina et al. 2003, Lupoglazoff et al. 2001, Tan et al. 2001), enhanced slow inactivation (Viswanathan et al. 2003, Wang et al. 2002), and entirely nonfunctional channels (Herfst et al. 2003, Schott et al. 1999). Mutations causing enhanced slow inactivation have generally been associated with AV conduction slowing (prolonged PR intervals), but no intraventricular or intraatrial conduction defect (normal P wave and QRS duration) (Viswanathan et al. 2003, Wang et al. 2002). In contrast, mutations leading to shifts in voltage dependence of activation and inactivation have often been associated with slow AV conduction, as well as delayed conduction throughout the atria and ventricles, including broad P waves, PR interval prolongation, and widening of the QRS complex (Bezzina et al. 2003, Lupoglazoff et al. 2001, Tan et al. 2001). Mutations that preferentially delay recovery from inactivation (via enhancing slow inactivation) could disproportionately affect cells with longer inherent AP duration (Purkinje cells). Hence, it is possible that excess slow inactivation, which would cause Na+ channels to recover from inactivation more slowly during diastole than do wild-type channels,
provides a mechanism whereby AV conduction is slowed in preference to atrial or ventricular conduction. In contrast, as in the case of G514C, mutations targeting the channel activation process could affect the myocardium more uniformly, as was observed. Consistent with this idea, it is noteworthy that H558R entirely eliminated the T512I effect on activation gating, but only partly corrected the slow inactivation defect (Viswanathan et al. 2003). As such, greater accumulation of Na+ channel slow inactivation upon successive stimuli in Purkinje cells— with their longer AP duration and smaller consequent diastolic interval— could lead to greater loss of Na+ channel function in these cells at rapid pacing rates, and thereby produce isolated AV conduction delay. Moreover, a premature stimulus could also further compromise the Purkinje diastolic interval and lead to a dramatic loss of Na+ current and result in all-or-none repolarization and conduction block (Figure 6).
Summary
It is becoming increasingly clear that even subtle changes in Na+ channel gating can dramatically affect cardiac rhythm. From functional studies of inherited mutations, it is evident that although in some cases the link between mutation-specific gating lesions and the clinical phenotype is direct, in other cases, the correlation is not straightforward. Yet-unrecognized factors such as humoral regulation, auxiliary subunits, and transcriptional regulation will surely play a role. Interestingly, heterogeneities in ion channel expression have been implicated as the major determinant underlying the functional properties of the varied cell types found in the myocardium (Schram et al. 2002). If mutationinduced gating defects are influenced by heterogeneities in Na+ channel expression, expression-related factors may prove to be critical in ultimately determining arrhythmia phenotype.
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