CHAPTER TWELVE
Sodium Channels, Cardiac Arrhythmia, and Therapeutic Strategy Dori Miller, Lili Wang, Juming Zhong1 Department of Anatomy, Physiology & Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Structure and Physiological Function of Cardiac Na+ Channels 3. Cardiac Diseases Associated With Abnormal Na+ Channels 3.1 Long QT syndrome 3.2 Brugada syndrome 3.3 Other cardiac problems 3.4 Mutations of b-subunits and other regulatory proteins 4. Conclusion Conflict of Interest References
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Abstract Cardiac sodium channels are transmembrane proteins distributed in atrial and ventricular myocytes and Purkinje fibers. A large and rapid Na+ influx through these channels initiates action potential and thus excitation–contraction coupling of cardiac cells. Cardiac sodium channel is composed of a pore-forming a-subunit and one or two accessory b-subunits. The cardiac a-subunit is encoded by gene SCN5A located on chromosome 3p21. There are four types of b-subunits identified so far, and b1 is the primary b-subunit in cardiac Na+ channels. The gene responsible for b1 subunits is SCNB. The expression of b-subunits together with a subunits enhances the Na+ current and modifies the channel activities. In addition, interactions of the cardiac Na+ channel with other proteins may facilitate the channel activity and membrane expression of the channel. Over the past two decades, molecular genetic studies have identified the linkage of gene mutations of the Na+ channel proteins and other regulatory proteins to many inherited arrhythmogenic diseases. The most common cardiac arrhythmogenic diseases associated with Na+ channelopathies are long QT syndrome (LQT3) and Brugada syndromes (BrSs). This chapter intends to summarize the current understanding of the normal sodium-channel structure and function, the gene mutation-associated cardiac arrhythmias, and the current diagnosis and management of these diseases. Advances in Pharmacology, Volume 70 ISSN 1054-3589 http://dx.doi.org/10.1016/B978-0-12-417197-8.00012-2
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1. INTRODUCTION The major function of the cardiac contractile pumping is to propel blood throughout the body. Continuous circulating blood delivers nutrients and oxygen to and removes wastes from each organ and also serves as the transporter for neurotransmitters and hormones between various regions of the body. The pumping action of the heart is initiated by the rapidly regulated delivery of the electric signals from the pacemaker, the sinus node, to the contractile proteins in the individual cardiac muscle cells. This complex sequence is referred to “excitation–contraction coupling” (Bers, 2002). At the single cell level, this process begins when an action potential depolarizes the plasma membrane of the cardiac cell. Membrane depolarization activates the voltage-gated calcium channels located mostly in the T-tubules, allowing Ca2+ entry from the extracellular space. This Ca2+ entry triggers large amount of Ca2+ release from the sarcoplasmic reticulum (SR). The combination of Ca2+ entry from the calcium channels and SR Ca2+ release increases the intracellular Ca2+ transient, allowing Ca2+ to bind to troponin C. Binding of Ca2+ with troponin C unmasks the myosin-binding site on the actin molecule. With the consumption of ATP, actin–myosin binding induces cell shorting, and contraction occurs. When Ca2+ is removed from the cytosol by several mechanisms including sarcolemmal and SR Ca2+ ATPase, sarcolemmal Na +/Ca2+ exchange, and mitochondrial Ca2+ uniport, Ca2+ dissociates from the troponin C and relaxation occurs (Bers, 2002). Normal mechanical function and heart rhythm depends on proper electric impulses throughout the myocardium. Generation of myocardial action potential reflects the sequential activation and inactivation of membrane ion channels (ref. Fig. 12.1). Ion channels are pores located in the plasma membrane of the cell and regulate the movement of specific ions. As action potentials differ in different regions of the heart, the following brief description focused on the working myocardium, including both atria and ventricles. Cardiac action potential occurs in five phases (0–4): phase 4 is the resting membrane potential, phase 0 is the rapid depolarization, phase 1 is the rapid repolarization, phase 2 is the plateau, and phase 3 is the final repolarization. Each phase is linked with the opening/closing of specific channels and influx/efflux of the associated ions. During phase 4, the cell membrane is permeable to K+ but both Na+ and Ca2+ channels are closed. When cardiac myocytes are excited by electric stimuli from the conducting cells or
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A
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Figure 12.1 Illustration of action potential, ECG, and associated currents. Shown is a normal action potential and the relative time specific readout from an ECG and ionic current. The dashed line represents changes seen due to abnormal Na+ channel function caused by LQTS. A ¼ action potential. B ¼ ECG. C ¼ Na+ current. D ¼ Ca2+ current. E ¼ K+ current. Dashed line ¼ LQT3.
adjacent cells, the membrane potential will rapidly depolarize (phase 0) due to the opening of fast, voltage-gated Na+ channels. Na+ influx is transient and lasts about 1–2 ms. A brief phase of rapid repolarization (phase 1) follows the peak of the action potential. This is due in part to the closing or inactivation of Na+ channels and in other part to the transient outward K+ current (Ito) and outward Cl current. The plateau or phase 2 of the cardiac action potential results from the balance of the inward and outward current crossing the cell membrane. The inward current is induced by the opening of the slow-activated Ca2+ channels, and the outward currents were carried by Cl- channels and various K+ channels. Repolarization of the plateau (phase 3) occurs due to the inactivation of slow inward Ca and Na+ channels and opening of various slow outward K channels. Other ion transporters
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including outward Cl channels, Na/K pump, and Na/Ca exchanger are all participated in the rapid repolarization. Finally, with the membrane permeation only to K+, membrane potential returns to the resting stage. As described in the preceding text, action potential is a complicated event that requires interplay between multiple voltage-gated ion channels and occurs due to the movement of ions across the sarcolemma. The primary voltage-gated ion channels involved in the cardiac electrophysiology include Na+, K+, and Ca2+ channels. Any change in these channels will induce improper electric property of cardiac muscle, thus causing abnormal cardiac function. This chapter will focus on the normal properties of the Na+ channel, genetic mutation of Na+ channel-induced cardiac problems, and therapeutic strategies.
2. STRUCTURE AND PHYSIOLOGICAL FUNCTION OF CARDIAC NA+ CHANNELS As mentioned previously, INa is critical to generating action potential and E–C coupling in working cardiac myocytes and Purkinje fibers. Transient opening of Na+ channels is responsible for the rapid upstroke of action potential. Fast inactivation of Na+ channels participates in the rapid repolarization of the membrane potential. Aberrations of these elements of Na fluxes in the cell are linked to loss-of-function (decreased peak/early INa) and gain-of-function (increased late INa) syndromes to be discussed in the succeeding text. The first Na+ channel was cloned in 1984 (Noda et al., 1984). Since then, studies have been focused on the structure–function properties of Na+ channels, mutation of channel gene-induced diseases, and potential treatment of these diseases. Voltage-gated Na+ channels are composed of a principal pore-forming subunit (a-subunit) and one or two smaller accessory subunits (b-subunits). When the a-subunit is expressed on its own, it possesses the functional activity for Na+ current, indicating a functional formation of the channels by a-subunit (Catterall, 1986). Na+ channels comprise a major gene family with nine types of a-subunits, which are encoded for by genes SCN1A–SCN11A. These different Na+ channel genes are expressed in various tissues and have different properties. The primary cardiac Na+ channel in humans, Nav1.5, is encoded for by the gene SCN5A located on chromosome 3p21 (George et al., 1995). Na+ channels are differentially expressed across the cardiac tissue. Nav1.5 is highly expressed in the Purkinje fibers, His bundle and bundle branches,
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atria, and ventricles, while less abundant in the atrioventricular nodes. Studies have shown that Nav1.5 are localized within cardiac tissue to the T-tubules, myocyte surface, and intercalated disks. This differential expression can have implications on therapeutic design. For example, studies show that Na+ channels in atria and ventricle respond differently (inactivation differences) to certain pharmacological agents such that treatment must be specific to the Na+ channels involved. The a-subunit of Nav1.5 consists of four homologous domains (DI– DIV), each of which contains six transmembrane segments (S1–S6). The amino acid residues that link the adjacent domains lie in the cytoplasmic side (Fig. 12.2). The amino acid residue linker between S5 and S6 of each domain, which has an extracellular loop dipping down into the membrane, serves as the ion pore. The S4 segment, which contains 6–8 charged residues, serves as the primary voltage sensor. The linker between S3 and S4 is located in the cytosol and serves as the key for the inactivation process (Catterall, 1986). The outer part of the Na+ channel pore is believed to be the loop between S5 and S6. This loop has a short segment (SS1) entering the membrane and another short segment (SS2) exiting the membrane and thus is
Figure 12.2 Schematic topology of cardiac sodium-channel Nav1.5. Na+ channel is composed a single a-subunit and one or two b-subunits. The a-subunit is the pore-forming subunit and consists of four homologous domains (DI–DIV). Each domain is composed of six segments (S1–S6). The linkage between S5 and S6 is considered the outer pore of the channel, where S4 is responsible for detecting voltage changes in the membrane. The cytoplasmic loop between DIII and DIV that contain residues of IFM (denoted by red (dark gray in print version) stars) is believed important for channel fast in activation. b-Subunits play a critical role in the regulation and membrane expression of Nav1.5.
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denoted the SS1–SS2 region. Studies using tetrodotoxin (TTX) and sitedirected mutagenesis determined that a negatively charged glutamate residue at position 387 that links S5 and S6 of domain I at the outside of the membrane is able to bind to the positively charged TTX. Binding of TTX to this site blocks Na+ current through this channel. Neutralization of E387 and other negatively charged residues adjacent to E387 abolished the Na+ current also. Mutation of each of the negatively charged residues at the similar location in other three domains also prevented TTX binding and Na+ current. This suggests that the SS1–SS2 region forms part of the outer pore of the Na+ channel. Furthermore, it is assumed that the SS1–SS2 regions of all four domains form as a ring of negative charges surrounding the external opening of the pore. In addition, the amino acid residues located in this region determine the high selectivity for sodium ions (Heinemann, Terlau, Stuhmer, Imoto, & Numa, 1992; Noda, Suzuki, Numa, & Stuhmer, 1989; Terlau et al., 1991). Activation of voltage-gated Na+ channels results from a protein conformation change driven by membrane potential change. The S4 on each domain serves as the voltage sensor. It contains six to eight positively charged residues, usually arginine or lysine, which are interposed by two nonpolar residues between them. Mutation of these charged residues with neutral or negatively charged residues reduces the voltage-dependent activation of the channel (Stuhmer et al., 1989). Catterall (1986) suggested a helical screw model of the S4 segment. This model suggests that the S4 adopts a a-helical structure in which the positively charged arginine residues are stabilized by interaction with negatively charged residues in adjacent transmembrane domains. In response to a voltage change, the S4 helix moves across the membrane in a spiral path and exchanges ion pairs between the positively charged S4 residues and fixed negative charges in the surrounding transmembrane segments. This movement of S4 on each domain together is able to change the pore conformation, thus opening the channels. Na+ channels display two modes of inactivation: A rapid decay of Na+ currents following short depolarization (within a few milliseconds) is considered fast inactivation. Slow inactivation occurs when cell membrane is depolarized for seconds or longer. These two modes of inactivation are mediated by different molecular mechanisms. Fast inactivation is possibly associated with the intracellular linker between domains III and IV. Early studies by Armstrong and colleagues (Armstrong & Bezanilla, 1977; Armstrong, Bezanilla, & Rojas, 1973; Bezanilla & Armstrong, 1977) indicated that intracellular application of the proteolytic enzyme pronase
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removed fast inactivation of Na+ channels in the squid axon, suggesting that the inactivation gate is located inside the cytoplasm and is accessible to the cytoplasmic enzymes. Using the antibodies specifically against various sites of the intracellular regions of Na+ channel, Vassilev, Scheuer, and Catterall (1988) demonstrated that antibodies directed against the region between domains III and IV prevented Na+ channel inactivation, while antibodies directed at other intracellular regions had no effect. The effect of these antibodies against the DIII–DIV linker on the channel inactivation is voltage-dependent. At negative membrane potentials, the antibody could bind rapidly and inhibited Na+ channel inactivation on depolarization. At more positive membrane potentials where Na+ channels are activated and then partially inactivated, the antibody bound much slower and was much less effective. These results suggest that the linker becomes inaccessible to antibody binding when the channel is inactivated. Subsequent studies using mutagenesis demonstrated that a cluster of three hydrophobic residues within the linker loop of domain III and IV plays an important role in the fast inactivation of Na+ channels. Mutation of the phenylalanine residue at position 1489 to glutamine completely blocks fast inactivation. Mutation of I1488 and M1490 to glutamine, the adjacent two residues of F1489, also slowed the inactivation but to a lesser extent (West et al., 1992). On the other hand, the charged residues within the loop are not responsible for fast inactivation (Patton, West, Catterall, & Goldin, 1992). These studies suggest that the IFM (1488–1490) residues within the linker loop of domains III and IV serve as a hydrophobic latch in the inner mouth of the pore and thus inactivate the channel. An intracellular binding site for the IFM latch seems located at the cytoplasmic ends of S5 and S6 (West et al., 1992). Slow inactivation is similar to fast inactivation but involves different structural elements. Intracellular application of proteolytic enzymes prevented fast inactivation, but did not affect slow inactivation (Armstrong & Bezanilla, 1977; Bezanilla & Armstrong, 1977). Mutations within the domain III and IV linker also had no effect on slow inactivation while successfully blocked the fast inactivation (Patton et al., 1992; West et al., 1992). On the other hand, mutations at the cytoplasmic ends of S5 and S6, which are thought to form the inner mouth of the pore, removed the slow inactivation. This suggests that slow inactivation may involve a conformation change at the inner mouth of the channel (Hayward, Brown, & Cannon, 1997). The b-subunit is composed of a single transmembrane domain with an extracellular N terminus and an intracellular C terminus and is heavily glycosylated. Four b-subunits (b1–b4) are identified so far. b1 is expressed in
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the heart and is encoded by gene SCNB located on chromosome 19q13 (Makita, Bennett, & George, 1994; Makita, Sloan-Brown, Weghuis, Ropers, & George, 1994). Other b-subunits are also expressed in cardiac tissue. It is proposed that Na+ channel is composed of a single a-subunit with one or two b-subunits to form heterodimeric and/or heterotrimeric complexes (Catterall, 2014; Meadows & Isom, 2005). The expression of b-subunits enhances the Na+ current and modifies the channel properties, especially channel inactivation (Catterall, 2014; Isom et al., 1992; Makita, Bennett, et al., 1994; Makita, Sloan-Brown, et al., 1994; Meadows & Isom, 2005).
3. CARDIAC DISEASES ASSOCIATED WITH ABNORMAL Na+ CHANNELS 3.1. Long QT syndrome Long QT syndrome (LQTS) is an inherited or acquired disorder that causes sudden death from cardiac arrhythmias, specifically torsade de pointes and ventricular fibrillation (VF), and the leading cause of sudden cardiac death in young people. Clinically, this problem is characterized by prolonged QT intervals on the surface electrocardiogram (ECG). Prolonged QT intervals reflect increased action potential durations and delayed ventricular repolarizations in cardiac myocytes and typically involve ventricular tachyarrhythmia. Not surprisingly, decreases in repolarizing outward K+ currents or increases in depolarizing inward Na+ or Ca2+ currents can lead to prolongation of the action potential and thus prolongation of the QT interval. Up to now, 13 different types of LQTS have been identified. Molecular genetic studies have demonstrated that three types of congenital LQTS, including LQT1, LQT2, and LQT3, constitute >75% of the clinical cases. While LQT1 and LQT2 are due to the loss of function of voltagegated K+ channels, LQT3 is associated with gain of function of SCNA5 mutations or Na channel proteins, usually resulting from the failure of Na channels to inactivate (Amin, Asghari-Roodsari, & Tan, 2010; Wang, Shen, Splawski, et al., 1995). LQT3 accounts for around 13% of all patients with LQTS and is associated with mutations in SCN5A. Study by Wang, Shen, Splawski, et al. (1995) was the first to report that mutations of SCN5A were associated with the LQTS. Single-strand conformation polymorphism and DNA sequence analyses revealed identical intragenic deletions of SCN5A in affected members of two unrelated LQT families. The deleted sequences reside in a region
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that is important for channel inactivation. Cycle sequencing experiments revealed the presence of a 9 bp deletion beginning at nucleotide 4661 of the cDNA. This deletion disrupts the coding sequence, resulting in a deletion of three conserved amino acids, Lys-1505-Pro-1506-GIn-1507 (KPQ), in the cytoplasmic linker between DIll and DIV. These data suggest that mutations in SCN5A cause chromosome 3-linked LQT and indicate a likely cellular mechanism for this disorder (Wang, Shen, Splawski, et al., 1995). Additionally, the same group identified more mutation sites in SCN5A, in affected members of four LQT families. These mutations include two identical intragenic deletions and two missense mutations. The location and character of these mutations suggest that this form of LQT results from a delay in cardiac sodium-channel fast inactivation or altered voltagedependence of inactivation (Wang, Shen, Li, et al., 1995). Subsequent studies by other groups identified more mutations of SCN5A that associated with LQT3 patients. Currently, 84 SCN5A mutations are known to be related to LQT3. Most of these mutations are missense mutations and are located in the intracellular regions. The transmembrane residues are less frequently affected. It is interesting to note that no LQT3 mutation was detected in the P-loops connecting S5 and S6, in S1 segments, and in the complete transmembrane part of domain 2. This suggests that either gating or inactivation defects leading to a clinically relevant gain of function cannot be achieved by mutating these regions or that respective gain-of-function mutations have not yet been discovered. In contrast to LQT3 mutations, the P-loops seem to be a preferred target for Brugada syndrome (BrS) and conduction disease mutations. Nearly half of the 84 known mutant channels have been studied by heterologous expression and electrophysiological measurements. Most of them are found to cause gain of function of sodium channels in cardiac tissues (Zimmer & Surber, 2008). This gain of function includes abnormal sustained or persistent current compared to wild-type Nav1.5, increased window current, slower inactivation, and faster recovery from inactivation. Persistent current (Isus) represents a continuous flow of Na+ ions through the channel pore during the AP plateau and repolarization phase and is assumed to cause a prolongation of the ventricular AP. This mechanism seems to be the primary cause of the disease, because most SCN5A mutations identified in LQT3 carriers resulted in this inactivation defect. A few examples include a de novo missense mutation (R1623Q, S4 segment of domain 4) identified in an infant Japanese girl with a severe form of LQT3 (Makita et al., 1998). When expressed in oocytes, mutant Na+ channels exhibited only minor
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abnormalities in channel activation but had significantly delayed macroscopic inactivation. Single-channel analysis revealed that R1623Q channels have significantly prolonged open times with bursting behavior (Makita et al., 1998). Another deletion in SCN5A, delQKP 1507–1509, in the DIII–DIV linker of the sodium channel was identified in a family with a typical LQT3 phenotype (Keller et al., 2003). When constructed in vitro and expressed in the tsA201 human cell line, these mutated channels indicated a persistent inward sodium current, which was nearly completely blocked by the sodium-channel blockers TTX and lidocaine. The deletion mutants also resulted in a significant shift of steady-state activation to more depolarized voltages. Lupoglazoff et al. (2001) found a respective positive correlation in patients carrying the V1777M mutation. The homozygous mutation caused a serious QT prolongation (526 ms), whereas the heterozygous mutation in the parents and siblings of the index patient resulted in borderline QTc intervals (415–442 ms). Heterologously expressed V1777M channels generated a pronounced persistent current, whereas simultaneous coexpression of wild-type and mutant channels reduced this current fraction to nearly 50%, which is consistent with the in vivo observation. A window current cannot be assumed because both steady-state activation and inactivation curves were shifted towards hyperpolarized potentials and accelerated recovery from inactivation was not reported (Lupoglazoff et al., 2001). Genetically engineered mice expressing mutant channels that produce large persistent currents also showed the typical features of LQT3 (Remme et al., 2006; Yong et al., 2007). The effect of a persistent current on cardiac AP has been also investigated by mathematical modeling methods (Clancy & Rudy, 2002). All these data together indicate the strict physiological consequence of a persistent Na+ inward current to cause LQT3 syndrome. However, a general correlation between the degree of the persistent current and the length of the QT interval cannot be established for all the known mutations. Mutations causing persistent current are mainly located in the regions involved in fast inactivation of sodium channels. As discussed previously, the inactivation gate in Na+ channels is formed by the DIII–DIV linker where the clustered hydrophobic amino acids isoleucine, phenylalanine, and methionine (IFM motif ) serve as a lid and flanking glycine and proline residues function as molecular hinges “hinged-lid model” (Catterall, 2000; Kellenberger, West, Catterall, & Scheuer, 1997; West et al., 1992). The inactivation gate receptor is formed by multiple peptide segments including amino acid residues in DIVS6 and in intracellular loops DIIIS4/S5 and DIVS4/S5 (Catterall, 2000; Smith & Goldin, 1997). Notably, this
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open-state inactivation is coupled to activation (Chanda & Bezanilla, 2002; Sheets, Kyle, Kallen, & Hanck, 1999). The most severe LQT3 phenotype is produced by a three amino acid deletion in the inactivation loop that links DIII and DIV, dKPQ1505–1507 (Bennett, Yazawa, Makita, & George, 1995; Chandra, Starmer, & Grant, 1998). Mutations in those inactivation gate receptor regions including the S4/S5 linkers in DIII and DIV as well as in DIVS6 destabilize the inactivated state and thus allow the mutant channels to reopen at depolarized potentials. In addition, because of the essential role of the segment 4 in DIV on coupling channel activation and fast inactivation, it is not surprising that mutations in this S4 helix also destabilize the inactivated state. These mutations include R1623Q, R1626P, R1644H, R1623L, and R1644C (Dumaine et al., 1996; Makita et al., 1998; Ruan, Liu, Bloise, Napolitano, & Priori, 2007; Wang, Yazawa, George, & Bennett, 1996). More recently, the first half of the intracellular C terminus region (part nearest to S6 in Fig. 12.1) was predicted to be composed of six helices (H1–H6) that physically interact predominantly with the motif PIPR in the DIII–DIV linker (Cormier, Rivolta, Tateyama, Yang, & Kass, 2002; Motoike et al., 2004). This interaction is thought to stabilize the inactivation gate-occluded channel (Motoike et al., 2004; Shin et al., 2004). Interestingly, a calmodulin-binding motif (IQ motif: IQxxxRxxxxR) in the proposed helix H6 is essential for this interaction, because both the removal of the IQ motif in a C terminal deletion variant (S1885Stop; Cormier et al., 2002) and the specific IQ modification (exchange for alanine; Tester, Will, Haglund, & Ackerman, 2005) resulted in significantly increased persistent currents. Consequently, mutations that eliminate the IQ motif (Q1909R and R1913H; Tester et al., 2005) should also result in a sustained Na+ inward current upon membrane depolarization. Other gain-of-function mechanisms in LQT3 include increased window current, a delayed onset of inactivation, faster recovery from inactivation, and higher peak current density. A window or overlap current results from the overlap of the steady-state inactivation and steady-state activation curves. This voltage range is very narrow for the normal sodium channels. Mutations of SCN5A often result in relative shifts of the steady-state inactivation and activation curves or in increased slope factors of these curves. Such alterations can increase both the critical voltage range and the magnitude of the resulting window current and prolong QT intervals in the absence of a persistent current. Mutations in this group include E1295K (Abriel et al., 2001), A1330P (Wedekind et al., 2001), A1330T (Smits et al., 2005), I1768V channels (Rivolta et al., 2002), and T1620K (Surber et al., 2008).
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Delayed onset of fast inactivation results in a decelerated decay of macroscopic currents. A slower current decay alone should not directly prolong the AP, but may affect voltage-dependent activity of other outward or inward currents that are crucial for AP duration. Examples of these mutations are A1330P (Wedekind et al., 2001), P1332L (Ruan et al., 2007), T1620K (Surber et al., 2008), and Y1795C (Rivolta et al., 2001). A faster recovery from inactivation is mostly associated with a larger inward Na+ current. Because the respective peak current occurred outside the overlap voltage of activation and inactivation, channel reopening occurs as a result of faster recovery from the inactivated state and generates a larger inward current during the ramp compared to wild-type channels. Some examples of this group of mutations include I1768V (Clancy, Tateyama, Liu, Wehrens, & Kass, 2003), A572D and G615E (Albert et al., 2008), and Y1795C (Rivolta et al., 2001). 3.1.1 Diagnosis of LQT3 The clinical manifestations and diagnosis of LQTS have been extensively described in many publications. Those readers who are interested in the details may find these reviews very informative (Schwartz & Ackerman, 2013; Schwartz, Crotti, & Insolia, 2012). Here, we will briefly describe those aspects important to distinguish LQT3 from other LQTS. Clinically, typical cardiac manifestations of LQTS are polymorphic ventricular tachycardia, a syncopal episode called torsades de pointes (TdP), which most of time is self-limiting and often degenerating into VF. These syncopal episodes of VF can occur without changes in heart rate and without specific sequences such as “short–long–short” interval, even though long pauses in LQTS patients increase the probability of TdP. While it had been known that although most patients would develop their symptoms under stress, in a minority of cases, these life-threatening cardiac events could occur at rest and/or during sleep. The reasons for these different patterns remained obscure until molecular biology allowed to researchers distinguish between different genotypes. Most of the events of LQT1 patients occur during exercise or stress. Conversely, most of the events of LQT2 patients occur during emotional stress such as auditory stimuli (sudden noises and telephone ringing, especially while at rest), while for LQT3 patients, they occur during sleep or at rest (Schwartz et al., 2012). LQTS is diagnosed with a prolonged interval between Q and T wave on the surface electrocardiogram. Traditionally, QT intervals longer than 440 milliseconds are considered prolonged, although this value varies among
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genders and ages. Diagnosis must exclude variations in heart rate, serum calcium, or the presence of QT-prolonging drugs. Some LQTS patients have sudden pauses in sinus rhythm exceeding 1.2 s that are not related to sinus arrhythmia and may contribute to the initiation of arrhythmias in LQTS patients. Their occurrence represents an important warning signal in LQT3 patients. In addition, various stress tests with ECG would sufficiently distinguish different type of LQTS, especially type 1 LQTS (Horner, Horner, & Ackerman, 2011; Schwartz & Crotti, 2011). When a patient is suspected of having LQTS by clinical tests mentioned in the preceding text, genetic testing will be a valuable step in the channelopathies of LQTS. Currently, about 80% of LQTS are associated with mutations in three LQTS susceptibility genes, KCNQ1 (IKs channel subunit also known as KvLQT1), KCNH2 (IKr channel subunit also known as HERG), and SCN5A. SCN5A gives rise to LQT3, which accounts for approximately 13% of all genotyped individuals with LQTS (Kapplinger et al., 2009; Wang, Shen, Splawski, et al., 1995).
3.1.2 Treatment of LQT3 LQTS are commonly treated with b-adrenergic blocking agents, left cardiac sympathetic denervation (LCSD), and the implantable cardioverter defibrillator (ICD). They are complemented currently by gene-specific approaches. Historically, b-adrenergic blockers have been used as Na+ channel agonists. Not all b-blockers are equally effective and thus this method has variable success. Common b-blockers used include propranolol, metoprolol, atenolol, and ranolazine. Each has varying effectiveness in controlling LQT3. Differences in blockage of late/sustained Na+ current may play a role in the different clinical efficacy of various b-blockers, and this effect is highest for propranolol, lower but present for nadolol, and completely absent for metoprolol. Given its direct late sodium current blocking properties, propranolol is probably the LQT3-preferred b-blocker (Besana, Wang, George, & Schwartz, 2012). In addition to these common b-blockers, specific sodium-channel blockers are also effective in the treatment of LQT3. This includes mexiletine, which has demonstrated a significant benefit in the treatment of LQT3 (Ruan et al., 2007; Wang et al., 2008). Ranolazine is a newer therapy used for the treatment of LQTS. This compound is a sodiumchannel blocker and specifically targets the late Na+ current but has shown to slightly elongate the QT interval due to minimal blocking of IK (Moss et al., 2008).
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LCSD is a surgical approach to remove the first four thoracic ganglia (T1–T4). The rationale for LCSD is largely based on its antifibrillatory effect and includes a major reduction in norepinephrine release at ventricular level with the absence of postdenervation supersensitivity and no reduction in heart rate (Collura, Johnson, Moir, & Ackerman, 2009). Most LQT3 patients, except the very high-risk symptomatic LQT3 infants, respond well to LCSD (Collura et al., 2009). The ICD is used mostly in cases when a patient has a documented cardiac arrest. There is a common consensus for immediately implanting an ICD to save the life when the patient has a cardiac shock or cardiac arrest. There are several criteria for LQTS patients. Primarily, this approach should be applied for those patients who survived a cardiac arrest and those patients who have high electric instability even with other therapy options (Schwartz & Ackerman, 2013).
3.2. Brugada syndrome BrS was first described in 1992 (Brugada & Brugada, 1992) as a hereditary disease that affects 4 in 10,000 people. The syndrome primarily causes right ventricular tachycardia or VF and presents ST segment elevation in the ECG. Patients usually die from sudden cardiac arrest during sleep. Clinically, the patients have normal heart structure and contractile function, but selfterminating VF in these patients results in symptoms of syncope, seizure, and sleep disturbance, as the arrhythmia is more frequent at night. The ECG characteristics exhibit day-to-day variation and may not always be present (Veltmann et al., 2006). BrS is believed the result of increased heterogeneity of the ion currents involved in the phase I repolarization of action potential in right ventricle. Mutations in 12 different genes encoding Na+ channels, Ca2+ channels, K+ channels, and other membrane proteins have been associated with BrS (Antzelevitch, 2012; Hsiao et al., 2013; Veerakul & Nademanee, 2012). Mutations of the Na+ channel are the most common cause of BrS and account for 15–30% of BrS cases. Chen et al. (1998) was the first to report that a mutation in the SCN5A gene was linked to BrS. They identified a missense mutation, a splice-donor mutation, and a frame-shift mutation in the coding region of SCN5A in three families with BrS. As of 2013, there are more than 200 known SCN5A mutations that associated BrS, and most of them are missense mutations. All the BrS mutations seem to be randomly distributed over the Nav1.5 but seem to cluster in the pore-forming part (Brugada, Brugada, & Roy, 2013; Zimmer & Surber, 2008).
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The functional characteristics of SCN5A mutations associated with BrS1 have been analyzed in mammalian cell lines and mouse models. In contrast to the SCN5A mutations in LQT3 patients that induce gain of function, most SCN5A mutations in BrS1 patients that are heterologously expressed are loss of function, leading to reduced Na+ current. Different types of lossof-function mechanisms were suggested, including expression of nonfunctional channels, decreased expression of total and/or membrane Na+ proteins, and altered gating properties of channel. In the first case, mutated Na+ channel proteins may pass the ER quality control system and traffic normally to the sarcolemma but form channels that conduct no or very small INa (nonfunctional). An example of this group is G1408R (Kyndt et al., 2001). Decreased membrane expression of Nav1.5 proteins results from premature degradation of the mutant proteins by the quality control system in the ER or defective trafficking to the membrane. Mutations of SCN5A with R1432G, G1743R, and T353I are identified to be associated with trafficking deficiency (Baroudi et al., 2001; Pfahnl et al., 2007; Valdivia et al., 2004). Expression may also be decreased because mutant Nav1.5 proteins fail to interact with b-subunits or regulatory proteins, which mediate their normal localization on the sarcolemma. The third mechanism (altered gating properties) comprises delayed activation (i.e., activation at more positive potentials), earlier inactivation (i.e., inactivation at more negative potentials), faster inactivation, and enhanced slow inactivation (Veerakul & Nademanee, 2012). Delayed activation, earlier inactivation, and faster inactivation reduce INa by decreasing the probability of the channels to reside in the activated state. Enhanced slow inactivation means that mutant channels preferentially enter into the slow inactivation state, which is associated with longer recovery times during the action potential. At fast heart rates, phase 4 becomes too short for such channels to recover completely from slow inactivation. This leads to an accumulation of the channels in the slow inactivation state and INa reduction. 3.2.1 Diagnosis of BrS Clinically, BrS patients have right ventricular tachycardia or VF. Selfterminating VF in these patients often results in symptoms of syncope, seizure, and sleep disturbance, as the arrhythmia is more frequent at night. ECG is the most common means for diagnosis. The Brugada consensus reports described the signature ECG and diagnostic criteria. In brief, there are three subtypes of ECG patterns. Type 1 occurs spontaneously and has elevated cove-shaped (>2 mm) ST segment followed by an inverted
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T wave. Type 2 presents a-high-takeoff J-point elevation and a gradually descending ST segment (>1 mm) and a positive or biphasic T wave. Type 3 can resemble that of type 1 or type 2 with
Sodium Channels, Cardiac Arrhythmia, and Therapeutic Strategy Dori Miller, Lili Wang, Juming Zhong1 Department of Anatomy, Physiology & Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Structure and Physiological Function of Cardiac Na+ Channels 3. Cardiac Diseases Associated With Abnormal Na+ Channels 3.1 Long QT syndrome 3.2 Brugada syndrome 3.3 Other cardiac problems 3.4 Mutations of b-subunits and other regulatory proteins 4. Conclusion Conflict of Interest References
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Abstract Cardiac sodium channels are transmembrane proteins distributed in atrial and ventricular myocytes and Purkinje fibers. A large and rapid Na+ influx through these channels initiates action potential and thus excitation–contraction coupling of cardiac cells. Cardiac sodium channel is composed of a pore-forming a-subunit and one or two accessory b-subunits. The cardiac a-subunit is encoded by gene SCN5A located on chromosome 3p21. There are four types of b-subunits identified so far, and b1 is the primary b-subunit in cardiac Na+ channels. The gene responsible for b1 subunits is SCNB. The expression of b-subunits together with a subunits enhances the Na+ current and modifies the channel activities. In addition, interactions of the cardiac Na+ channel with other proteins may facilitate the channel activity and membrane expression of the channel. Over the past two decades, molecular genetic studies have identified the linkage of gene mutations of the Na+ channel proteins and other regulatory proteins to many inherited arrhythmogenic diseases. The most common cardiac arrhythmogenic diseases associated with Na+ channelopathies are long QT syndrome (LQT3) and Brugada syndromes (BrSs). This chapter intends to summarize the current understanding of the normal sodium-channel structure and function, the gene mutation-associated cardiac arrhythmias, and the current diagnosis and management of these diseases. Advances in Pharmacology, Volume 70 ISSN 1054-3589 http://dx.doi.org/10.1016/B978-0-12-417197-8.00012-2
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1. INTRODUCTION The major function of the cardiac contractile pumping is to propel blood throughout the body. Continuous circulating blood delivers nutrients and oxygen to and removes wastes from each organ and also serves as the transporter for neurotransmitters and hormones between various regions of the body. The pumping action of the heart is initiated by the rapidly regulated delivery of the electric signals from the pacemaker, the sinus node, to the contractile proteins in the individual cardiac muscle cells. This complex sequence is referred to “excitation–contraction coupling” (Bers, 2002). At the single cell level, this process begins when an action potential depolarizes the plasma membrane of the cardiac cell. Membrane depolarization activates the voltage-gated calcium channels located mostly in the T-tubules, allowing Ca2+ entry from the extracellular space. This Ca2+ entry triggers large amount of Ca2+ release from the sarcoplasmic reticulum (SR). The combination of Ca2+ entry from the calcium channels and SR Ca2+ release increases the intracellular Ca2+ transient, allowing Ca2+ to bind to troponin C. Binding of Ca2+ with troponin C unmasks the myosin-binding site on the actin molecule. With the consumption of ATP, actin–myosin binding induces cell shorting, and contraction occurs. When Ca2+ is removed from the cytosol by several mechanisms including sarcolemmal and SR Ca2+ ATPase, sarcolemmal Na +/Ca2+ exchange, and mitochondrial Ca2+ uniport, Ca2+ dissociates from the troponin C and relaxation occurs (Bers, 2002). Normal mechanical function and heart rhythm depends on proper electric impulses throughout the myocardium. Generation of myocardial action potential reflects the sequential activation and inactivation of membrane ion channels (ref. Fig. 12.1). Ion channels are pores located in the plasma membrane of the cell and regulate the movement of specific ions. As action potentials differ in different regions of the heart, the following brief description focused on the working myocardium, including both atria and ventricles. Cardiac action potential occurs in five phases (0–4): phase 4 is the resting membrane potential, phase 0 is the rapid depolarization, phase 1 is the rapid repolarization, phase 2 is the plateau, and phase 3 is the final repolarization. Each phase is linked with the opening/closing of specific channels and influx/efflux of the associated ions. During phase 4, the cell membrane is permeable to K+ but both Na+ and Ca2+ channels are closed. When cardiac myocytes are excited by electric stimuli from the conducting cells or
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A
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Figure 12.1 Illustration of action potential, ECG, and associated currents. Shown is a normal action potential and the relative time specific readout from an ECG and ionic current. The dashed line represents changes seen due to abnormal Na+ channel function caused by LQTS. A ¼ action potential. B ¼ ECG. C ¼ Na+ current. D ¼ Ca2+ current. E ¼ K+ current. Dashed line ¼ LQT3.
adjacent cells, the membrane potential will rapidly depolarize (phase 0) due to the opening of fast, voltage-gated Na+ channels. Na+ influx is transient and lasts about 1–2 ms. A brief phase of rapid repolarization (phase 1) follows the peak of the action potential. This is due in part to the closing or inactivation of Na+ channels and in other part to the transient outward K+ current (Ito) and outward Cl current. The plateau or phase 2 of the cardiac action potential results from the balance of the inward and outward current crossing the cell membrane. The inward current is induced by the opening of the slow-activated Ca2+ channels, and the outward currents were carried by Cl- channels and various K+ channels. Repolarization of the plateau (phase 3) occurs due to the inactivation of slow inward Ca and Na+ channels and opening of various slow outward K channels. Other ion transporters
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including outward Cl channels, Na/K pump, and Na/Ca exchanger are all participated in the rapid repolarization. Finally, with the membrane permeation only to K+, membrane potential returns to the resting stage. As described in the preceding text, action potential is a complicated event that requires interplay between multiple voltage-gated ion channels and occurs due to the movement of ions across the sarcolemma. The primary voltage-gated ion channels involved in the cardiac electrophysiology include Na+, K+, and Ca2+ channels. Any change in these channels will induce improper electric property of cardiac muscle, thus causing abnormal cardiac function. This chapter will focus on the normal properties of the Na+ channel, genetic mutation of Na+ channel-induced cardiac problems, and therapeutic strategies.
2. STRUCTURE AND PHYSIOLOGICAL FUNCTION OF CARDIAC NA+ CHANNELS As mentioned previously, INa is critical to generating action potential and E–C coupling in working cardiac myocytes and Purkinje fibers. Transient opening of Na+ channels is responsible for the rapid upstroke of action potential. Fast inactivation of Na+ channels participates in the rapid repolarization of the membrane potential. Aberrations of these elements of Na fluxes in the cell are linked to loss-of-function (decreased peak/early INa) and gain-of-function (increased late INa) syndromes to be discussed in the succeeding text. The first Na+ channel was cloned in 1984 (Noda et al., 1984). Since then, studies have been focused on the structure–function properties of Na+ channels, mutation of channel gene-induced diseases, and potential treatment of these diseases. Voltage-gated Na+ channels are composed of a principal pore-forming subunit (a-subunit) and one or two smaller accessory subunits (b-subunits). When the a-subunit is expressed on its own, it possesses the functional activity for Na+ current, indicating a functional formation of the channels by a-subunit (Catterall, 1986). Na+ channels comprise a major gene family with nine types of a-subunits, which are encoded for by genes SCN1A–SCN11A. These different Na+ channel genes are expressed in various tissues and have different properties. The primary cardiac Na+ channel in humans, Nav1.5, is encoded for by the gene SCN5A located on chromosome 3p21 (George et al., 1995). Na+ channels are differentially expressed across the cardiac tissue. Nav1.5 is highly expressed in the Purkinje fibers, His bundle and bundle branches,
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atria, and ventricles, while less abundant in the atrioventricular nodes. Studies have shown that Nav1.5 are localized within cardiac tissue to the T-tubules, myocyte surface, and intercalated disks. This differential expression can have implications on therapeutic design. For example, studies show that Na+ channels in atria and ventricle respond differently (inactivation differences) to certain pharmacological agents such that treatment must be specific to the Na+ channels involved. The a-subunit of Nav1.5 consists of four homologous domains (DI– DIV), each of which contains six transmembrane segments (S1–S6). The amino acid residues that link the adjacent domains lie in the cytoplasmic side (Fig. 12.2). The amino acid residue linker between S5 and S6 of each domain, which has an extracellular loop dipping down into the membrane, serves as the ion pore. The S4 segment, which contains 6–8 charged residues, serves as the primary voltage sensor. The linker between S3 and S4 is located in the cytosol and serves as the key for the inactivation process (Catterall, 1986). The outer part of the Na+ channel pore is believed to be the loop between S5 and S6. This loop has a short segment (SS1) entering the membrane and another short segment (SS2) exiting the membrane and thus is
Figure 12.2 Schematic topology of cardiac sodium-channel Nav1.5. Na+ channel is composed a single a-subunit and one or two b-subunits. The a-subunit is the pore-forming subunit and consists of four homologous domains (DI–DIV). Each domain is composed of six segments (S1–S6). The linkage between S5 and S6 is considered the outer pore of the channel, where S4 is responsible for detecting voltage changes in the membrane. The cytoplasmic loop between DIII and DIV that contain residues of IFM (denoted by red (dark gray in print version) stars) is believed important for channel fast in activation. b-Subunits play a critical role in the regulation and membrane expression of Nav1.5.
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denoted the SS1–SS2 region. Studies using tetrodotoxin (TTX) and sitedirected mutagenesis determined that a negatively charged glutamate residue at position 387 that links S5 and S6 of domain I at the outside of the membrane is able to bind to the positively charged TTX. Binding of TTX to this site blocks Na+ current through this channel. Neutralization of E387 and other negatively charged residues adjacent to E387 abolished the Na+ current also. Mutation of each of the negatively charged residues at the similar location in other three domains also prevented TTX binding and Na+ current. This suggests that the SS1–SS2 region forms part of the outer pore of the Na+ channel. Furthermore, it is assumed that the SS1–SS2 regions of all four domains form as a ring of negative charges surrounding the external opening of the pore. In addition, the amino acid residues located in this region determine the high selectivity for sodium ions (Heinemann, Terlau, Stuhmer, Imoto, & Numa, 1992; Noda, Suzuki, Numa, & Stuhmer, 1989; Terlau et al., 1991). Activation of voltage-gated Na+ channels results from a protein conformation change driven by membrane potential change. The S4 on each domain serves as the voltage sensor. It contains six to eight positively charged residues, usually arginine or lysine, which are interposed by two nonpolar residues between them. Mutation of these charged residues with neutral or negatively charged residues reduces the voltage-dependent activation of the channel (Stuhmer et al., 1989). Catterall (1986) suggested a helical screw model of the S4 segment. This model suggests that the S4 adopts a a-helical structure in which the positively charged arginine residues are stabilized by interaction with negatively charged residues in adjacent transmembrane domains. In response to a voltage change, the S4 helix moves across the membrane in a spiral path and exchanges ion pairs between the positively charged S4 residues and fixed negative charges in the surrounding transmembrane segments. This movement of S4 on each domain together is able to change the pore conformation, thus opening the channels. Na+ channels display two modes of inactivation: A rapid decay of Na+ currents following short depolarization (within a few milliseconds) is considered fast inactivation. Slow inactivation occurs when cell membrane is depolarized for seconds or longer. These two modes of inactivation are mediated by different molecular mechanisms. Fast inactivation is possibly associated with the intracellular linker between domains III and IV. Early studies by Armstrong and colleagues (Armstrong & Bezanilla, 1977; Armstrong, Bezanilla, & Rojas, 1973; Bezanilla & Armstrong, 1977) indicated that intracellular application of the proteolytic enzyme pronase
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removed fast inactivation of Na+ channels in the squid axon, suggesting that the inactivation gate is located inside the cytoplasm and is accessible to the cytoplasmic enzymes. Using the antibodies specifically against various sites of the intracellular regions of Na+ channel, Vassilev, Scheuer, and Catterall (1988) demonstrated that antibodies directed against the region between domains III and IV prevented Na+ channel inactivation, while antibodies directed at other intracellular regions had no effect. The effect of these antibodies against the DIII–DIV linker on the channel inactivation is voltage-dependent. At negative membrane potentials, the antibody could bind rapidly and inhibited Na+ channel inactivation on depolarization. At more positive membrane potentials where Na+ channels are activated and then partially inactivated, the antibody bound much slower and was much less effective. These results suggest that the linker becomes inaccessible to antibody binding when the channel is inactivated. Subsequent studies using mutagenesis demonstrated that a cluster of three hydrophobic residues within the linker loop of domain III and IV plays an important role in the fast inactivation of Na+ channels. Mutation of the phenylalanine residue at position 1489 to glutamine completely blocks fast inactivation. Mutation of I1488 and M1490 to glutamine, the adjacent two residues of F1489, also slowed the inactivation but to a lesser extent (West et al., 1992). On the other hand, the charged residues within the loop are not responsible for fast inactivation (Patton, West, Catterall, & Goldin, 1992). These studies suggest that the IFM (1488–1490) residues within the linker loop of domains III and IV serve as a hydrophobic latch in the inner mouth of the pore and thus inactivate the channel. An intracellular binding site for the IFM latch seems located at the cytoplasmic ends of S5 and S6 (West et al., 1992). Slow inactivation is similar to fast inactivation but involves different structural elements. Intracellular application of proteolytic enzymes prevented fast inactivation, but did not affect slow inactivation (Armstrong & Bezanilla, 1977; Bezanilla & Armstrong, 1977). Mutations within the domain III and IV linker also had no effect on slow inactivation while successfully blocked the fast inactivation (Patton et al., 1992; West et al., 1992). On the other hand, mutations at the cytoplasmic ends of S5 and S6, which are thought to form the inner mouth of the pore, removed the slow inactivation. This suggests that slow inactivation may involve a conformation change at the inner mouth of the channel (Hayward, Brown, & Cannon, 1997). The b-subunit is composed of a single transmembrane domain with an extracellular N terminus and an intracellular C terminus and is heavily glycosylated. Four b-subunits (b1–b4) are identified so far. b1 is expressed in
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the heart and is encoded by gene SCNB located on chromosome 19q13 (Makita, Bennett, & George, 1994; Makita, Sloan-Brown, Weghuis, Ropers, & George, 1994). Other b-subunits are also expressed in cardiac tissue. It is proposed that Na+ channel is composed of a single a-subunit with one or two b-subunits to form heterodimeric and/or heterotrimeric complexes (Catterall, 2014; Meadows & Isom, 2005). The expression of b-subunits enhances the Na+ current and modifies the channel properties, especially channel inactivation (Catterall, 2014; Isom et al., 1992; Makita, Bennett, et al., 1994; Makita, Sloan-Brown, et al., 1994; Meadows & Isom, 2005).
3. CARDIAC DISEASES ASSOCIATED WITH ABNORMAL Na+ CHANNELS 3.1. Long QT syndrome Long QT syndrome (LQTS) is an inherited or acquired disorder that causes sudden death from cardiac arrhythmias, specifically torsade de pointes and ventricular fibrillation (VF), and the leading cause of sudden cardiac death in young people. Clinically, this problem is characterized by prolonged QT intervals on the surface electrocardiogram (ECG). Prolonged QT intervals reflect increased action potential durations and delayed ventricular repolarizations in cardiac myocytes and typically involve ventricular tachyarrhythmia. Not surprisingly, decreases in repolarizing outward K+ currents or increases in depolarizing inward Na+ or Ca2+ currents can lead to prolongation of the action potential and thus prolongation of the QT interval. Up to now, 13 different types of LQTS have been identified. Molecular genetic studies have demonstrated that three types of congenital LQTS, including LQT1, LQT2, and LQT3, constitute >75% of the clinical cases. While LQT1 and LQT2 are due to the loss of function of voltagegated K+ channels, LQT3 is associated with gain of function of SCNA5 mutations or Na channel proteins, usually resulting from the failure of Na channels to inactivate (Amin, Asghari-Roodsari, & Tan, 2010; Wang, Shen, Splawski, et al., 1995). LQT3 accounts for around 13% of all patients with LQTS and is associated with mutations in SCN5A. Study by Wang, Shen, Splawski, et al. (1995) was the first to report that mutations of SCN5A were associated with the LQTS. Single-strand conformation polymorphism and DNA sequence analyses revealed identical intragenic deletions of SCN5A in affected members of two unrelated LQT families. The deleted sequences reside in a region
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that is important for channel inactivation. Cycle sequencing experiments revealed the presence of a 9 bp deletion beginning at nucleotide 4661 of the cDNA. This deletion disrupts the coding sequence, resulting in a deletion of three conserved amino acids, Lys-1505-Pro-1506-GIn-1507 (KPQ), in the cytoplasmic linker between DIll and DIV. These data suggest that mutations in SCN5A cause chromosome 3-linked LQT and indicate a likely cellular mechanism for this disorder (Wang, Shen, Splawski, et al., 1995). Additionally, the same group identified more mutation sites in SCN5A, in affected members of four LQT families. These mutations include two identical intragenic deletions and two missense mutations. The location and character of these mutations suggest that this form of LQT results from a delay in cardiac sodium-channel fast inactivation or altered voltagedependence of inactivation (Wang, Shen, Li, et al., 1995). Subsequent studies by other groups identified more mutations of SCN5A that associated with LQT3 patients. Currently, 84 SCN5A mutations are known to be related to LQT3. Most of these mutations are missense mutations and are located in the intracellular regions. The transmembrane residues are less frequently affected. It is interesting to note that no LQT3 mutation was detected in the P-loops connecting S5 and S6, in S1 segments, and in the complete transmembrane part of domain 2. This suggests that either gating or inactivation defects leading to a clinically relevant gain of function cannot be achieved by mutating these regions or that respective gain-of-function mutations have not yet been discovered. In contrast to LQT3 mutations, the P-loops seem to be a preferred target for Brugada syndrome (BrS) and conduction disease mutations. Nearly half of the 84 known mutant channels have been studied by heterologous expression and electrophysiological measurements. Most of them are found to cause gain of function of sodium channels in cardiac tissues (Zimmer & Surber, 2008). This gain of function includes abnormal sustained or persistent current compared to wild-type Nav1.5, increased window current, slower inactivation, and faster recovery from inactivation. Persistent current (Isus) represents a continuous flow of Na+ ions through the channel pore during the AP plateau and repolarization phase and is assumed to cause a prolongation of the ventricular AP. This mechanism seems to be the primary cause of the disease, because most SCN5A mutations identified in LQT3 carriers resulted in this inactivation defect. A few examples include a de novo missense mutation (R1623Q, S4 segment of domain 4) identified in an infant Japanese girl with a severe form of LQT3 (Makita et al., 1998). When expressed in oocytes, mutant Na+ channels exhibited only minor
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abnormalities in channel activation but had significantly delayed macroscopic inactivation. Single-channel analysis revealed that R1623Q channels have significantly prolonged open times with bursting behavior (Makita et al., 1998). Another deletion in SCN5A, delQKP 1507–1509, in the DIII–DIV linker of the sodium channel was identified in a family with a typical LQT3 phenotype (Keller et al., 2003). When constructed in vitro and expressed in the tsA201 human cell line, these mutated channels indicated a persistent inward sodium current, which was nearly completely blocked by the sodium-channel blockers TTX and lidocaine. The deletion mutants also resulted in a significant shift of steady-state activation to more depolarized voltages. Lupoglazoff et al. (2001) found a respective positive correlation in patients carrying the V1777M mutation. The homozygous mutation caused a serious QT prolongation (526 ms), whereas the heterozygous mutation in the parents and siblings of the index patient resulted in borderline QTc intervals (415–442 ms). Heterologously expressed V1777M channels generated a pronounced persistent current, whereas simultaneous coexpression of wild-type and mutant channels reduced this current fraction to nearly 50%, which is consistent with the in vivo observation. A window current cannot be assumed because both steady-state activation and inactivation curves were shifted towards hyperpolarized potentials and accelerated recovery from inactivation was not reported (Lupoglazoff et al., 2001). Genetically engineered mice expressing mutant channels that produce large persistent currents also showed the typical features of LQT3 (Remme et al., 2006; Yong et al., 2007). The effect of a persistent current on cardiac AP has been also investigated by mathematical modeling methods (Clancy & Rudy, 2002). All these data together indicate the strict physiological consequence of a persistent Na+ inward current to cause LQT3 syndrome. However, a general correlation between the degree of the persistent current and the length of the QT interval cannot be established for all the known mutations. Mutations causing persistent current are mainly located in the regions involved in fast inactivation of sodium channels. As discussed previously, the inactivation gate in Na+ channels is formed by the DIII–DIV linker where the clustered hydrophobic amino acids isoleucine, phenylalanine, and methionine (IFM motif ) serve as a lid and flanking glycine and proline residues function as molecular hinges “hinged-lid model” (Catterall, 2000; Kellenberger, West, Catterall, & Scheuer, 1997; West et al., 1992). The inactivation gate receptor is formed by multiple peptide segments including amino acid residues in DIVS6 and in intracellular loops DIIIS4/S5 and DIVS4/S5 (Catterall, 2000; Smith & Goldin, 1997). Notably, this
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open-state inactivation is coupled to activation (Chanda & Bezanilla, 2002; Sheets, Kyle, Kallen, & Hanck, 1999). The most severe LQT3 phenotype is produced by a three amino acid deletion in the inactivation loop that links DIII and DIV, dKPQ1505–1507 (Bennett, Yazawa, Makita, & George, 1995; Chandra, Starmer, & Grant, 1998). Mutations in those inactivation gate receptor regions including the S4/S5 linkers in DIII and DIV as well as in DIVS6 destabilize the inactivated state and thus allow the mutant channels to reopen at depolarized potentials. In addition, because of the essential role of the segment 4 in DIV on coupling channel activation and fast inactivation, it is not surprising that mutations in this S4 helix also destabilize the inactivated state. These mutations include R1623Q, R1626P, R1644H, R1623L, and R1644C (Dumaine et al., 1996; Makita et al., 1998; Ruan, Liu, Bloise, Napolitano, & Priori, 2007; Wang, Yazawa, George, & Bennett, 1996). More recently, the first half of the intracellular C terminus region (part nearest to S6 in Fig. 12.1) was predicted to be composed of six helices (H1–H6) that physically interact predominantly with the motif PIPR in the DIII–DIV linker (Cormier, Rivolta, Tateyama, Yang, & Kass, 2002; Motoike et al., 2004). This interaction is thought to stabilize the inactivation gate-occluded channel (Motoike et al., 2004; Shin et al., 2004). Interestingly, a calmodulin-binding motif (IQ motif: IQxxxRxxxxR) in the proposed helix H6 is essential for this interaction, because both the removal of the IQ motif in a C terminal deletion variant (S1885Stop; Cormier et al., 2002) and the specific IQ modification (exchange for alanine; Tester, Will, Haglund, & Ackerman, 2005) resulted in significantly increased persistent currents. Consequently, mutations that eliminate the IQ motif (Q1909R and R1913H; Tester et al., 2005) should also result in a sustained Na+ inward current upon membrane depolarization. Other gain-of-function mechanisms in LQT3 include increased window current, a delayed onset of inactivation, faster recovery from inactivation, and higher peak current density. A window or overlap current results from the overlap of the steady-state inactivation and steady-state activation curves. This voltage range is very narrow for the normal sodium channels. Mutations of SCN5A often result in relative shifts of the steady-state inactivation and activation curves or in increased slope factors of these curves. Such alterations can increase both the critical voltage range and the magnitude of the resulting window current and prolong QT intervals in the absence of a persistent current. Mutations in this group include E1295K (Abriel et al., 2001), A1330P (Wedekind et al., 2001), A1330T (Smits et al., 2005), I1768V channels (Rivolta et al., 2002), and T1620K (Surber et al., 2008).
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Delayed onset of fast inactivation results in a decelerated decay of macroscopic currents. A slower current decay alone should not directly prolong the AP, but may affect voltage-dependent activity of other outward or inward currents that are crucial for AP duration. Examples of these mutations are A1330P (Wedekind et al., 2001), P1332L (Ruan et al., 2007), T1620K (Surber et al., 2008), and Y1795C (Rivolta et al., 2001). A faster recovery from inactivation is mostly associated with a larger inward Na+ current. Because the respective peak current occurred outside the overlap voltage of activation and inactivation, channel reopening occurs as a result of faster recovery from the inactivated state and generates a larger inward current during the ramp compared to wild-type channels. Some examples of this group of mutations include I1768V (Clancy, Tateyama, Liu, Wehrens, & Kass, 2003), A572D and G615E (Albert et al., 2008), and Y1795C (Rivolta et al., 2001). 3.1.1 Diagnosis of LQT3 The clinical manifestations and diagnosis of LQTS have been extensively described in many publications. Those readers who are interested in the details may find these reviews very informative (Schwartz & Ackerman, 2013; Schwartz, Crotti, & Insolia, 2012). Here, we will briefly describe those aspects important to distinguish LQT3 from other LQTS. Clinically, typical cardiac manifestations of LQTS are polymorphic ventricular tachycardia, a syncopal episode called torsades de pointes (TdP), which most of time is self-limiting and often degenerating into VF. These syncopal episodes of VF can occur without changes in heart rate and without specific sequences such as “short–long–short” interval, even though long pauses in LQTS patients increase the probability of TdP. While it had been known that although most patients would develop their symptoms under stress, in a minority of cases, these life-threatening cardiac events could occur at rest and/or during sleep. The reasons for these different patterns remained obscure until molecular biology allowed to researchers distinguish between different genotypes. Most of the events of LQT1 patients occur during exercise or stress. Conversely, most of the events of LQT2 patients occur during emotional stress such as auditory stimuli (sudden noises and telephone ringing, especially while at rest), while for LQT3 patients, they occur during sleep or at rest (Schwartz et al., 2012). LQTS is diagnosed with a prolonged interval between Q and T wave on the surface electrocardiogram. Traditionally, QT intervals longer than 440 milliseconds are considered prolonged, although this value varies among
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genders and ages. Diagnosis must exclude variations in heart rate, serum calcium, or the presence of QT-prolonging drugs. Some LQTS patients have sudden pauses in sinus rhythm exceeding 1.2 s that are not related to sinus arrhythmia and may contribute to the initiation of arrhythmias in LQTS patients. Their occurrence represents an important warning signal in LQT3 patients. In addition, various stress tests with ECG would sufficiently distinguish different type of LQTS, especially type 1 LQTS (Horner, Horner, & Ackerman, 2011; Schwartz & Crotti, 2011). When a patient is suspected of having LQTS by clinical tests mentioned in the preceding text, genetic testing will be a valuable step in the channelopathies of LQTS. Currently, about 80% of LQTS are associated with mutations in three LQTS susceptibility genes, KCNQ1 (IKs channel subunit also known as KvLQT1), KCNH2 (IKr channel subunit also known as HERG), and SCN5A. SCN5A gives rise to LQT3, which accounts for approximately 13% of all genotyped individuals with LQTS (Kapplinger et al., 2009; Wang, Shen, Splawski, et al., 1995).
3.1.2 Treatment of LQT3 LQTS are commonly treated with b-adrenergic blocking agents, left cardiac sympathetic denervation (LCSD), and the implantable cardioverter defibrillator (ICD). They are complemented currently by gene-specific approaches. Historically, b-adrenergic blockers have been used as Na+ channel agonists. Not all b-blockers are equally effective and thus this method has variable success. Common b-blockers used include propranolol, metoprolol, atenolol, and ranolazine. Each has varying effectiveness in controlling LQT3. Differences in blockage of late/sustained Na+ current may play a role in the different clinical efficacy of various b-blockers, and this effect is highest for propranolol, lower but present for nadolol, and completely absent for metoprolol. Given its direct late sodium current blocking properties, propranolol is probably the LQT3-preferred b-blocker (Besana, Wang, George, & Schwartz, 2012). In addition to these common b-blockers, specific sodium-channel blockers are also effective in the treatment of LQT3. This includes mexiletine, which has demonstrated a significant benefit in the treatment of LQT3 (Ruan et al., 2007; Wang et al., 2008). Ranolazine is a newer therapy used for the treatment of LQTS. This compound is a sodiumchannel blocker and specifically targets the late Na+ current but has shown to slightly elongate the QT interval due to minimal blocking of IK (Moss et al., 2008).
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LCSD is a surgical approach to remove the first four thoracic ganglia (T1–T4). The rationale for LCSD is largely based on its antifibrillatory effect and includes a major reduction in norepinephrine release at ventricular level with the absence of postdenervation supersensitivity and no reduction in heart rate (Collura, Johnson, Moir, & Ackerman, 2009). Most LQT3 patients, except the very high-risk symptomatic LQT3 infants, respond well to LCSD (Collura et al., 2009). The ICD is used mostly in cases when a patient has a documented cardiac arrest. There is a common consensus for immediately implanting an ICD to save the life when the patient has a cardiac shock or cardiac arrest. There are several criteria for LQTS patients. Primarily, this approach should be applied for those patients who survived a cardiac arrest and those patients who have high electric instability even with other therapy options (Schwartz & Ackerman, 2013).
3.2. Brugada syndrome BrS was first described in 1992 (Brugada & Brugada, 1992) as a hereditary disease that affects 4 in 10,000 people. The syndrome primarily causes right ventricular tachycardia or VF and presents ST segment elevation in the ECG. Patients usually die from sudden cardiac arrest during sleep. Clinically, the patients have normal heart structure and contractile function, but selfterminating VF in these patients results in symptoms of syncope, seizure, and sleep disturbance, as the arrhythmia is more frequent at night. The ECG characteristics exhibit day-to-day variation and may not always be present (Veltmann et al., 2006). BrS is believed the result of increased heterogeneity of the ion currents involved in the phase I repolarization of action potential in right ventricle. Mutations in 12 different genes encoding Na+ channels, Ca2+ channels, K+ channels, and other membrane proteins have been associated with BrS (Antzelevitch, 2012; Hsiao et al., 2013; Veerakul & Nademanee, 2012). Mutations of the Na+ channel are the most common cause of BrS and account for 15–30% of BrS cases. Chen et al. (1998) was the first to report that a mutation in the SCN5A gene was linked to BrS. They identified a missense mutation, a splice-donor mutation, and a frame-shift mutation in the coding region of SCN5A in three families with BrS. As of 2013, there are more than 200 known SCN5A mutations that associated BrS, and most of them are missense mutations. All the BrS mutations seem to be randomly distributed over the Nav1.5 but seem to cluster in the pore-forming part (Brugada, Brugada, & Roy, 2013; Zimmer & Surber, 2008).
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The functional characteristics of SCN5A mutations associated with BrS1 have been analyzed in mammalian cell lines and mouse models. In contrast to the SCN5A mutations in LQT3 patients that induce gain of function, most SCN5A mutations in BrS1 patients that are heterologously expressed are loss of function, leading to reduced Na+ current. Different types of lossof-function mechanisms were suggested, including expression of nonfunctional channels, decreased expression of total and/or membrane Na+ proteins, and altered gating properties of channel. In the first case, mutated Na+ channel proteins may pass the ER quality control system and traffic normally to the sarcolemma but form channels that conduct no or very small INa (nonfunctional). An example of this group is G1408R (Kyndt et al., 2001). Decreased membrane expression of Nav1.5 proteins results from premature degradation of the mutant proteins by the quality control system in the ER or defective trafficking to the membrane. Mutations of SCN5A with R1432G, G1743R, and T353I are identified to be associated with trafficking deficiency (Baroudi et al., 2001; Pfahnl et al., 2007; Valdivia et al., 2004). Expression may also be decreased because mutant Nav1.5 proteins fail to interact with b-subunits or regulatory proteins, which mediate their normal localization on the sarcolemma. The third mechanism (altered gating properties) comprises delayed activation (i.e., activation at more positive potentials), earlier inactivation (i.e., inactivation at more negative potentials), faster inactivation, and enhanced slow inactivation (Veerakul & Nademanee, 2012). Delayed activation, earlier inactivation, and faster inactivation reduce INa by decreasing the probability of the channels to reside in the activated state. Enhanced slow inactivation means that mutant channels preferentially enter into the slow inactivation state, which is associated with longer recovery times during the action potential. At fast heart rates, phase 4 becomes too short for such channels to recover completely from slow inactivation. This leads to an accumulation of the channels in the slow inactivation state and INa reduction. 3.2.1 Diagnosis of BrS Clinically, BrS patients have right ventricular tachycardia or VF. Selfterminating VF in these patients often results in symptoms of syncope, seizure, and sleep disturbance, as the arrhythmia is more frequent at night. ECG is the most common means for diagnosis. The Brugada consensus reports described the signature ECG and diagnostic criteria. In brief, there are three subtypes of ECG patterns. Type 1 occurs spontaneously and has elevated cove-shaped (>2 mm) ST segment followed by an inverted
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T wave. Type 2 presents a-high-takeoff J-point elevation and a gradually descending ST segment (>1 mm) and a positive or biphasic T wave. Type 3 can resemble that of type 1 or type 2 with
