Thomas J. Hund and Peter J. Mohler

Chapter Outline

Na_v Channel Activity in Health and Disease

Over the course of the action potential, Na⁺ channels undergo a remarkable series of conformational changes that are essential for the rapid and controlled influx of Na⁺ ions that supports the action potential upstroke (detailed in Chapter 1).²³ Recent work to crystallize the bacterial Na⁺ channel Na_vAb (forms as a tetramer of identical subunits akin to most K⁺ channels) has added to decades of important research on the complex Na_v structure–function relationship governing channel activity.^24,25 Briefly, in response to membrane depolarization, channels first activate rapidly as the four charged S4 segments (see Figure 9-1) move outward and open the channel pore.²⁶ Channel activation is followed almost immediately by rapid inactivation caused by further outward movement of the S4 sensors in DIII and DIV and blocking of the channel pore by the DIII-DIV linker that likely depends on the interaction between hydrophobic residues (isoleucine-phenylalanine-methionine, IFM) in the III-IV linker and multiple sites in the linkers between S4 and S5 in DIII and DIV and the cytoplasmic end of S6 in DIV.^27–29 Rapid inactivation is followed by transition into several different slow inactivation states that are controlled, in part, by P segments linking S5 and S6 segments that form the inner channel pore (see Figure 9-1). Importantly, disruption of Na⁺ channel gating at any step during this highly coordinated set of movements may result in inappropriate (elevated, reduced) current and give rise to arrhythmias.

Dysfunction in Na_v1.5 activity has been identified as a potential cause of arrhythmia in several forms of congenital and acquired disease (see Figure 9-1). Classic examples of the link between inherited defects in Na_v1.5 function and disease may be found in two inherited arrhythmia syndromes closely linked to mutations in SCN5A: Brugada syndrome and LQT3. An overview of molecular basis for these diseases is provided here; both are addressed in detail in later chapters. Brugada syndrome is an autosomal, dominant cardiac arrhythmia syndrome characterized by ST-segment elevation in the right precordial leads (V₁-V₃) and sudden death in the absence of overt structural disease.^30,31 The syndrome is caused by loss-of-function variants in SCN5A (although identified in only about 20% of Brugada cases³¹) that result in loss of Na⁺ current early in the action potential because of a loss of functional channel expression/localization or defects in channel gating.^20,32,33 The substrate for Brugada syndrome arrhythmia is thought to arise in the epicardium of the right ventricle where high expression of transient outward K⁺ current I_to may shift the balance of current to favor exaggerated phase 1 repolarization (notch) and even loss of the action potential plateau.^34,35 Loss-of-function SCN5A variants have also been associated with isolated cardiac conduction disease characterized by slow conduction throughout the heart (even requiring pacemaker implantation) without the repolarization abnormalities or ventricular tachyarrhythmia observed in Brugada syndrome.³⁶ In general, gene variants that give rise to cardiac conduction disease produce a less severe effect on channel function than those associated with Brugada syndrome, by introducing a secondary gain-of-function change in Na_v activity (e.g., depolarizing shift in voltage dependence of inactivation) that partially balances the primary loss-of-function change (e.g., depolarizing shift in voltage dependence of activation).

Although Brugada syndrome is a disease associated with Na_v loss-of-function, LQT3 represents the other extreme and illustrates how Na_v gain-of-function may also produce arrhythmia. A heterogeneous autosomal dominant genetic disease, LQT3 is associated with abnormal QT-interval prolongation on the electrocardiogram, syncope, polymorphic ventricular tachycardia, and sudden death.¹⁴ Variants in SCN5A are the cause of LQT3, which is associated with increased risk of cardiac events during rest and decreased efficacy of β-blocker therapy.^14,37,38 At least 50 variants in SCN5A have been identified as causal for LQT3, and although these variants are scattered throughout the cytoplasmic face of the channel (with a cluster in the S4 segments), they primarily produce an increase in Na⁺-channel current (gain-of-Na_v function) by disrupting rapid inactivation, by shifting the voltage-dependence of activation or inactivation, or by altering recovery from inactivation. A prototypical case study of the causal link between SCN5A mutations and LQT3 comes from the first reported LQT3 mutation resulting in three–amino acid deletion in the DIII-DIV linker that is important for rapid channel inactivation (ΔKPQ, see Figure 9-1).³⁸ The variant ΔKPQ allele disrupts rapid inactivation, producing a small (~0.5% of peak) but sustained Na⁺ current during the action potential plateau that shifts the balance of current to prolong action potential and increase the likelihood of arrhythmogenic afterdepolarizations.^38,39 Although LQT3 and Brugada syndrome are often presented as two very different faces of Na_v dysfunction, overlap syndromes have been reported, with features of both diseases showing QT prolongation at slow rates coupled with ST elevation during exercise. Notably, the 1795insD mutation (see Figure 9-1) interrupts rapid inactivation to enhance persistent Na⁺ current and prolong action potential duration, especially at slow pacing rates, but it also enhances an intermediate inactivation state that compromises Na_v recovery between stimuli, leading to loss of current at fast rates.^40,41 Thus, as discussed before, for cardiac conduction disease, a single molecular defect may alter channel activity at more than one phase (e.g., rapid and slow inactivation processes) in the gating cycle, giving rise to a complex disease phenotype.

Studies of rare congenital conditions such as LQT3 and Brugada syndrome have generated important insight into Na_v channel function and have increased our understanding of arrhythmogenesis in the more complex setting of acquired heart disease, where Na_v channel dysfunction is a common finding. In heart failure, for example, an increase in persistent (late) Na⁺ current has been observed both in patients and animal models of human disease.42–46 Similar to LQT3, in heart failure it is likely that increased late Na⁺ current delays action potential repolarization and promotes pro-arrhythmogenic afterdepolarizations.⁴⁵ Increased Na⁺ entry via the late current may also disrupt normal Ca²⁺ homeostasis and promote mechanical dysfunction and progression of disease.⁴⁷ The underlying mechanism for changes in Na_v function in heart failure is unknown but is most likely not a result of changes in expression of Na_v α or β-subunits.^42,45 In light of known defects in β-adrenergic and calcium-dependent signaling pathways in heart failure, it is likely that defects in channel posttranslational modification play a role in Na_v dysfunction. Regardless of Na⁺ current mechanism, mounting studies support the late Na⁺ current as a viable therapeutic target (e.g., inhibition with ranolazine) for preventing arrhythmias and progression of disease in heart failure.^48–51

There have also been many studies on the link between defects in Na_v function and arrhythmias after myocardial infarction, where reentrant arrhythmias are highly localized to the highly remodeled tissue surrounding the infarct (border zone). In particular, in the canine heart, dramatic electrical and structural remodeling has been identified coupled with anisotropic conduction and reentrant arrhythmias in the border zone region. Conduction through this region is highly irregular, characterized by slow and discontinuous conduction.⁵² Myocytes isolated from the border zone regions display a characteristic time course of changes in ion channel activity and action potential, including significant decrease in peak Na⁺ current, altered subcellular localization, decreased channel availability, and slow recovery from inactivation.^53–55 Computational studies demonstrate that measured defects in Na_v channel activity prolong refractoriness, decrease conduction velocity, and increase susceptibility to initiation of reentrant arrhythmias.^53,56–59 Although the precise mechanism underlying abnormal Na_v function after myocardial infarction is likely multifactorial and not fully understood, roles for increased oxidative stress and/or increased CaMKII-dependent channel phosphorylation have been identified.^57,60 Interestingly, adenoviral overexpression of skeletal muscle Na_v channel, with inactivation shifted to more depolarized potentials compared with cardiac isoform, has been shown to improve conduction and suppress arrhythmias in the canine after myocardial infarction.⁶¹ It is intriguing to consider the possibility that targeting channel regulatory pathways (e.g., CaMKII) may be a second, and conceivably more practical, method to alter channel availability for therapeutic benefit.

Na_V Channel Trafficking and Stabilization in Health and Disease

As outlined before, Na_v function is critical for normal heart function, and alteration of Na_v activity is a hallmark of many forms of inherited and acquired diseases. Despite major advances, the mechanistic link among specific molecular defects, Na_v channel dysfunction, and arrhythmias associated with many human arrhythmia variants and in common disease remains elusive. Mounting evidence, in particular from human arrhythmia variants in genes encoding ion channel accessory proteins (e.g., adapter and scaffolding proteins, channel subunits, chaperones), highlight the importance of proper Na_v channel localization within specific cellular domains for normal heart function.62–69

Voltage-gated Na_v channels are targeted to specific membrane domains in cardiomyocytes (Figure 9-3). Residing primarily at cell ends in the region of the intercalated disc (ID), Na_v channels are also found at secondary sites at lateral and transverse-tubule membranes (see Chapter 18). Although the cardiac isoform Na_v1.5 largely comprises the Na_v population at the ID, neuronal isoforms (e.g., Na_v1.1, Na_v1.3, Na_v1.6) are found almost exclusively at transverse tubules.^12,13,70 Although the functional consequences for differential targeting of Na_v isoforms within the cardiomyocyte remain unclear, computational studies demonstrate that concentration of Na_v channels at the ID (Na_v1.5), where cells are electrically and mechanically coupled, supports electrical impulse propagation.^71,72 Likewise, a role for the neuronal isoforms (largely outside the ID) has been explored using low doses of tetrodotoxin (TTX) (insufficient to block TTX-resistant Na_v1.5). These experiments show that although neuronal channels are not necessary for conduction under normal conditions, they may play modulatory roles in excitation-contraction coupling and ventricular function.^12,13

An important outstanding question is how are Na_v channels differentially targeted and regulated within the cardiomyocyte? The answer may be found in the growing family of adapter, accessory, cytoskeletal and regulatory proteins—including α1-syntrophin, ankyrin-G, βIV-spectrin, glycerol-3-phosphate dehydrogenase 1–like protein, MOG1, Nedd4-2, SAP97, and calmodulin—that reside in macromolecular complexes with Na_v channels (see Figure 9-2).^4,5,16–22 Recent studies demonstrate that interactions between Na_v and these related proteins: (1) regulate channel function, (2) differ depending on local membrane domain, and (3) are altered in disease.

The Na_v1.5 α-subunit associates with one or more β-subunits (β1-β4). These β-subunits are type I integral membrane proteins with an extracellular N-terminus containing an immunoglobulin domain with homology to domains found in cell adhesion molecules, a single transmembrane domain, and a cytoplasmic C-terminal sequence.⁷³ Differential localization of β-subunits has been reported in mouse heart, with co-localization of β1 and β2 found at the intercalated disc and T-tubules, β3 at T-tubules, and β4 at the intercalated disc.¹² Beta-subunits are reported to alter Na_v1.5 membrane expression and gating. Importantly, human mutations in Na_v β-subunits have been linked to diseases and conditions, including Brugada syndrome (SCN1B and SCN3B), long QT (SCN4B), cardiac conduction disease (SCN1B), and atrial fibrillation (SCN1B, SCN2B and SCN3B).^4,67,73–76

The ankyrin family of multifunctional adapter proteins (e.g., ankyrin-R, ankyrin-B, and ankyrin-G expressed in the heart) coordinates macromolecular complexes at specific membrane domains in cardiomyocytes. The most studied ankyrin in the heart, ankyrin-B, is involved in targeting of the Na⁺/Ca²⁺ exchanger, Na⁺/K⁺ adenosine triphosphatase (ATPase), Kir6.2, InsP₃ receptor, and protein phosphatase 2A to transverse tubules.77–80 Ankyrin-G, on the other hand, is concentrated at the cardiomyocyte ID, where it associates directly with Na_v1.5 through binding to a conserved motif in the DII-DIII linker (see Figures 9-2 and 9-3).^19,20,81 Interfering with ankyrin-G/Na_v1.5 interaction, either through knockdown of ankyrin-G or expression of mutant Na_v1.5 lacking ankyrin-binding activity, disrupts Na_v1.5 membrane, targeting resulting in loss of Na⁺ current.^19,20 Recently, ankyrin-G has been identified in a functional macromolecular complex in vivo with Na_v1.5, the actin-associated polypeptide βIV-spectrin, and calmodulin kinase II (CaMKII) for targeting and regulation of Na_v function and cell membrane excitability.⁸² Ankyrin-G also has been shown to associate and co-localize with desmosome and gap junction proteins plakophilin-2 and connexin43,⁸³ supporting the notion that ankyrin-G is an important nodal point for coordinating mechanical and electrical signaling at the cardiomyocyte ID. Importantly, defects in ankyrin-based pathways have been identified as the underlying cause for abnormal channel targeting and arrhythmias in congenital and acquired forms of cardiac disease.^{20,62,84–86} In the case of Na_v1.5, a human SCN5A variant linked to Brugada syndrome (E1053K) is located in the ankyrin-binding motif of Na_v1.5 and disrupts ankyrin-binding activity in vitro (see Figure 9-1).²⁰

Na_v channels display unique biophysical properties based on their specific membrane localization.⁷⁰ Consistent with these findings, work from several groups has recently shown that Na_v1.5 complexes with distinct partners at the ID versus lateral membranes.⁸⁷ In addition to ankyrin-G, Na_v1.5 has been found to associate with synapse-associated protein SAP97, a member of the membrane-associated guanylate kinase family, at the cardiomyocyte ID (see Figure 9-3).⁸⁷ This interaction depends on the terminal three amino acids of the Na_v1.5 C-terminus (see Figure 9-2), and knockdown of SAP97 leads to a decrease in I_Na and reorganization of Na_v1.5 in adult cardiomyocytes. Na_v1.5 is also found, albeit to a much lower degree, at the lateral membrane and is likely regulated by interaction with the dystrophin-syntrophin complex through direct binding of Na_v1.5 C-terminus with a PDZ domain in syntrophin.^88–91 Na⁺ current and Na_v1.5 expression are decreased in mice lacking dystrophin (mdx^5cv), and human mutations in SNTA1 (encodes α1-syntrophin) have been identified as the cause of increased Na⁺ current in congenital long QT syndrome, and sudden infant death syndrome.^90–92,87 It will be interesting in the future to determine the precise role of Na_v1.5 interacting partners at the intercalated disc and lateral membrane in targeting, retention, and function of the channel.

Na_v Channel Posttranslational Regulation in Health and Disease

Tight spatial and temporal control of local signaling domains is essential for proper regulation and activity of Na_v channels in cardiomyocytes. Importantly, changes in posttranslational modification of membrane proteins are associated with increased susceptibility to congenital and acquired arrhythmia.90,93–95 In particular, Na_v channel activity is heavily regulated by posttranslational modification, including phosphorylation by protein kinase C,^96–98 protein kinase A (PKA),^99–102 CaMKII,^82,103–106 and glycosylation.^4,107 Important in the setting of heart disease, β-adrenergic stimulation is known to increase I_Na through PKA-direct and PKA-indirect modulation.^99–101,108 As discussed before, increased I_Na (late) likely contributes to arrhythmogenesis in heart failure. In parallel, β-adrenergic stimulation is enhanced in failing hearts. Therefore, it is interesting to consider the possibility that defects in Na_v1.5 posttranslational modification downstream of β-adrenergic stimulation may underlie I_Na dysfunction in heart failure. Although PKA directly phosphorylates Na_v1.5 at specific residues in the DI-DII linker (see Figure 9-2),¹⁰² studies with PKA inhibitors suggest that this accounts for only part of the effect of β-adrenergic stimulation on Na_v function.^101,109 PKA-independent effects likely involve regulation of Na_v1.5 surface expression by the stimulatory G protein α-subunit (Gαs) and caveolin-3 (CAV3).^101,109,110 Human gene variants in CAV3 have been linked to increased late (persistent) I_Na and arrhythmias in long QT syndrome type 9.⁶⁵ Although the link between mutant CAV3 and changes in Na_v function remain unknown, computational studies suggest that disruption of CAV3/Gαs interaction may alter the dynamics of caveolae membrane fusion to give rise to increased late Na⁺ current.¹¹¹

Na_v channels are also regulated by Ca²⁺/CaM-dependent pathways, which are known to be dysregulated in disease.112,113 CaM interacts directly with multiple Na_v isoforms, including Na_v1.5 via a C-terminal IQ motif and a domain in the DIII-DIV loop with variable results.^113–119 Although the net effect of CaM on channel activity likely depends on factors such as specific isoform and experimental conditions, binding of CaM to the IQ motif of human Na_v1.5 has been shown to enhance slow component of inactivation,¹¹³ with other studies showing CaM-dependent effects on steady-state inactivation.^105,114,118 Interestingly, the human mutation A1924T in the Na_v1.5 IQ motif (see Figure 9-1) results in Brugada syndrome and alters channel activity by interrupting CaM binding to Na_v1.5.^113–115 More recently, a role for Ca²⁺/CaM regulation of Na_v1.5 via CaMKII-dependent phosphorylation has been described.^{82,104–106,112} Specifically, initial studies showed that overexpression of CaMKIIδc in rabbit (adenoviral, acute) and mouse (transgenic, chronic) ventricular cardiomyocytes shifted steady-state inactivation in the hyperpolarizing direction (decreased availability), increased intermediate activation, slowed recovery from inactivation, and enhanced late Na⁺ current.¹⁰⁴ At the cellular level, CaMKIIδ overexpression resulted in accumulation of intracellular Na⁺ and prolongation of APD, which translates to increased QRS (marker for slow intraventricular conduction) and QT intervals on the electrocardiogram, as well as increased susceptibility to ventricular arrhythmias.¹⁰⁴ It is now clear that CaMKII phosphorylates the channel at multiple sites in the DI-DII linker (including S516, S571, T594) (see Figure 9-2) to alter steady-state channel availability, recovery from inactivation and persistent current.^82,105,106 Furthermore, CaMKII-dependent regulation of Na_v1.5 depends on direct interaction with the actin-associated polypeptide βIV-spectrin, which targets CaMKII with Na_v1.5 to the cardiomyocyte intercalated disc.⁸² Loss of spectrin/CaMKIIδ interaction disrupts CaMKII regulation of Na_v1.5 and abnormal cell membrane excitability. Future studies should determine the broader role for βIV-spectrin in coordinating Na_v macromolecular complexes at the cardiomyocyte intercalated disc and its role in arrhythmogenesis.

Summary and Future Directions

The vertebrate heart has evolved, highly specialized pathways for targeting and regulation of Na_v channels, reflecting their central role in control of cardiac excitation-contraction at baseline and in disease. Proper localization of Na_v within local signaling domains is essential for normal membrane excitability and heart function. Importantly, mounting evidence demonstrates that defects in local signaling and regulation of Na_v channels underlie abnormal cell excitability and arrhythmia in heart disease, including human heart failure. As we learn more about the constituency, localization and function of specific Na_v macromolecular complexes within the cardiomyocyte, we anticipate the discovery of new therapeutic targets and strategies for preventing arrhythmias and improving heart function in human heart disease patients.

Acknowledgments

This work was supported by the National Institutes of Health grants HL084583 and HL083422 (to P.J.M.) and HL096805 (to T.J.H.), the Pew Scholars Trust (to P.J.M.), the American Heart Association (to P.J.M.), the Saving Tiny Hearts Society (to P.J.M.), the Gilead Sciences Research Scholars Program (to T.J.H.), and the Fondation Leducq Award to the Alliance for Calmodulin Kinase Signaling in Heart Disease (to P.J.M.).

References