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