Voltage-Gated Sodium Channel β1/β1B Subunits Regulate Cardiac Physiology and Pathophysiology

Cardiac myocyte contraction is initiated by a set of intricately orchestrated electrical impulses, collectively known as action potentials (APs). Voltage-gated sodium channels (NaVs) are responsible for the upstroke and propagation of APs in excitable cells, including cardiomyocytes. NaVs consist of a single, pore-forming α subunit and two different β subunits. The β subunits are multifunctional cell adhesion molecules and channel modulators that have cell type and subcellular domain specific functional effects. Variants in SCN1B, the gene encoding the Nav-β1 and -β1B subunits, are linked to atrial and ventricular arrhythmias, e.g., Brugada syndrome, as well as to the early infantile epileptic encephalopathy Dravet syndrome, all of which put patients at risk for sudden death. Evidence over the past two decades has demonstrated that Nav-β1/β1B subunits play critical roles in cardiac myocyte physiology, in which they regulate tetrodotoxin-resistant and -sensitive sodium currents, potassium currents, and calcium handling, and that Nav-β1/β1B subunit dysfunction generates substrates for arrhythmias. This review will highlight the role of Nav-β1/β1B subunits in cardiac physiology and pathophysiology.


INTRODUCTION
The heart contracts to pump blood throughout the body. It consists of specialized cells called cardiac myocytes (CMs), and contraction of CMs is initiated by electrical impulses called action potentials (APs) (Nerbonne and Kass, 2005). Cardiac APs are generated and propagated through the coordinated signaling of ion channels. Upon membrane depolarization, voltage-gated sodium channels (Na V s) activate and inactivate rapidly to allow sodium influx (Hille and Catterall, 2012). This is responsible for the rising phase and propagation of the AP in mammalian CMs (Nerbonne and Kass, 2005). Na V s are heterotrimeric transmembrane proteins consisting of one pore-forming α and two β subunits (Catterall, 2000). Na V -β subunits are expressed in mammalian heart (Isom et al., 1992;Makita et al., 1994) and their functional loss can result in electrical abnormalities that predispose patients to arrhythmias. Variants in the gene SCN1B, encoding the splice variants Na V -β1 and Na V -β1B, are implicated in a variety of inherited pathologies including epileptic encephalopathy (O'Malley and Isom, 2015), Brugada syndrome (BrS) (Watanabe et al., 2008;Hu et al., 2012), long-QT syndrome (LQTS) (Riuró et al., 2014), atrial arrhythmias (Watanabe et al., 2009), and sudden infant death syndrome (SIDS) (Hu et al., 2012) (Figure 1, Table 1). Remarkably, regardless of disease etiology, patients with SCN1B mutations have an increased risk of sudden death. Classically, the Na V -β subunits were characterized as modulators of the Na V ion-conducting pore. However, from research over the past two decades, we know that Na V -β subunits are dynamic, FIGURE 1 | SCN1B variants are linked to epilepsy syndromes and cardiac conduction diseases. SCN1B encodes Na v -β1 (left) and its secreted splice variant Na v -β1B (right). Sites for β1-α interaction, ankyrin binding, phosphorylation, and proteolytic cleavage are indicated. Ig: immunoglobulin. Human disease variants in β1 or β1B are indicated in yellow and are described in Table 1. Adapted from O'Malley and Isom (2015). multifunctional proteins that play important roles in cardiac physiology (O'Malley and Isom, 2015). Here, we will focus our review on the current understanding of Na V -β1/β1B function in CMs and discuss disease implications.

NA V S ARE DIFFERENTIALLY EXPRESSED IN CARDIAC MYOCYTES
To understand Na v -β subunit physiology in heart, one must first consider the associated Na v -α subunits. Na v 1.5 is the predominantly expressed Na V -α in CMs and the primary contributor to recorded sodium current (I Na ) density (Rogart et al., 1989;Gellens et al., 1992;Catterall, 2000;Maier et al., 2002). Na v 1.5 is a "tetrodotoxin resistant (TTX-R)" channel (Catterall et al., 2005), in contrast to "TTX-sensitive (TTX-S)" channels, e.g., Na V s normally found in brain, for which TTX has nanomolar affinity (Catterall et al., 2005). TTX has micromolar affinity for Na v 1.5 due to the presence of a cysteine residue in the selectivity filter in a position that is otherwise filled by an aromatic amino acid in TTX-S channels (Satin et al., 1992). TTX-S channels, Na v 1.1, Na v 1.3, and Na v 1.6, are expressed in heart as well as in brain Lopez-Santiago et al., 2007). They are preferentially localized in the transverse tubules (T-tubules) (Malhotra et al., , 2002Lopez-Santiago et al., 2007) where they are postulated to function in excitationcontraction coupling (Maier et al., 2002) (Figure 2).
Na v 1.5 contributes to at least two distinct multiprotein complexes in ventricular CMs, one at the lateral membrane containing dystrophin and syntrophin (Figure 2), and the other at the ID involving the membrane-associated guanylate kinase (MAGUK) protein adapter protein, synapse-associated protein 97 (SAP97), and ankyrin-G (Petitprez et al., 2011) ( Figure 3C). In heterologous cells, surface expression of Na v 1.5 is regulated by its interaction with SAP97 via a PDZdomain (post-synaptic density protein-PSD95, disc large tumor suppressor-Dlg1, zonula occludens1-ZO1). Either the truncation of the fourth domain of Na v 1.5 (Shy et al., 2014) or depletion of SAP97 (Matsumoto et al., 2012) results in reduced channel cell surface expression, with a subsequent decrease of I Na .
Na v 1.5 also interacts with fibroblast growth factor homologous factor 1B (FHF1B) (Liu et al., 2003), calmodulin (Kim et al., 2004;Young and Caldwell, 2005), Nedd4like-ubiqutin-protein ligases (Van Bemmelen et al., 2004;Rougier et al., 2005), and is phosphorylated by Fyn (Ahern et al., 2005), a src family tyrosine kinase, all of which are involved in the regulation of channel subcellular localization and activity ( Figure 3C). Taken together, these results accentuate the idea that cardiac Na V s associate with protein complexes that are specific to subcellular domains, and these interactions are critical to cardiac physiology. Undoubtedly, changes in one component of a given complex results in significant consequences to overall cardiac excitability and synchrony.
FIGURE 2 | TTX-S Na v s are localized to T-tubules. TTX-S Na v s, including Na v 1.1, Na v 1.3, and Na v 1.6, are located at the T-tubules of CMs where they are thought to participate in the regulation of excitation-contraction coupling. Non-phosphorylated Na v -β1 subunits are co-localized with TTX-S Na v -α subunits at the T-tubules where they play roles in calcium signaling and homeostasis. Na v 1.5 is localized at the lateral membrane as well as the ID (Figure 3). At the lateral membrane, Na v 1.5 is complexed with syntrophin and dystrophin. Abbreviations: L-type calcium channel (LTCC), phospholamban (PLB), ryanodine receptor (RyR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), sodium-calcium exchanger (NCX), transverse tubules (T-tubule).
Canonically, Na V -βs are known as modulators of Na V electrophysiological properties and cell surface expression (Brackenbury and Isom, 2011). Heterologous expression systems and mouse models have shown that Na V -βs modulate Na V − αs in cell type specific manners, thus the Na V α/β subunit composition of a given cell confers unique biophysical properties that can be finely tuned (Calhoun and Isom, 2014). Not surprisingly, Na V -β1 modulation of Na v 1.5 varies depending on the system studied. In Xenopus oocytes, the amplitude of Na v 1.5 expressed I Na increases with increasing amounts of β1 mRNA (Qu et al., 1995). Antisense-mediated post-transcriptional silencing of Scn1b in H9C2, a CM line, alters TTX-S and TTX-R FIGURE 3 | VGSC complexes at the cardiac intercalated disk. CMs associate at the ID, where Na v 1.5, Nav-β1 subunits, adherens junctions, gap junctions, and desmosomes define intercellular communication. (A) Associated CMs. (B) Proposed model of the GJ plaque, perinexus, and perinexus edge. Nav-β1 subunits at the ID are tyrosine phosphorylated, possibly through Fyn kinase activation, and may function in cell-cell adhesion in the perinexus and perinexus edge. (C) At the ID, Nav1.5 associates with a multi-protein complex (also see Table 2). The S4 segment of each Na v -α subunit homologous domain forms the voltage sensing domain (VSD) and segments 5 and 6 in each domain create the ion-conducting pore. Three hydrophobic amino acids, IFM, form the inactivation gate. Abbreviations: Ankyrin-G (AnkG), Calmodulin (Cal), Ca 2+ /calmodulin-dependent protein kinase II (CaMKIIδ), Fibroblast growth factor homologous factor 1B (FHF1B), N-Cadherin (N-Cad), Nedd4-like-ubiqutin-protein ligases (Nedd4), Synapse-associated protein 97 (SAP97).
Na v -α mRNA and protein expression, resulting in decreased I Na (Baroni et al., 2014). In contrast, Scn1b null mouse CMs have increased expression of Scn3a and Scn5a, along with increased TTX-S and TTX-R I Na (Lopez-Santiago et al., 2007). In heterologous cells, Na V -β1 expression results in slight changes in Na v 1.5 I Na , but significant effects on voltage-dependence and channel kinetics. In Tsa201 cells transfected with Na v 1.5, coexpression of Na V -β1 positively shifts the voltage-dependence of inactivation . Co-expression of Na V -β1 with Na v 1.5 in Xenopus oocytes causes a depolarizing shift in steady-state inactivation compared with WT alone (Zhu et al., 2017), suggesting that β1 may allow the α subunit voltagesensing domains to recover more rapidly to the resting state. Thus, Na V -β1 may initiate fine-tuned acute and chronic feedback mechanisms that differentially control expression and function of Na V -αs in the heart.
Na v -β1B is expressed in fetal brain and in heart at all developmental time points. When expressed alone or in the Stability and anchoring to the cell membrane Petitprez et al., 2011;Matsumoto et al., 2012 presence of TTX-S Na v -αs in a heterologous expression system, Na v -β1B is secreted (Patino et al., 2011). Secreted Na v -β1B functions as a CAM ligand to promote signal transduction in cultured neurons (Patino et al., 2011). In contrast, Na V -β1B is retained at the cell surface when co-expressed with Na v 1.5 (Patino et al., 2011) and Na v -β1B co-expression increases I Na density compared to Na V 1.5 alone (Watanabe et al., 2008). The disease variant, β1B-G257R (Figure 1, Table 1), causes Na v -β1B to be retained inside the cell, resulting in a functional null phenotype (Patino et al., 2011). The variant, β1B-W179X (Figure 1, Table 1), fails to increase Na v 1.5 I Na density, suggesting that it may also be a functional null mutation (Watanabe et al., 2008). A number of Na v -β1B variants have now been linked to cardiac arrhythmias (Figure 1, Table 1), thus this subunit is critical to cardiac physiology. While the Na v -αs are known to form and function as monomers, recent evidence suggests that they can also form dimers, and that dimerization is mediated through an interaction site within the first intracellular loop (Clatot et al., 2017). Na V -α dimers display coupled gating properties, which are mediated through the action of 14-3-3 proteins (Clatot et al., 2017). The 14-3-3 family of proteins is important for the regulation of cardiac I Na , and disrupted 14-3-3 expression may exert pro-arrhythmic effects on cardiac electrical properties (Allouis et al., 2006;Sreedhar et al., 2016). The functional importance of cardiac Na V -α dimerization may be to target and enhance the density of channels at specific subcellular domains. Na v 1.5-R1432G, a surface localization defective SCN5A mutant, displays a dominant negative effect on WT Na v 1.5, but only in the presence of Na V -β1 (Mercier et al., 2012). Thus, Na V -β1 may normally mediate physical interactions between Na v 1.5 dimers, however further research must be performed.

NA V -βS DO MORE THAN MODULATE I NA
Na V -β subunits are multifunctional (O'Malley and Isom, 2015). In addition to modulating channel gating and cell surface expression/localization, Na V -βs are Ig superfamily cell adhesion molecules (CAMs) that facilitate cell-cell communication and initiate intracellular signaling cascades. Na V -β1 and -β2 participate in trans-homophilic cell adhesion, resulting in the recruitment of ankyrin-G to the plasma membrane at sites of cell-cell contact (Malhotra et al., 2000). Importantly, this occurs both in the presence and absence of Na V -α, at least in vitro. Na V -β1 and -β2 also participate in cell-matrix adhesion, binding tenascin-R and tenascin-C to modulate cell migration (Srinivasan et al., 1998;Xiao et al., 1999). The Na V -β3 amino acid sequence is most similar to Na V -β1 compared to the other Na V -β subunits (Morgan et al., 2000). While Na V -β3 does not function as a CAM when expressed in Drosophila S2 cells, as shown for Na V -β1 and -β2 (Chen et al., 2012), it does so in mammalian cells where trans homophilic adhesion was shown to require an intact Cys2-Cys24 disulfide bond (Yereddi et al., 2013). Na V -β function, localization, and expression are regulated by multiple post-translational modifications including phosphorylation, glycosylation, and proteolytic cleavage (Calhoun and Isom, 2014). All Na V -βs have highly glycosylated N-terminal domains, containing 3 to 4 N-linked glycosylation sites each (Isom et al., 1992;McCormick et al., 1998;Johnson et al., 2004), and these modifications contribute to cell surface expression and channel modulation (Johnson et al., 2004). Lastly, Na V -βs are targets for sequential proteolytic cleavage by α-secretase/BACE1 and γ-secretase, resulting in the release of N-terminal and C-terminal domains (Wong et al., 2005). These cleavage products may have important physiological effects on transcriptional regulation of Na V -α subunit genes. For example, γ-secretase cleavage of Na V -β2 in neurons in vitro leads to translocation of the intracellular domain to the nucleus, where it increases SCN1A mRNA expression and Na v 1.1 protein (Kim et al., 2007).
Of the five Na v -β subunits, Na v -β1 has been the most studied in terms of its CAM function. In the heart, Na V -β1 ID localization suggests a role in cardiac cell-cell contact. Scn1b and Scn5a have overlapping temporal and spatial expression profiles during heart development (Domínguez et al., 2005). In ventricular CMs, Na V -β1 is co-localized at the ID (Kaufmann et al., 2010) with Na v 1.5 (Maier et al., 2004), as well as at the T-tubules with TTX-S channels Lopez-Santiago et al., 2007). Recent evidence suggests that Na V -β1-mediated cell-cell adhesion may occur at the perinexal membrane, and this putative interaction can be acutely inhibited by βadp1, a novel peptide mimetic of the Na V -β1 CAM domain (Veeraraghavan et al., 2016). Dose-dependent administration of βadp1 decreased cellular adhesion in Na V -β1-overexpressing fibroblasts. 75% of βadp1-treated hearts exhibited spontaneous ventricular tachycardias, revealing preferential slowing of transverse conduction. These data support a role for trans Na V -β1-mediated cell-cell adhesion at the perinexal membrane and suggest a role for adhesion in conduction (Figure 3B). Because a large proportion of SCN1B disease variants affect the Ig domain (Figure 1), it is likely that disruption of Na V -β1-mediated cellcell adhesion contributes to disease mechanisms and, if so, that restoring adhesion may be a future therapeutic target.
The Na V -β1 intracellular domain can be phosphorylated at tyrosine (Y) residue 181 (Malhotra et al., 2002(Malhotra et al., , 2004McEwen et al., 2004), possibly through activation of Fyn kinase (Brackenbury et al., 2008;Nelson et al., 2014) (Figure 1). β1Y181E, a phosphomimetic, participates in cell adhesion but does not interact with ankyrin or modulate I Na , suggesting that Y181 is an important regulatory point for cytoskeletal association and channel modulation (Malhotra et al., 2002). In CMs, tyrosine-phosphorylated Na V -β1 and non-phosphorylated Na V -β1 are differentially localized to subcellular domains where they interact with specific cytoskeletal and signaling proteins (Malhotra et al., 2004). At the T-tubules, non-phosphorylated Na V -β1 interacts with TTX-S Na V s and ankyrin-B (Figure 2) (Malhotra et al., 2004). In contrast, tyrosine-phosphorylated Na V -β1 is localized to the ID where it interacts with Na v 1.5 and N-cadherin (Figures 3B,C) (Malhotra et al., 2004). We do not yet know whether phosphorylation targets Na V -β1 to specialized subcellular regions or whether Na V -β1 is differentially phosphorylated upon arrival. Phosphorylation may be a signaling mechanism by which cells regulate the density and localization of Na V -β1, and by association Na v -αs, to specific subcellular domains. In summary, Na V -β1 subunits serve as critical links between the extracellular and intracellular signaling environments of cells through ion channel modulation as well as cell-cell adhesion.

NA V -β1 MODULATES POTASSIUM CHANNELS
Na V -β1 can interact with and modulate voltage-gated potassium channels (K v s) in addition to Na V s. K v -α subunits assemble as tetramers that normally associate with modulatory Kv-β subunits (Snyders, 1999). The K v 4.x subfamily of channels express rapidly activating, inactivating, and recovering cardiac transient outward currents (I to ) (Snyders, 1999). Co-expression of Na V -β1 with K v 4.3 results in a ∼four-fold increase in I to density (Deschênes and Tomaselli, 2002). Additionally, Na V -β1 alters the voltagedependence and kinetics of channel gating compared to K v 4.3 expressed alone (Deschênes and Tomaselli, 2002). Importantly, Na V -β1 associates with K v 4.2 and enhances its surface expression (Marionneau et al., 2012). Whole-cell voltage-clamp recordings obtained from cells expressing K v 4.2 with Na V -β1 resulted in higher I to densities compared to K v 4.2 alone (Marionneau et al., 2012). Na V -β1 can also interact with and modulate K v 1 (K v 1.1, K v 1.2, K v 1.3, or K v 1.6) and K v 7 (K v 7.2) channels (Nguyen et al., 2012). Lastly, Na V -β1B can also associate with K v 4.3, resulting in increased I to (Hu et al., 2012). Thus, K v currents can be modulated by Na V -β subunits, at least in heterologous expression systems. Transfection of neonatal rat ventricular myocytes with siRNA targeting Na V -β1 significantly reduced the expression of K v 4.x protein and reduced both I Na and I to (Deschênes et al., 2008), suggesting that Na V -β1 can modulate K v currents in the heart in vivo.
The inward rectifier current I K1 , expressed by Kir2.1, is critical for setting the resting membrane potential and modulating the late-phase of repolarization and AP duration in CMs (Nerbonne and Kass, 2005). Similar to Na v 1.5, Kir2.x channels contain a C-terminal PDZ-binding domain which mediates interaction with SAP97 and syntrophin (Matamoros et al., 2016). It is thought that Kir2.x channels associate in microdomains that include caveolin 3, Na v 1.5, SAP97, and syntrophin (Vaidyanathan et al., 2013). Na v 1.5 interacts with α1syntrophin via an internal N-terminal PDZ-like binding domain in addition to the C-terminal PDZ-binding domains (Matamoros et al., 2016). Importantly, Na v 1.5-β1 co-expression increases Kir2.1 and Kir2.2, but not Kir2.3, currents, again suggesting that these channels are functionally linked and that Na v -β1 is critical to the formation of multi-ion channel complexes.

IN VIVO ROLES OF SCN1B
Animal models have been instrumental in understanding the role of Scn1b in cardiac excitability. Scn1b deletion in mice results in severe seizures, ventricular arrhythmias, and sudden death prior to weaning (Chen, 2004). Scn1b null ventricular CMs exhibit prolonged AP repolarization, increased Scn5a/Na v 1.5 gene and protein expression, increased Scn3a expression, increased transient and persistent I Na density, and prolonged QT and RR intervals (Lopez-Santiago et al., 2007). In agreement with an adhesive role for β1, cytoskeletal disruption in CMs also results in increased persistent I Na (Undrovinas et al., 1995). Consistent with this, ventricular CMs isolated from cardiac-specific Scn1b null mice have increased I Na density, increased susceptibility to polymorphic ventricular arrhythmias, and altered intracellular calcium handling that is TTX-S (Lin et al., 2015). These data indicate that loss of Scn1b expression is arrhythmogenic, mediated by altered ion channel gene and protein expression, I Na , I K , and calcium handling. Cardiac specific Scn1b deletion increases the duration of calcium signaling, resulting in delayed afterdepolarizations (Lin et al., 2015). It will be interesting to determine if expression of disease-associated SCN1B variants leads to dysfunctional ryanodine receptor signaling, which can also result in altered levels of intracellular calcium and the generation of arrhythmias (Bers, 2008;Fearnley et al., 2011;Glasscock, 2014).
The cardiac AP relies on the orchestration of multiple ion channels in concert. Na V -β1 is an important modulator of Na vα as well as some K V -and Kir-α subunits. Na V and K channels may be functionally linked through Na V -β1/β1B, and if so, defects in this mechanism may contribute to cardiac disease. It will be critical to determine the physiological effects of Na V -β1 interaction with other K channels, calcium channels, or other calcium-handling proteins at the T-tubules. It is intriguing to consider that Na V -β1 may serve as a central communication hub between sodium, potassium, and calcium channel families to coordinate depolarization, repolarization, and calcium signaling in CMs.

SCN1B AND HUMAN DISEASE
SCN1B variants are implicated in a variety of inherited pathologies, including epileptic encephalopathy and cardiac arrhythmias (O'Malley and Isom, 2015) (Figure 1, Table 1). The epileptic encephalopathy Dravet syndrome is linked to heterozygous variants in SCN1A leading to haploinsufficiency in most patients, however, a subset of patients has SCN1B homozygous loss-of-function variants (Patino et al., 2009). The leading cause of mortality in Dravet syndrome is Sudden Unexpected Death in Epilepsy (SUDEP) (Nobili et al., 2011;Kalume, 2013;Devinsky et al., 2016). SCN1B variants are also linked to inherited cardiac arrhythmia syndromes that increase the risk of sudden death, including BrS (Hu et al., 2012), LQTS (Riuró et al., 2014), atrial arrhythmias (Watanabe et al., 2009), and SIDS (Hu et al., 2012). Diagnostic overlap between epilepsy and cardiac conduction disease can confound causative links between the two phenotypes (Ravindran et al., 2016). Cardiac conduction abnormalities can be poorly recognized in patients with epilepsy and vice versa (Zaidi et al., 2000). A retrospective electrocardiography study revealed that abnormal ventricular conduction was more common in SUDEP cases than in epileptic controls (Chyou et al., 2016). We propose that variants in SCN1B, including those linked to epilepsy, predispose patients to compromised cardiac electrical abnormalities. Thus, cardiovascular evaluation may be helpful in treating epileptic encephalopathy patients.

SUMMARY
Na V -β1 and -β1B are multifunctional molecules that associate with Na v and K channels, cytoskeletal proteins, CAMs, and extracellular matrix molecules in the heart and brain. In addition, Na V -β1/β1B modulate multiple ionic currents, channel expression levels, and channel subcellular localization. Thus, it is not surprising that variants in SCN1B are linked to devastating cardiac and neurological diseases with a high risk of sudden death. In the field of cardiac physiology, important questions remain regarding specific cardiac Na V -β1 binding partners, potential effects of Na V -β1 on calcium-handling, the potential role of Na V -β1 in Na v 1.5 dimerization, and the mechanism of phosphorylation events that affect Na V -β1 targeting to and association with subcellular domain specific signaling complexes at the ID, lateral membrane, and T-tubules. Understanding the functions of Na V -β1 within these protein complexes will help to elucidate underlying mechanisms of cardiac arrhythmias and associated sudden death, as well as lead to the discovery of novel biomarkers and therapeutic targets for human disease.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
This work was funded by NIH R37-NS-076752 to LI and by T32GM00776737 to LI on which NE is a trainee.