A privileged role for neuronal Na⁺ channels in regulating ventricular [Ca²⁺] and arrhythmias

Tight regulation of membrane potential and Ca²⁺ dynamics in cardiac myocytes is achieved by the spatial relationships between the proteins involved. Because, for example, a Ca²⁺-dependent ion channel will respond more readily to Ca²⁺ passing through an adjacent channel than to one located a few micrometers away, ventricular myocytes arrange many of the critical proteins into microdomains, thereby enabling adequate coupling of these processes. Transverse tubules (T-tubules)—protrusions of the sarcolemmal membrane into the cell interior—contain numerous microdomains known as “couplons” (Ríos et al., 2015). In these couplons, LTCCs are brought into close proximity with RyRs, the Ca²⁺ release channels of the SR, and additional relevant ion transport proteins.

The complexity of coupling between Ca²⁺ and membrane potential has important implications for the maintenance of stable cardiac electrical activity. In particular, dangerous and potentially lethal ventricular arrhythmia syndromes can result when ion transport is disrupted by mutations, drugs, changes in gene expression, or pathological posttranslational modifications. Indeed, two of the most well-studied congenital arrhythmia syndromes, long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT), can result from mutations in a wide range of ion channels and Ca²⁺-handling proteins. At present, 15 forms of LQTS (Bohnen et al., 2017) and 5 forms of CPVT (Landstrom et al., 2017) have been described. These two disorders show clear differences in clinical presentation and, at the cellular level, different mechanisms are thought to underlie “triggered activity”—i.e., the inappropriate membrane depolarizations that initiate arrhythmias. Specifically, triggered activity in LQTS is often considered to be purely voltage driven (Weiss et al., 2010); prolonged repolarization allows LTCCs to recover from inactivation, and reactivation of these channels during the repolarization phase can cause a secondary action potential upstroke known as an early afterdepolarization (EAD). In contrast, arrhythmias in CPVT are thought to arise from a different form of triggered activity, namely, delayed afterdepolarizations (DADs). These events result from spontaneous, aberrant release of Ca²⁺ from the SR after the cell membrane has fully repolarized (Sobie et al., 2017).

Although this general paradigm fits the majority of clinical and cellular observations, recent work suggests that, under some conditions, the picture may be more nuanced. In particular, arrhythmia susceptibility in LQTS might not always result from purely voltage-dependent mechanisms, but can sometimes depend on Ca²⁺-voltage coupling (Terentyev et al., 2014). Along these lines, Koleske et al. (2018) provide important new insight. The results presented in this study show how the action potential prolongation seen in certain models of LQTS can lead to SR [Ca²⁺] overload and arrhythmogenic spontaneous Ca²⁺ release. The data also offer novel suggestions regarding the spatial relationships between ion transport proteins. Specifically, the authors suggest that nNa_V channels may have a privileged role that makes them attractive drug targets for these disorders.

The current study builds on previously published results from the same research group. In 2016, many of the same investigators used a mouse model of RyR dysfunction to show that catecholamine-stimulated Na⁺ influx through nNa_Vs can trigger arrhythmias (Radwański et al., 2016). In the current study (Koleske et al., 2018), the authors expand on this finding in an effort to uncover the underlying mechanisms. One of the major strengths of this study is the comprehensive approach taken by the authors. Multiple combinations of pharmacological perturbations were performed, both membrane potential and intracellular [Ca²⁺] were simultaneously monitored, and cellular as well as organ-level physiology were assessed.

The following general experimental strategy was used: A cellular environment encouraging arrhythmic dynamics (through EADs and/or DADs) was created, then Na⁺ channels were blocked systematically to pinpoint the key player(s) contributing to triggered activity. First, in WT mouse ventricular myocytes, the authors induced a proarrhythmic LQTS phenotype using 4-Aminopyridine (4AP), an inhibitor of transient outward current (I_to), accompanied by β-adrenergic stimulation with isoproterenol (ISO). Inhibition of I_to, the major repolarizing current in these cells, increases action potential duration (APD), leading to enhanced Ca²⁺ entry through LTCCs and elevated SR Ca²⁺ load (Devenyi and Sobie, 2016). ISO also augments Ca²⁺ entry through LTCCs and Ca²⁺ uptake into the SR, further elevating the SR load. The combination of these two perturbations by Koleske et al. (2018) caused the cells to exhibit frequent triggered activity and aberrant intracellular Ca²⁺ release. Three Na_V-blocking agents were then applied to assess the potential role of nNa_Vs. Although the global Na⁺ channel blocker lidocaine did not reduce the frequency of either triggered activity or aberrant Ca²⁺ release, the nNa_V-specific blockers riluzole and 4,9-anhydro-tetrodotoxin (4,9-ah-TTX) significantly reduced both. None of the three Na_V blockers reversed the action potential prolongation induced by 4AP+ISO treatment, indicating that the antiarrhythmic effects of the drugs do not simply result from changes in action potential shape.

After these initial results suggesting that nNa_V blockade can inhibit arrhythmic behaviors, the authors investigated two additional experimental models to obtain further support, with a particular focus on unstable intracellular Ca²⁺ dynamics in the form of DAD-inducing Ca²⁺ waves. In the first model, APD prolongation was induced by nonselective augmentation of Na⁺ influx with Anemone toxin II. They found that riluzole or 4,9-ah-TTX treatment largely reduced the frequency of EADs and DADs, and abolished the LQT phenotype. This assessment was repeated for a model in which the LQT phenotype was induced with the LTCC-augmenting drug Bay K 8644, and consistent results were observed.

The last two sets of experiments were performed in myocytes from mice with cardiac-specific deletion of calsequestrin (CASQ2), the primary SR Ca²⁺ buffer. Mutations in CASQ2 account for a recessive form of CPVT (Faggioni et al., 2012), and the CASQ2-null mouse has been a reliable CPVT model for more than a decade (Knollmann et al., 2006). Although the substantial action potential prolongation induced by the pharmacological treatments is not typical of the CPVT phenotype, these experiments may nevertheless help to provide insight into heart failure. This condition is associated with both action potential prolongation and unstable SR Ca²⁺ release (Bers et al., 2006), although many additional alterations contribute to the phenotype.

Overall, the study demonstrated a protective, antiarrhythmic effect of nNa_V blockade in three separate experimental models of mouse ventricular arrhythmia. These results suggest a privileged role of neuronal Na_V channels in mediating cellular triggered activity and arrhythmia via a mechanism that involves Ca²⁺-voltage coupling.

How can we understand the results presented by Koleske et al. (2018), which suggest that Na⁺ influx through nNa_Vs plays a particularly important role in determining arrhythmic behavior? A straightforward potential explanation is illustrated schematically in Fig. 1 A, which shows the subcellular location of nNa_Vs at an influential position near the couplon. Classical cardiac Na⁺ channels, cNa_Vs (i.e., Na_V1.5), are for simplicity shown in the T-tubule membrane but distant from the couplon, although localization and functional data suggest that these channels cluster in the intercalated disk regions between adjacent myocytes (Lin et al., 2011; Westenbroek et al., 2013). The arrangements illustrated in Fig. 1 A imply that augmentation of either nNa_Vs or cNa_Vs would lead to action potential prolongation, which would then increase Ca²⁺ influx and diastolic SR [Ca²⁺] load. However, selective augmentation of nNa_Vs would be predicted to cause more pronounced effects than selective augmentation of cNa_Vs, as illustrated by the hypothetical action potential, intracellular [Ca²⁺], and SR [Ca²⁺] time courses shown in Fig. 1 B. Because nNa_Vs are located in much closer proximity to the couplon than cNa_Vs, a local increase in [Na⁺] near the couplon would reduce the efflux of Ca²⁺ through NCX. This would allow more local Ca²⁺ to be taken into the SR by the SR Ca²⁺ ATPase pump, leading to a greater increase in SR [Ca²⁺] load than would occur in the absence of elevated local [Na⁺]. Elevated SR [Ca²⁺] load caused by nNa_V augmentation would increase the risk of spontaneous SR Ca²⁺ release and DADs. This model can also explain why, when SR [Ca²⁺] is increased through various interventions, selective blockade of nNa_Vs (as in the experiments of Koleske et al., 2018) reduce the frequency of both Ca²⁺ waves and triggered activity. In contrast, nonspecific Na⁺ channel blockade using lidocaine did not correct triggered activity, suggesting that local increases in Na⁺ near other Na⁺ channels, i.e., cNa_Vs, do not significantly affect Ca²⁺ efflux through NCX.

A potentially important protein that is not depicted in Fig. 1 A is Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). This kinase can phosphorylate both RyRs and nNa_Vs, increasing their open probability (Wehrens et al., 2004) and augmenting the current through these channels (Radwański et al., 2016), respectively. Prior studies have suggested that a deleterious feedback loop involving elevated intracellular [Na⁺], intracellular [Ca²⁺], and CaMKII can be proarrhythmic in mouse myocytes (Morotti et al., 2014). Because agonists such as isoproterenol lead to activation of CaMKII in addition to the classical downstream effector protein kinase A (Curran et al., 2007; Gutierrez et al., 2013; Poláková et al., 2015), the proarrhythmic state seen during β-adrenergic stimulation is likely to involve CaMKII activation in addition to the steps outlined above. Indeed, Radwański et al. (2016) previously showed that CaMKII inhibition can be beneficial during conditions associated with increased Na⁺ flux through nNa_Vs.

In addition to supporting the findings of the present study, the conceptual model shown in Fig. 1 A is consistent with recent work by other groups. For instance, when the cell membrane is depolarized and intracellular [Ca²⁺] is low, NCX operates in reverse mode to import Ca²⁺ and export Na⁺. Although early studies showed that this Ca²⁺ entry pathway is inefficient at triggering SR Ca²⁺ release when operating in isolation (Sipido et al., 1997), more recent investigations have demonstrated that LTCCs, NCXs, and nNa_Vs can interact to boost the trigger for SR Ca²⁺ release (Sobie et al., 2008; Larbig et al., 2010; Chu et al., 2016). Indeed, selective blockade of nNa_Vs with nanomolar concentrations of TTX can inhibit SR Ca²⁺ release, suggesting involvement of these isoforms (Torres et al., 2010). This can only occur if these ion transport pathways are in reasonably close proximity, as also suggested by Koleske et al. (2018).

The results of this study (Koleske et al., 2018) also improve our understanding of cellular arrhythmia initiation mechanisms under pathological conditions. As mentioned above, studies into LQTS have generally focused on arrhythmias initiated by EADs (Weiss et al., 2010), a process that can be understood without considering SR Ca²⁺ release. However, a series of recent investigations have suggested that, at least under some conditions, Ca²⁺ overload and spontaneous SR Ca²⁺ release can contribute to the arrhythmic phenotype in various forms of LQTS. This can take the form of triggered activity initiated by spontaneous Ca²⁺ waves and DADs, as reported here. However, this unstable Ca²⁺ release can be proarrhythmic in other ways. Secondary spontaneous Ca²⁺ releases during the action potential can prolong the repolarization phase, thereby making EADs caused by LTCC reactivation more likely (Edwards et al., 2014; Terentyev et al., 2014). Additionally, unstable spontaneous Ca²⁺ release can encourage the formation of spatially discordant alternans, patterns of voltage and [Ca²⁺] at the tissue level that can predispose hearts to arrhythmias (Liu et al., 2018). Overall, these recent studies paint a picture that spontaneous SR Ca²⁺ release is a critical proarrhythmic signal not only in CPVT, as is well established, but also in LQTS, where its role has been less appreciated. The current study (Koleske et al., 2018) makes a significant addition to this emerging paradigm, emphasizing the importance of SR Ca²⁺release in LQTS and drawing additional mechanistic links between the two arrhythmia disorders.

As with all important investigations, the study by Koleske et al. (2018) suggests avenues for future research. One will be to determine the precise roles played by different nNa_V isoforms in promoting proarrhythmia. The schematic in Fig. 1 groups all nNa_Vs into a single category because most of the results presented by Koleske et al. (2018) were not aimed at distinguishing between nNa_V isoforms. However, the antiarrhythmic effectiveness of 4,9-ah-TTX, which preferentially blocks Na_v1.6, suggests a critical role for this isoform. This is consistent with prior studies (Noujaim et al., 2012; Westenbroek et al., 2013), including one by the same group (Radwański et al., 2016), indicating that Na_V1.6 may be more important in ventricular pathophysiology than other neuronal isoforms such as Na_V1.1 and Na_V1.3. Additional studies will be required, perhaps with cardiac-specific knockout mice (Noujaim et al., 2012), to fully elucidate the roles of the various isoforms.

A second issue to address in future work concerns the applicability of the results, obtained using mice, to arrhythmias in humans and larger animals. The mouse action potential is not only much shorter, but has a fundamentally different shape compared with action potentials seen in larger mammals. Repolarization in mouse myocytes depends primarily on transient outward current (I_to), whereas the rapid and slow delayed rectifier currents, I_Kr and I_Ks, are critical in humans, dogs, and rabbits. In contrast, the primary depolarizing currents, I_Na (carried by Na_V1.5) and I_CaL (carried by LTCCs), are similar in mice and humans. This means that mouse myocytes can serve as reasonable experimental models for LQT3 (caused by mutations in Na_V1.5) or LQT8 (caused by mutations in LTCCs), whether these models are genetic (Fredj et al., 2006; Drum et al., 2014) or pharmacological, as used here. Mice are much less useful for investigations of LQT1 or LQT2, caused by mutations in the genes responsible for I_Ks and I_Kr, respectively. Future studies using larger animals should address whether the proarrhythmic Na⁺–Ca²⁺ signaling described in this paper plays an important role in LQT1 and/or LQT2. For such studies that compare results obtained in different experimental models, novel computational approaches to translate perturbation responses across cell types are likely to be extremely useful (Gong and Sobie, 2018).

Overall, the study by Koleske et al. (2018) provides important novel insight into the role played by nNa_Vs in creating proarrhythmic conditions in ventricular myocytes. The results emphasize the importance of spatial microdomains, in which ion channels, pumps, and transporters can closely interact, and they improve our understanding of the potential proarrhythmic consequences of Ca²⁺-voltage coupling. The study is sure to inspire future investigations and may provide a roadmap for the development of novel antiarrhythmic therapeutics.

Research in E.A. Sobie’s laboratory is supported by the National Institutes of Health (grants U01 HL136297, U54 HG008098, and P50 GM071558), the National Science Foundation (grant MCB 1615677), and the American Heart Association (grant 15GRNT25490006). J.Q.X. Gong is supported by a predoctoral fellowship from the American Heart Association (grant 17PRE33670541). D.C. Jones was supported by the Postbaccalaureate Research Education Program of the National Institute of General Medical Sciences (training grant R25 GM064118).

The authors declare no competing financial interests.

Eduardo Ríos served as editor.