Post-translational modifications of the cardiac Na channel: contribution of CaMKII-dependent phosphorylation to acquired arrhythmias

Abstract

The voltage-gated Na channel isoform 1.5 (Na_V1.5) is the pore forming α-subunit of the voltage-gated cardiac Na channel, which is responsible for the initiation and propagation of cardiac action potentials. Mutations in the SCN5A gene encoding Na_V1.5 have been linked to changes in the Na current leading to a variety of arrhythmogenic phenotypes, and alterations in the Na_V1.5 expression level, Na current density, and/or gating have been observed in acquired cardiac disorders, including heart failure. The precise mechanisms underlying these abnormalities have not been fully elucidated. However, several recent studies have made it clear that Na_V1.5 forms a macromolecular complex with a number of proteins that modulate its expression levels, localization, and gating and is the target of extensive post-translational modifications, which may also influence all these properties. We review here the molecular aspects of cardiac Na channel regulation and their functional consequences. In particular, we focus on the molecular and functional aspects of Na channel phosphorylation by the Ca/calmodulin-dependent protein kinase II, which is hyperactive in heart failure and has been causally linked to cardiac arrhythmia. Understanding the mechanisms of altered Na_V1.5 expression and function is crucial for gaining insight into arrhythmogenesis and developing novel therapeutic strategies.

the cardiac Na channel, encoded by the gene SCN5A, is activated at negative membrane voltages and is responsible for the generation of the rapid upstroke of the cardiac action potential (AP). The α-subunit of the voltage-gated cardiac Na channel isoform (Na_V1.5) forms a macromolecular complex through interactions with many accessory proteins (75), regulatory proteins (132), and other ion channels (77, 105) that alter its expression, trafficking, localization, and gating [reviewed in Abriel (1)] (Fig. 1). Recent evidence also suggests that multiple pools of Na_V1.5 may exist in cardiomyocytes, and these channel populations may be differentially regulated (94, 109). Whereas most of the Na channels inactivate rapidly (within a few milliseconds), prolonged membrane depolarization during the AP plateau causes slow/intermediate (hundreds of milliseconds) inactivation of some channels, which recover to a closed conformation as the cell membrane repolarizes during diastole. In physiological situations, Na channel activation and inactivation properties are tightly regulated, thus maintaining cardiac excitability and ensuring propagation of the electrical impulse. However, alterations in channel function (e.g., due to gene mutations or post-translational modifications) may profoundly affect cardiac electrophysiology and arrhythmogenesis. Herein, we review post-translational regulation of Na_V1.5. While we describe the molecular and functional aspects of post-translational regulation by PKA, PKC, oxidative stress, Ca, and CaM, we focus on Na channel phosphorylation by Ca/CaM-dependent protein kinase II (CaMKII). We further use CaMKII modulation as a model to understand how Na channel dysfunction may be implicated in acquired arrhythmias.

Fig. 1.

Na channel as a macromolecular signaling complex. AnKG, ankyrin-G; FHF1B, fibrobast growth factor homologous factor 1; MOG1, multicopy suppressor of Gsp1; Nedd4, neural precursor cell expressed developmentally downregulated protein 4; C, catalytic; R, regulatory; P, phosphorylation; Cav3, caveolin-3; IFM, Ile-Phe-Met; ROS, reactive oxygen species; Mito, mitochondria; NOS, nitric oxide synthase; PDZ, postsynaptic density protein/Drosophila disk large tumor suppressor/zonula occludens-1; SNTA1, α₁-syntrophin.

Na Channel Alterations in Inherited SCN5A Channelopathies and Acquired Diseases

A number of mutations in the gene SCN5A have been described, which cause enhanced or reduced channel function and are linked to cardiac disorders including long QT syndrome (LQTS), Brugada syndrome (BrS), conduction defects, sinus dysfunction, and familial atrial fibrillation (AF) (101). These have tremendously aided our understanding of the regulation of expression, localization, and function of Na_V1.5 and suggest that its role is not limited to AP initiation and conduction, but it may also have more subtle functions and be involved in repolarization abnormalities. In addition, mutations disrupting Na channel protein interactions (within the macromolecular signaling complex modulating the localization and biophysical properties of Na_V1.5) have also been associated with LQTS and BrS [reviewed by Wilde and Brugada (132)].

These channelopathies provided useful models for understanding the molecular mechanisms whereby pharmacological interventions or disease states provoke cardiac arrhythmias. For example, LQT3 syndrome is caused by gain-of-function defects in Na channel inactivation, which lead to delays in ventricular repolarization that result in the prolongation of the QT interval. Numerous mutations induce a sustained (late Na current, I_Na,L) component of inward current (∼1% of the peak current) throughout the depolarization period. The role of this small current in prolonging repolarization and generating cardiac arrhythmias has been validated in quantitative models of the AP (27) and is further illustrated in the next subsection.

On the other hand, BrS is associated with loss-of-function mutations and is characterized by normal QT intervals and ST segment elevation in the right precordial leads, which mimics ischemic ECG manifestations and predisposition to malignant ventricular tachyarrhythmias. The pathophysiological mechanism is thought to involve heterogeneous loss of the AP dome between the epicardium and endocardium, which creates a transmural voltage gradient during ST segment (36, 137) or conduction delay in the right ventricular outflow tract (76). SCN5A mutations have also been reported leading to combinations of these phenotypes, known as overlap syndromes (100). Intriguingly, a single human mutation at 1795InsD in SCN5A is linked to simultaneous LQT3 and BrS features (124). Mutation-induced slowing of I_Na recovery from inactivation, increase in intermediate inactivation, and hyperpolarizing shift in channel availability all reduce I_Na (loss of function) and underlie the BrS-like symptoms (slow conduction) of patients at higher heart rates. In addition, this 1795InsD mutation causes an increase in I_Na,L. At slow heart rates where I_Na recovery from inactivation may be more complete and APs are intrinsically longer, I_Na,L-dependent AP prolongation is responsible for LQTS (28).

Emerging evidence also suggests that Na channel gating alterations are involved in widespread acquired diseases, e.g., drug-induced LQTS, cardiac ischemia, heart failure (HF), and AF. For example, CaMKII, which is upregulated and more active in HF (5), has been shown to regulate Na channel gating and almost exactly phenocopies the spectrum of gating changes seen for the combined LQTS/BrS phenotype due to 1795InsD (see Fig. 2). Also, the generation of reactive oxygen species (ROS) in conditions of increased oxidative stress (such as during ischemia, HF, and AF) correlates with CaMKII-mediated reduced I_Na availability, leading to impairment of cardiac conduction following myocardial infarction (25, 46) and enhancement of late I_Na and consequent arrhythmias (128). In this context, post-translational modifications of Na_V1.5 may constitute an acquired arrhythmogenic Na channel defect that could affect millions of people (as compared with the relatively small number of individuals affected by the mechanistically informative genetic Na channel mutations).

Fig. 2.

Effects of CaMKIIδ_C hyperactivity (or mutation 1795insD) on Na current (I_Na) gating. SSI and activation voltage dependence (A), entry of Na channels into intermediate inactivation (B), recovery from inactivation (C), and late I_Na (I_Na,L; D) are assessed with the voltage clamp protocols shown in insets in control (solid lines) and CaMKIIδ_C overexpression/1795insD mutation (dashed lines).

Late Na Current

The late Na current is carried by a fraction of Na channels that remain active throughout the AP plateau, rather than quickly inactivating as the majority of Na channels do. These channels undergo special modes of gating (burst and late-scattered openings) that confer I_Na,L slow inactivation and voltage dependence similar to the fast component (71). I_Na,L is increased in a number of LQT3 mutations and acquired diseases, including HF, where its inactivation kinetics are even slower than normal hearts (69, 71). I_Na,L alterations may be acute and result from post-translational modification of Na_V channels, or chronic, and involve changes in expression of Na_V and its modulatory proteins.

The molecular details of I_Na,L and its modulation are not fully understood. A significant (∼50%) contribution of noncardiac Na channel isoforms to I_Na,L has been reported in healthy canine myocytes (15), and it has been proposed that its enhancement in pathological conditions could be due to an increase in neuronal Na channel isoforms with higher fractional I_Na,L (135). In fact, specific Na_V1.8 inhibition in mice and rabbits is antiarrhythmic (138). Modulation of Na channels could also underlie augmentation of I_Na,L in disease. For example, Maltsev et al. (66) showed that the β₁-subunit (but not β₂) modulates I_Na,L produced by heterologously expressed Na_V1.5 by slowing its decay and increasing its amplitude relative to the peak current. Recently, they showed that reduced expression of either β₁- or β₂-subunits caused a significant loss of function or gain of function of I_Na,L, respectively, in both normal and failing canine myocytes (78). However, there are contrasting results on the modulation of I_Na,L by β-subunits in heterologous and native systems and whether they modulate similarly cardiac and noncardiac isoforms. Furthermore, interaction with the cytoskeleton, regulatory kinases (as discussed here) and phosphatases, trafficking proteins, and extracellular matrix proteins may all modulate I_Na,L in different pathological conditions (71).

Although small in magnitude compared with the peak Na current (∼1%), I_Na,L may disrupt the delicate balance between depolarizing and repolarizing currents during the AP plateau, leading to AP prolongation and increasing the propensity to ventricular arrhythmia (79). Indeed, a pathological increase in I_Na,L has been associated with arrhythmogenic phenotypes in inherited and acquired cardiac diseases including LQT3, HF, myocardial infarction, and AF. Physiological I_Na,L has been shown to contribute to reverse-rate dependence of AP duration (APD) and beat-to-beat variability caused by K current inhibitors in rabbit hearts (134). It has also been suggested that its inhibition may diminish the rate-dependent prolongation of the APD/QT interval caused by either drugs or pathological conditions that decrease rapidly activating K current and may decrease the occurrence of slow rate- or pause-triggered cardiac arrhythmias. Conversely, I_Na,L enhancement in HF has been predicted to exacerbate reverse frequency dependence of APD (115), which may increase the proarrhythmic risk. In addition, the increased Na influx via I_Na,L may partly be responsible for altered global Na and Ca homeostasis in cardiac hypertrophy (29) and failure (111), although the precise mechanism (longer APD vs. higher intracellular Na concentration) and the quantitative aspects of I_Na,L contribution are debatable (38, 128). In fact, a tetrodotoxin-sensitive increase in intracellular Na concentration has been reported in HF even in resting cardiac myocytes (where the Na channels are supposedly closed) (31).

Post-translational Modulation of Na_V1.5

Regulation by PKA and caveolin-3 complex.

Initial reports on β-adrenergic regulation of cardiac I_Na were controversial. Several groups reported a decrease of cardiac I_Na in response to PKA activation (22). However, subsequent studies convincingly demonstrated that PKA potentiates I_Na, possibly involving both a fast saturable and a slow unsaturable component. The fast component involves direct channel phosphorylation events regulating the kinetics and voltage dependence of channel gating. The most consistent fast effects were a negative shift in both steady-state inactivation (SSI) and activation and slowed recovery from inactivation (less available but more active channels) (141). As proposed by Zhou et al. (141), the negative shift in inactivation and use of more depolarized holding potentials (where Na channels may not have been fully available) could explain the discrepancies in early results. Subsequent studies that used more negative holding potentials (to ensure full availability of Na channels) observed potentiation of I_Na. These observations may have important physiological consequences for the increase in cardiac conduction velocity observed with sympathetic stimulation (where I_Na is thought to be increased) and the genesis of re-entrant arrhythmias in ischemic myocardium, which is often associated with depolarized diastolic membrane potentials (86).

The slow component of PKA-dependent I_Na potentiation is due to enhanced trafficking and insertion of additional functional channels into the membrane, as revealed in a series of studies with recombinant channels expressed in oocytes by the Murray group (41, 140, 141). Although it was demonstrated that the I–II cytoplasmic linker loop is critical for this effect (141), mutation of five putative PKA sites in this loop (including S483/S571/S593) failed to abolish PKA-dependent potentiation of rat I_Na (107, 140). Murphy and colleagues identified two sites on rat Na_V1.5 at positions S526 (525 in human) and S529 (528 in human) that are phosphorylated by PKA both in vitro and in vivo (83) (Fig. 1). PKA potentiation of I_Na was subsequently determined to be dependent on PKA phosphorylation of S525 and S528 and the presence of three endoplasmic reticulum (ER) retention signals on the I–II cytoplasmic linker loop (140). A S525A/S528A double phosphomutant or mutation of all three putative ER retention signals (with Arg-X-Arg at 533–535 playing the most prominent role) abolished PKA effects on I_Na (140). This favors a model whereby PKA phosphorylation of S525 and S528 (and possibly other proteins) recruits binding of protein partners that mask the ER retention signals of intracellular channel reserves to facilitate forward trafficking of channels to the membrane.

This concept of intracellular storage pools of Na channels was originally proposed by Catterall and colleagues (106) for neurons (where PKA has opposite effects). It is supported by studies in mammalian cells, including dog cardiomyocytes, which demonstrated that intracellular pools of Na_V1.5 exist in the ER and subsarcolemmal space that can be recruited to the membrane in response to PKA activation (41, 142). The intracellular pool hypothesis is further substantiated by the observation that Na_V1.5 and β-adrenergic receptors colocalize to caveolin domains that participate in membrane trafficking (139). Indeed, it has been shown that Na_V1.5 associates with caveolin 3 (Cav3, Fig. 1) (139), and this interaction facilitates direct (PKA independent) Gα_s protein stimulation of cardiac I_Na (62, 89). Furthermore, mutations in Cav3 that disrupt its interaction with Na_V1.5 are reported to result in increased I_Na,L, which is the basis for arrhythmogenic LQT9 (123).

Regulation by PKC and glycerol-3-phosphate dehydrogenase 1-like protein complex.

Analogous to neuronal Na_V1.2 channels, PKC decreases cardiac I_Na in native and heterologous cell systems (98). This I_Na decrease is dependent on voltage and phosphorylation of S1505 (rodent; S1503 human) in the Na_V1.5 III–IV inactivation loop (Fig. 1) (99). In cell attached current recordings of Chinese hamster cells expressing rat Na_V1.5, the PKC activator 1-oleoyl-2-acetylglycerol (10 μM) resulted in a voltage-dependent decrease in I_Na and a 15mV negative shift in SSI that were reversed by a PKC inhibitor peptide or mutation of S1505 to a nonphosphorylatable alanine (99). The combined effects of a decrease in maximal I_Na and a negative shift of the SSI curve likely explain the voltage dependence of PKC-induced I_Na reduction, with a greater decrease observed at depolarized potentials.

The importance of PKC in regulating I_Na was further demonstrated through the discovery that inherited mutations in glycerol-3-phosphate (G3P) dehydrogenase 1-like protein (GPD1L) are linked to the reduction in I_Na amplitude seen in some forms of BrS [A280V (61)] and Sudden Infant Death Syndrome [mutation E83K (121)] [see (132) for review]. Although the function of GPD1L is unknown, the related enzyme GPD1 catalyzes the NAD⁺-dependent reversible conversion of G3P to dihydroxyacetone phosphate and is an important metabolic link between glycolysis and triglyceride synthesis. The A280V and E83K mutations cause a loss of GPD1L enzymatic activity and decrease I_Na when coexpressed with wild-type (WT) Na_V1.5 in HEK293 cells (120). Importantly, both WT and mutant GPD1L-glutathione-S-transferase (GST) fusions pull down Na_V1.5, which suggests that the effect of GPD1L mutations on I_Na is through enzymatic function and not binding. The proposed pathway involves PKC, because when WT GPD1L and Na_V1.5 are coexpressed in HEK293 cells, 1-oleoyl-2-acetylglycerol or G3P (a GPD1 substrate) cause I_Na reduction, which is reversed with the serine/threonine kinase inhibitor staurosporine or mutation of S1503 to nonphosphorylatable alanine. Thus loss of function of GPD1L activity results in accumulation of G3P and phospholipid that activate PKC to phosphorylate S1503 and decrease I_Na (120). Interestingly, NADH mass action block of GPD1L also decreased I_Na, while there was no significant change with NAD⁺.

Studies by Liu et al. (60) further suggested that I_Na might be regulated directly by pyridine nucleotides, such as NADH. They found a twofold increase in intracellular NADH concentration when the A280V GPD1L mutant was adenovirally transfected into HEK293 cells stably expressing Na_V1.5. Moreover, there was a dose-dependent decrease in I_Na with intracellular application of NADH delivered by patch pipette. The effect of NADH was rapid (suggesting a post-translational effect), did not appreciably alter I_Na gating or mRNA levels and was reproducible in neonatal myocytes. The NADH-induced I_Na decrease required both PKC and ROS, and ROS was downstream of PKC indicating the channel may be directly oxidized. The reported PKC effects (and the GPD1L-dependent pathway) connect the metabolic state of the cell to I_Na and cardiac excitability and may be crucial in cardiac ischemia and failure, which are characterized by increased metabolic stress (11, 112) and PKC upregulation (130). Of clinical relevance, mouse hearts perfused with external lactate/pyruvate to increase intracellular NADH concentration had enhanced inducible ventricular tachycardia with programmed electrical stimulation. Conversely, increased extracellular NAD⁺ concentration was antiarrhythmic in SCN5A^+/− heterozygous mice that have decreased I_Na and increased propensity to inducible ventricular tachycardia (60).

GPD1L mutations are associated with a PKC-dependent decrease in the number of functional channels at the membrane. Mutation A280V was shown to decrease cell surface expression of Na_V1.5 by immunocytochemistry and confocal microscopy (61). Additionally, flow cytometry measurements of HEK293 cells showed decreased Na_V1.5 cell surface expression when coexpressed with A280V or E83K mutant GPD1L (120). This was reversed by mutation of S1503 on Na_V1.5 to alanine or addition of staurosporine to inhibit PKC.

Analogous PKC-dependent effects were obtained independent of GPD1L mutations. Hallaq et al. (40) monitored the movement of green fluorescent protein (GFP)-tagged Na_V1.5 (COOH-terminus) in HEK293 cells compared with hemagglutinin-tagged Na_V1.5 immobilized at the membrane using confocal microscopy (40). In response to PKC activators, they observed a decrease in Na_V1.5-GFP at the membrane, which was blocked by PKC inhibition, mutation of S1503 to alanine, or ROS inhibition. Fluorescence recovery after photobleach studies further identified that channels have decreased mobility within the membrane with PKC stimulation and, conversely, an increase with PKA stimulation. Peak I_Na was decreased at all test potentials after a 30-min exposure to the PKC activator phorbol 12-myristate 13-acetate (40). In contrast to the above experiments with relative long (30 min) drug exposures, Liu et al. (60) did not see a change in Na_V1.5 surface expression, assayed by biotinylation and Western blot of surface Na_V1.5 protein or confocal experiments of Na_V1.5-GFP at the membrane, upon acute (2–10 min) treatment with pyruvate/lactate (to increase NADH) or phorbol 12-myristate 13-acetate (to activate PKC) (60). Decreased Na_V1.5 cell surface expression and hence functional I_Na may be particularly relevant for chronic channel remodeling in HF, where PKC is increased (130).

Regulation by oxidation/nitrosylation: mitochondria and syntrophin complex.

Metabolic state is intimately coupled to I_Na function and relevant to our understanding of arrhythmogenesis in ischemia and HF (11, 86, 112), where both NADH and ROS are increased. In an elegant study, the Dudley group determined that mitochondria are the main source of the NADH-dependent ROS that decreases I_Na in HEK293 cells and rat neonatal ventricular myocytes (59). With the use of specific inhibitors of enzymes in the mitochondrial electron transport chain, the mechanism was further refined by identifying complexes I and III as the main sources of NADH-dependent ROS and the mitochondrial inner membrane anion channel as the main site of ROS release (59).

The studies of Liu and colleagues suggest that NADH directly activates PKC to increase mitochondrial ROS. ROS may have direct effects on the channel or may act through other downstream effectors [e.g., constitutive activation of CaMKII (34), lipoxidation products (84)]. Direct oxidation of methionine or cysteine residues on Na_V1.5 is associated with I_Na,L resulting from slowed inactivation and a hyperpolarizing shift in availability that decreases peak I_Na (129). In a study of several Na_V isoforms (Na_V1.2, 1.5, 1.7, 1.4), mutation of multiple methionine residues to leucines, including M1305L on the Ile-Phe-Met (IFM) inactivation motif, significantly attenuated but did not completely abolish the ROS-induced slowing of inactivation (53). The decrease in peak I_Na observed with ROS may also be influenced by the recent observation that ROS can negatively regulate SCN5A gene expression by promoting nuclear localization of the Foxo1 transcription factor and its binding to the SCN5A promoter to suppress transcription (72). This may have implications for the Na_V1.5 mRNA decrease observed in some disease models (see Tables).

Endogenous sources of ROS other than mitochondria also exist in cardiac myocytes, including nitric oxide (NO) produced by NO synthases (NOSs). Interestingly, NO has been associated with an increase in I_Na,L that is the proposed arrhythmogenic mechanism behind a missense mutation in syntrophin, A390V, causing LQT12 (116). α₁-Syntrophin is a dystrophin-associated scaffold protein that contains multiple protein interaction motifs. It associates with the neuronal NOS (nNOS), the plasmalemmal Ca-ATPase (PMCA), and the COOH-terminus of Na_V1.5 through postsynaptic density protein/Drosophila disk large tumor supressor/zonula occludens-1 protein (PDZ) domain interactions. Coimmunoprecipitations from mouse cardiac homogenates or pull downs with GST tagged Na_V1.5 COOH-terminus as bait suggest nNOS, PMCA, α₁-syntrophin, and Na_V1.5 interact to form a signaling complex (see Fig. 1). The A390V mutation on syntrophin is in the binding region for PMCA. In HEK293 cells, expression of this mutant syntrophin disrupted pull down of PMCA and a biotin switch assay revealed increased direct S-nitrosylation of Na_V1.5. Thus it was determined that A390V syntrophin fails to associate with PMCA4b, thus releasing PMCA4b-dependent inhibition of nNOS and promoting S-nitrosylation of Na_V1.5. NO increases peak and late I_Na, and this was reversed by inhibiting NOS and recapitulated with the NO donors (+)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) and S-nitrosoglutathione (GSNO). The positive shift in inactivation and increased window current (gain-of-function effects) induced by the A390V syntrophin mutant may explain the I_Na alterations. Furthermore, a recent study suggests that the molecular mechanism for a mutation in Cav3 (F97C) resulting in increased late I_Na and LQT9 (see Regulation by PKA and Cav3 complex) may also involve increased direct S-nitrosylation of Na_V1.5 (24) analogous to the above A390V syntrophin mutant that results in LQT12.

In contradiction to the above, Ahmmed et al. (4) showed that extracellularly applied NO in native guinea pig and mouse cardiomyocytes decreased I_Na through combined cGMP (PKG) and cAMP (PKA) pathways (4). However, release of caged intracellular NO in ventricular myocytes increased I_Na,L independent of guanlyate cyclase in another study (2). Thus direct oxidation or S-nitrosylation of Na_V1.5 may be partly responsible for the increased I_Na,L observed by some groups in diseased human or animal cardiac tissue (see Table 1).

Table 1.

I_Na changes in heart disease

Model	Species	Cell Type	INa, A/F	SSI	SSA	INaL	INa Kinetics	SCN5A mRNA	Nav1.5 Protein	Comments (References)
HCM	Human	V				↑		↔	↔	Mild to moderate HF, NYHA class II–III (29)
CM/CHD ± HF	Human	V		↔	↔		Development and recovery from inactivation ↔			Window current detected, no Δ in HF (102)
Explanted HF	Human	LV						↔		Myocytes from midmyocardial wall (50)
Explanted HF	Human	LV, RV						↑		(91)
Explanted end-stage HF	Human	LV, RV						↓		No Δ with LVADs; CaMKIIγ and δ ↔ in HF LVs; CaMKIIδ ↑ twofold in RV; CaMKIIδ ↓ to 40% of controls with LVAD (17)
Explanted HF	Human	V						↑Truncation ↓native		↓ protein expression and current in heterologous system with truncation variants (108)
Arrhythmogenic cardiomyopathy	Human	LV, RV							↓ ICC	(87)
Explanted/pacing	Human/dog	V	↓	↔	↔	↑		↔		No Δ in Nav 1.1, 1.3, β1- and β2-subunits; ↑ cell capacitance in HF (119)
Explanted/pacing	Human/dog	V				↑	Slower INaL decay			(69, 70)
Multiple coronary microembolizations	Dog	LV	↓	↔	↔	↑		↔	↓	↓ Peak reversed by carvedilol; no Δ in β1 mRNA, β1 or β2 protein; ranolazine decreased APD, EADs, APD beat-to-beat variability and dispersion (68, 118)
Pacing-induced HF	Dog	V	↔	↔			Rate and voltage dependence of INa decay ↔			Ito downregulation explains APD prolongation (51)
Ventricular tachypacing	Dog	P						↓	↓	Slowed conduction and AP rate of rise (64)
5-day infarction/ischemia	Dog	EBZ	↓↓	←	↔←		Slower recovery from inactivation and enhanced development of inactivation		Abnormal Nav1.5 cell surface staining	Reentrant excitation; ventricular tachycardia; slowed AP rate of rise (9, 10, 97)
Pressure/volume overload	Rabbit	V	↔		↔					↑Heart weight, longer QRS duration, slower conduction velocity (131)
Aortic banding	Guinea pig	LV	↑	↔	↔		Time course of INa inactivation and recovery ↔			Increase in INa density with cardiac hypertrophy (at 4 wk) is attenuated with progression to cardiac failure (at 8 wk) (3)
Post-MI	Rat	LV					Slower decay	↔		18% TTX-sensitive component of APD prolongation; ↑Nav1.1 mRNA and protein expression (45)
Siderotic heart disease	Gerbil/neonatal rat	V	↓	←	↔		Time course of INa activation and inactivation ↔; slower recovery from inactivation		↔	↔Single channel currents, ↓AP overshoot and duration (57)
RV pressure overload	Rat	LV, RV	↔		↑RV			↔		CTEPH model; reduced CX43; trend to INa density ↓ in both LV and RV (42)
Volume overload	Rat	SAN						↓	↓	Nav1.1 and Nav1.6 primary isoforms, Nav1.5 and Nav1.7 weakly expressed, Nav1.2 and Nav1.3 not expressed; ↓ HR and ↑ sinus node recovery time (32)

APD, action potential (AP) duration; CM, cardiomyopathy; CHD, congenital heart disease; CTEPH, chronic thromboembolic pulmonary hypertension; CX43, connexin 43; EADs, early afterdepolarizations; EBZ, epicardial border zone; HCM, hypertrophic cardiomyopathy; HF, heart failure; HR, heart rate; ICC, immunocytochemistry; I_Na, Na current; I_Na,L, late I_Na; I_to, transient outward K current; LV, left ventricle; LVAD, LV assist device; MI, myocardial infarction; Na_V1.5, voltage-gated Na channel isoform 1.5; NYHA, New York Heart Association; P, Purkinje; RV, right ventricle; SAN, sinoatrial node; SSA, steady-state activation; SSI, steady-state inactivation; TTX, tetrodotoxin; V, ventricle.

Regulation by CaM.

The regulation of Na_V by Ca, either directly or indirectly through CaM or CaMKII, has been a fervent area of research with groups reporting conflicting results. CaM was the first of these studied. Yeast two-hybrid screens demonstrated that CaM directly interacts with an IQ motif in the COOH-terminus of neuronal Na_V1.2 (81) and cardiac Na_V1.5 (113). Additionally, GST-fusion proteins of the WT COOH-terminal domain of both Na_V1.5 and the skeletal muscle Na_V1.4 pull-down CaM (30), demonstrating CaM binding to these regions, and mutation of the IQ domain prevents CaM pull down (30, 54). As with Ca_V1.2 (95), the Na_V COOH-terminus binds both Ca/CaM and apoCaM with different affinities (20, 54). In whole cell patch-clamp experiments of TSA201 cells transfected with Na_V1.5, Tan et al. (113) showed that a peptide antagonist of Ca-dependent CaM binding (peptide 290–309) delivered by patch pipette caused a +6 mV depolarizing shift in inactivation (CaM hyperpolarizes SSI) and decreased intermediate inactivation. Deschenes et al. (30), however, showed that CaM shifted SSI to negative potentials in both a mouse myoblast cell line and HEK293 cells expressing Na_V1.4, but observed no effect of CaM on Na_V1.5. Thus the effects of CaM may be Na_V isoform dependent.

Na_V1.5 may also be directly regulated by Ca. A pair of EF hand-like domains are present in the Na_V1.5 COOH-terminus just upstream from the IQ domain (Glu1773-Asp1852) (133). Enhancement of intrinsic tyrosine or tryptophan residue fluorescence in the IQ or EF hand-like domains, respectively, was used as an assay for Ca binding the COOH-terminus (54). It was found that Ca binds to CaM in complex with the Na_V1.5 (or Na_V1.2) COOH-terminus but not to the COOH-terminus itself. This is in direct contradiction to studies by the Balser and Chazin groups that report the first EF hand-like domain in the COOH-terminus directly binds Ca, which destabilizes I_Na inactivation and shifts SSI to depolarized potentials (133). Nuclear magnetic resonance studies by the same group revealed the local structure of the COOH-terminal EF hand-like domain is capable of binding Ca (21).

Differences in experimental design, cell types, Ca buffering conditions, and the addition of fluoride to internal pipette solutions likely all contribute to these discrepant early studies. In particular, fluoride is a known Ca chelator and phosphatase inhibitor that could unmask basal phosphorylation effects. Additionally, peptide 290–309, which was used in early studies to disrupt CaM binding (113, 133), was originally developed as a CaMKII inhibitor that disrupts Ca/CaM-dependent activation of the kinase (93). This raises the interesting possibility that the effects of CaM reported in these studies may have intersected with CaMKII effects by inhibiting basal CaMKII phosphorylation. In fact, exogenous Ca/CaM enhanced slow inactivation (a reported effect of CaMKII) in one study using fluoride and peptide 290–309 (113), but not in another study that omitted these (30). Despite the discrepancies above, arrhythmogenic mutations in or around the COOH-terminal IQ and EF hand-like domains support a critical role for these domains in regulation of I_Na inactivation (26).

So what is CaM doing on the Na_V COOH-terminus? In contrast to earlier studies, more recent studies have implicated the COOH-terminus in stabilization of the fast inactivation gate through interaction with the III–IV loop (82) and IQ-bound Ca/CaM binding this same loop (96). Inherited mutations in both the III–IV linker and COOH-terminus that are associated with I_Na,L and arrhythmia (26) support the novel idea that CaM could potentially influence fast inactivation (103). A GST construct comprising the III–IV loop pulls down a Na_V1.5 COOH-terminus/CaM complex in Ca but not in EGTA solution (54). This interaction required CaM and was absent in an IQ/AA mutant, although CaM was not pulled down, suggesting it may be displaced. Recently, the Ahern group (103) captured a crystal structure of the Na_V1.5 COOH-terminus in complex with the III–IV linker via a Ca/CaM bridge. They further identified two aromatic tyrosine residues (Tyr1494/95) on the III–IV loop, which are critical for its interaction with the C-lobe of CaM (104). It is interesting to speculate that the Ahern crystal structure may have captured a snapshot of Ca/CaM bound transiently to the III–IV loop before it is displaced to allow fast inactivation to proceed normally through interactions with the COOH-terminus and occlusion of the α-pore. Displaced Ca/CaM may function to activate CaMKII or remove latent regulation of I_Na inactivation by apoCaM bound to the IQ motif (16). These questions remain unresolved.

What are the potential functional consequences of CaM binding the COOH-terminus and how does this relate to the III–IV loop? Electrophysiological studies of I_Na in Na_V1.5 COOH-terminal (including the IQ motif) LQTS mutants reveal I_Na,L potentially related to perturbed interactions of the COOH-terminus with the III–IV loop, i.e., fast inactivation gate (37, 54). This might explain the arrhythmogenic phenotype in inherited mutations involving the IQ motif (and possibly also the III–IV loop). As for the potential role of CaM to modulate fast inactivation in physiological conditions, one hypothesis is that high-frequency stimulation that elevates the local [Ca] around the channel results in resident apo-CaM binding Ca, CaM lobe switching, and destabilization of inactivation (122) (see Fig. 1). Ca remains bound to CaM to disrupt the interaction of the COOH-terminus with the III–IV loop and impair fast inactivation. Thus the availability curve is shifted to depolarized potentials and more Na channels will be available to drive the next AP upstroke. Although purely speculative, such a mechanism for channel facilitation may be particularly important for repetitive firing during rapid heart rates, e.g., in the response to exercise. It is intriguing to consider the possibility that CaM serves a physiological role in transducing Ca signals to enhance Na channel availability through impairment of fast inactivation, whereas CaMKII plays a pathophysiological role in enhancing slow inactivation and stabilizing the inactivated state (as illustrated below).

Regulation by CaMKII.

A number of studies have aimed at elucidating the functional effects of CaMKII-mediated Na_V1.5 phosphorylation (14). In HEK293 cells expressing Na_V1.5, inhibition of CaM kinase with 10 μM KN93 (but not its inactive analog KN92) slowed I_Na decay, shifted SSI to depolarized potentials, and slowed entry into inactivated states (30). On the other hand, 100 nM autocamtide-2-related inhibitory peptide (AIP) in the pipette had no effect on gating, but intracellular [AIP] might have been limiting (IC₅₀ for CaMKII inhibition ∼40 nM). As discussed below, these CaMK effects are consistent with more recent studies analyzing CaMKII effects on I_Na gating.

In a seminal study by Wagner et al. (126), CaMKII was shown to associate with and phosphorylate Na_V1.5 in cardiac myocytes. CaMKIIδ_C overexpression, either chronically in transgenic (TG) mice or by acute adenoviral overexpression in rabbit, caused simultaneous gain- and loss-of-function effects on native I_Na. Specifically, in myocytes CaMKII shifted SSI to negative potentials (Fig. 2A), enhanced accumulation of intermediate/slow inactivation (Fig. 2B), slowed recovery from inactivation (Fig. 2C), slowed fast I_Na inactivation, and increased I_Na,L (Fig. 2D). Moreover, all of these effects could be acutely reversed with CaMKII inhibition, thus strongly arguing for specific CaMKII-dependent modulation of cardiac Na channels. CaMKIIδ_C overexpressing mice also displayed enhanced propensity to tachyarrhythmias. Importantly, these effects phenocopy the inherited 1795insD mutation on Na_V1.5 that in patients results in combined LQTS and BrS effects (124). Moreover, Maltsev et al. (67) tested the inhibition of CaMKII and found that CaMKII slows the decay of I_Na,L, more so in failing versus nonfailing canine myocytes.

Aiba et al. (6) subsequently used GST fusion constructs of the intracellular regions of Na_V1.5 to narrow down CaMKII-dependent phosphorylation to the I–II loop (6). However, the reported I_Na functional effects of CaMKII phosphorylation (positive shift in SSI, faster recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased I_Na,L) were largely inconsistent with those described by Wagner et al. (126) and other groups (8, 47). These discrepancies may be due to Aiba and colleagues' use of the CaMKIIα isoform (vs. CaMKIIδ_C), delivery method (dialysis via patch pipette vs. adenovirus or transgenesis), different Ca buffering, and use of fluoride in pipette solutions.

Through alanine scanning of phosphorylation sites fitting the traditional CaMKII phosphorylation motif, RXXS/T, Hund et al. (47) identified S571 as a CaMKII target site in vitro (47). In a HEK293 heterologous cell system expressing Na_V1.5, CaMKII shifts the SSI of WT Na_V1.5 to negative potentials. This effect on channel inactivation [observed in all studies, except Aiba et al. (6)] was abolished when S571 was mutated to a nonphosphorylatable alanine and mimicked when S571 was mutated to a phosphomimetic glutamate residue. Furthermore, the authors demonstrated a role for β_IV-spectrin in targeting CaMKII to Na_V1.5 at the intercalated discs. In a more recent study by the same group (56), it was further demonstrated that S571 is phosphorylated in the border zone of infarcted canine hearts, is slightly increased in human HF, and is phosphorylated in mouse hearts following acute stimulation by isoproterenol (with phosphatase inhibitors), but not in mice overexpressing the CaMKII inhibitor AC3I. Two rare, negatively charged arrhythmogenic point mutations at A572D and Q573E functionally mimic the effect of CaMKII phosphorylation at the adjacent S571 residue, suggesting negative charge within this region may confer similar effects on channel biophysics. Although a direct link between S571 and increased I_Na,L has not yet been established, prolonged APs and triggered activity were observed with arrhythmia variants A572D and Q573E, expressed in neonatal myocytes and simulation studies. However, a study by Tester et al. (114) demonstrated that the inherited A572D mutation, although originally identified in LQTS genetic screens, is not functionally different from WT channels and is not proarrhythmic by itself.

Our group was the first to show that autophosphorylated CaMKII binds the I–II loop, and we confirmed this loop as the primary CaMKII phosphorylation target in vitro (8). Using peptide spot arrays of the entire I–II loop, we systematically identified CaMKII phosphorylation sites by P32 incorporation with in vitro kinase assays. We found that S516 and T594 are specifically phosphorylated by CaMKII but found no evidence for phosphorylation at the S571 site identified in the Hund study. In HEK293 cells expressing Na_V1.5α, we demonstrated that CaMKIIδ_C shifts I_Na SSI to negative potentials and enhances accumulation of intermediate inactivation (consistent with the previously reported CaMKIIδ_C effects). These effects are reversed by AIP or mutation of any of the reported CaMKII targets (T594, S516, or S571) to nonphosphorylatable alanine. T594E and S516E, but not S571E, charged glutamate phosphomimetics recapitulate CaMKII effects even in the absence of exogenous CaMKII and presence of AIP. Interestingly, a recent phosphoproteomic study suggested that S571 is phosphorylated at baseline in unstimulated mouse Na_V1.5α, whereas phosphorylation at S516 and T594 was not seen (74). CaMKII phosphorylation at T594 and S516 awaits further confirmation in native myocytes, and more studies are needed to understand the relative contributions of T594, S516, and S571 to I_Na gating changes in pathological conditions.

Functional Consequences for Acquired Arrhythmias: CaMKII as a Model

Because CaMKII can cause both LQTS-like I_Na,L and loss-of-function BrS-like effects, we sought to investigate how its effects contribute to arrhythmogenesis (38). Indeed, acquired forms of altered Na_V1.5 function attributable to post-translational modifications (e.g., phosphorylation or oxidation) may increase the risk of arrhythmia and sudden cardiac death during ischemia-reperfusion or HF. Furthermore, CaMKII phosphorylates numerous Ca handling proteins as well as sarcolemmal K channels (Fig. 4) (13). This in turn can influence myocyte Ca regulation and synergize with I_Na gating effects in arrythmogenesis.

Fig. 4.

Arrhythmogenic mechanisms of CaMKIIδ_C-mediated I_Na regulation. Gain (+) and loss (−) of function effects of CaMKIIδ_C phosphorylation are indicated with upward and downward triangles, respectively. NCX, Na/Ca exchanger; NKA, Na-K-ATPase; Na_V1.5, voltage-gated Na channel isoform 1.5; RyR, ryanodine receptor; PLB, phospholamban; SERCA, sarco(endo)plasmic reticulum Ca ATPase; APD, AP duration.

CaMKII-mediated gain-of-function effects, i.e., the late or persistent inward current, may delay repolarization, prolong APD (Fig. 3B), and alter intracellular Na and Ca homeostasis, potentially predisposing to arrhythmogenesis [via early and delayed afterdepolarizations (EADs and DADs, respectively); Figs. 3, A and B, and 4]. Our modeling study (38) predicted that CaMKII-mediated enhancement in I_Na,L could cause AP prolongation, especially at slow pacing rates (consistent with a LQT3 phenotype). A mechanistic link between H₂O₂ (CaMKII mediated)-dependent increase of late I_Na, AP prolongation, and EADs has been established in experimental (136) and simulation (43) studies. Edwards et al. (33) reported that myocytes from TG mice overexpressing CaMKIIδ_C (before pronounced HF remodeling) are susceptible to EADs, which are dependent on sarcoplasmic reticulum (SR) Ca release caused by pronounced isoproterenol-induced increases in Ca transient amplitude and APD. Computational modeling also suggested that the mechanism of AP prolongation and EAD initiation involves recruitment of I_Na,L secondary to SR Ca release-dependent augmentation of inward Na/Ca exchange current. Prolonged APs and cellular arrhythmias (both EADs and DADs) have been reported in human cardiomyocytes from patients with hypertrophic cardiomyopathy and attributed to CaMKII-mediated I_Na,L increase (29). Disrupting spectrin-mediated molecular anchoring of CaMKII at Na_V1.5 has been shown to remove the effects of CaMKII on I_Na and reduce EAD propensity (47). By enhancing SR Ca loading and promoting spontaneous intracellular Ca waves, H₂O₂-induced EADs were also shown to cause DADs (136). Recently, the generation of ROS under conditions of increased oxidative stress, such as during HF, was shown to correlate with CaMKII-mediated augmentation of late I_Na and consequent arrhythmias (128).

Fig. 3.

Consequences of CaMKIIδ_C hyperactivity on cardiac function. A: delayed afterdepolarizations (DADs). B: early afterdepolarizations (EADs). C: transmural dispersion of repolarization. D: slowing of action potential (AP) rate of rise and decreased AP amplitude. Endo, endocardium; Epi, epicardium; bpm, beats/min.

The loss-of-function effects (decreased availability, enhanced intermediate inactivation, and slowed recovery there from) may favor a BrS-like phenotype (Fig. 4). Our model (38) predicted that these alterations induced by CaMKII could reduce the velocity of AP upstroke (Fig. 3D) and slow conduction and are likely to be exacerbated at high heart rates (Fig. 3D) whereby the diastolic interval is reduced and Na channels have less time to recover. Increased post-repolarization refractoriness in the infarct border zone [where CaMKII activity is enhanced (25, 46)], attributed to I_Na remodeling, has been proposed as a mechanism for slow conduction and arrhythmogenesis (18). Indeed, simulations showed CaMKII hyperactivity in the infarct border zone, due primarily to increased oxidation, causes I_Na-mediated reduction of AP upstroke (46) and conduction velocity (25), increase in effective refractory period, and increased susceptibility to formation of conduction block at the border zone margin, which predisposes to reentrant arrhythmias (25).

CaMKII also mediates Ca current facilitation, which enhances peak L-type Ca current and slows inactivation (gain of function) and tends to prolong APD (along with I_Na,L) (38). CaMKII also alters both fast and slow transient outward K current (I_to; enhanced recovery from inactivation) and inward rectifier K current (127) in ways that shorten APD (Fig. 4). Simulations predicted that with transmural heterogeneity of I_to and I_to downregulation in HF, the net effect of CaMKII would be to shorten epicardial APD and prolong endocardial APD, thus accentuating the normal transmural dispersion of repolarization (Fig. 3C) (38), an effect known to be proarrhythmic (7).

As discussed, recent computational myocytes models have incorporated elements of the CaMKII signaling cascade and provided new insights regarding the role of CaMKII in regulating cardiomyocyte contractility and excitability in health and disease. Whereas much progress has been made to determine and model the kinetics of phosphorylation of various CaMKII targets (110), the kinetics of CaMKII-mediated phosphorylation and dephosphorylation of Na channels is unknown. Thus these models are somewhat limited in assuming steady-state fractional phosphorylation of Na channels rather than dynamic transitions between unphosphorylated and phosphorylated Na channels (43). However, the dynamics of CaMKII-dependent Na channel phosphorylation may be critical for its arrhythmogenic consequences and associated potential treatment. Recently, Moreno et al. (80) developed a computational approach to study the interaction of kinetics of the blockers flecainide and lidocaine with cardiac Na channels. They used the model to predict the drug effects on human ventricular cellular and tissue electrical activity and in the setting of one common arrhythmia trigger, spontaneous ventricular ectopy. The model predicted that clinically relevant concentrations of the antiarrhythmic drugs flecainide and lidocaine would exacerbate, rather than ameliorate, arrhythmia. On the other hand, therapies that target late I_Na, such as block by ranolazine, may have promise in treating patients that are at risk for arrhythmia (65, 125).

Pathological Changes in Na Current

Studies of the I_Na changes occurring in cardiac pathologies/disease (summarized in Tables 1 and 2) are less abundant than those characterizing inherited Na_V1.5 channelopathies. This is largely due to the difficulty in obtaining human donor transplant tissue suitable for electrophysiology (or biochemistry), as well as the cost and complexity of animal models. Moreover, the results of such studies are often variable and difficult to interpret. For example, human explant studies have wide variability in age, sex, ethnicity, severity of disease, and low sample number and are further complicated by patient history (e.g., pharmacological treatments that can alter I_Na). Animal models (Table 1) may have important species differences, and TG mice with altered genetics (Table 2) may have compensatory ion channel remodeling effects that complicate the interpretation of I_Na functional effects.

Table 2.

I_Na changes in TG mice with HF

Model	Cell Type	INa, A/F	SSI	SSA	INaL	INa Kinetics	SCN5A mRNA	Nav1.5 Protein	Comments (References)
CaMKIIδc-OE	V	↔	←	↔	↑	Slower recovery from inactivation and enhanced development of inactivation		↑	Trend to ↓ INa density; TG mice exhibit longer QRS intervals and monomorphic and polymorphic VT upon programmed electrical stimulation (126)
Muscle LIM protein MLP−/−	V	↓	←	→		Slowed inactivation		↓	↑ APD and EAD propensity, ↓ AP amplitude and rate of depolarization, lower Nav1.5 protein molecular weight (deglycosylation) (117)
Unilateral nephrectomy/DOCA implantation, salt water substitution	V	↓					↔	↔	Reducing mitochondrial ROS by application of NAD+, mitoTEMPO, PKC inhibitors, or PKA activators, restored INa (58)
Inducible CX43-KO	LV, RV	↓						↓	Slower conduction and longer QRS in pacing induced VT+ mice (49)
PKP2 ± ARVC	V	↓	←	↔		Slower recovery from inactivation		↔ ICC	Abnormal ultrastructure; exaggerated INa decrease and slowed conduction w/flecainide, arrhythmia and SCD (19)
ACE 8/8-OE		↔	↔ ←	←					Cardia-specific (α-MHC) overexpression; results in fourfold increase in ANG II (48, 52)
Calcineurin-OE	V	↓↓	↔	↔		↔		↔ ICC	Hypertrophy, ↓ rate of AP rise, progressive heart block, SCD (39)
Snail ± OE/DCM	V	↓					↓		DCM, ECG abnormalities, conduction defects; Homozygous mice lethal (44)
CSQ-OE	V	↓↓	→	→					DCM, hypertrophy, ↑ cell capacitance, ↑ PR and QT interval, conduction block (55)
Mdx (5cv)	V	↓					↔	↓	Dystrophin deficient; impaired conduction (35)
AnkB−/− KO	V	↓	←	←	↑	Slowed recovery from inactivation			Prolonged APD; impaired QT-rate adaptation; slowed HR; longer single Na channel mean open time; late single channel openings (23)

ACE, angiotensin-converting enzyme; ANG II, angiotensin II; ARVC, arrhythmogenic RV cardiomyopathy; CSQ, calsequestrin; DCM, dilated cardiomyopathy; KO, knockout; MHC, myosin heavy chain; MLP, muscle LIM protein; OE, overexpression; PKP2, plakophilin-2; TG, transgenic; VT, ventricular tachycardia.

Na channel expression and functional alterations in HF are complex and may affect cardiac electrophysiology in various ways. Despite the above limitations, a few common observations in the reported results emerge. First, many studies report a decrease in functional I_Na density (9, 10, 57, 68, 97, 118, 119). Second, I_Na,L has been reported in both canine (68–70, 118) and human HF (119), as well as in failing mouse cardiomyocytes (126). Both observations could be the result of various complex post-translational modifications of Na_V1.5, including phosphorylation by CaMKII. Moreover, both would be expected to contribute to arrhythmias as illustrated in Figs. 3 and 4 for CaMKII-dependent Na_V1.5 gain- and loss-of-function effects: the reduced I_Na could lead to conduction slowing and ventricular arrhythmias based on reentrant mechanisms, whereas increased I_Na,L may delay repolarization, prolong APD, alter intracellular Na and Ca homeostasis, and potentially predispose to arrhythmogenesis (EADs, DADs). As reported for K channels (73), a decrease in functional current density could be the result of either a decrease in peak conductance, an active decrease in channel number at the membrane, or myocyte hypertrophy without compensatory increases in channel expression (85). Most studies do not distinguish between these. Thus the decrease in I_Na current densities reported in Tables 1 and 2 must be interpreted cautiously. However, a few studies have reported changes in Na_V1.5 protein and/or mRNA levels (Tables 1 and 2) that coexist and are consistent with a decrease in I_Na density. More controlled experiments in animal disease models are needed to further refine the molecular mechanisms of I_Na changes in cardiac disease and their relative contributions to arrhythmias.

Summary and Concluding Remarks

We reviewed here the molecular and functional aspects of cardiac Na channel modulation and its causal link to cardiac arrhythmias. Interestingly, PKA, PKC, and CaMKII similarly shift I_Na availability to negative potentials. Although not yet identified, it is probable that additional putative PKC sites exist on the Na_V1.5 I–II loop (as for PKA and CaMKII) that also decrease availability. Thus kinase phosphorylation may act in a concerted manner through the I–II loop to enhance inactivation under various stimuli. A negative shift in I_Na availability would be particularly detrimental in ischemic conditions, which are associated with depolarized resting membrane potentials (86).

Chronic phosphorylation by either PKA or PKC alters channel surface levels, but in antagonistic ways. In the case of PKA, the increase in myocardial conduction velocity associated with sympathetic stimulation implicates an increase in channel density at the membrane. In contrast, PKC decreases the number of functional channels at the membrane, and this may be particularly relevant for chronic channel remodeling as in HF. In fact, PKC isoforms are upregulated with increased activity in HF (88, 130). More studies are needed to confirm this regulation in cardiomyocytes and determine if CaMKII similarly alters channel surface expression.

Additionally, PKC regulation appears to be intimately intertwined with metabolism, since mutations in GPD1L that increase both NADH and ROS decrease I_Na in a PKC-dependent manner. It is still unclear which mode of PKC activation (increased G3P and phospholipids or direct activation by NADH) is the most relevant, but there is good evidence that direct phosphorylation of Na_V1.5 by PKC at S1503 (and likely other sites) decreases I_Na. Likewise, the exact mode of regulation that leads to the decrease in I_Na observed with PKC activation is unclear (ROS vs. direct PKC phosphorylation). Is the effect of ROS direct or indirect via activation of other downstream proteins, such as CaMKII? Indeed, it is conceivable that increased PKC activity fuels an increase in mitochondrial ROS, which constitutively activate CaMKII to regulate I_Na. Both CaMKII and PKC appeared to be involved in the Ca-dependent increase in I_Na,L observed in rabbit cardiomyocytes (63).

Interestingly, the PKA phosphorylation sites at S525/S528 and the ER retention motif at 533–535 all neighbor a CaMKII phosphorylation site identified at S516 (8) and a methylated Arg at position 513 (12) (of the same CaMKII motif). The close proximity of these PKA and CaMKII phosphorylation motifs and their similar functional consequences for channel inactivation suggest that this region of the I–II loop is important for channel gating and conformationally/structurally accessible to regulation in the fully folded channel complex. Although the Catterall group (92) recently solved the crystal structure for a bacterial voltage-gated Na channel, this channel lacks the regulatory cytoplasmic loops present on mammalian channels. Regarding CaMKII sites identified on Na_V1.5 thus far, a recent phosphoproteomic study of unstimulated, native mouse Na_V1.5α showed basal phosphorylation of S571 and no phosphorylation at T594 or S516 (74). Although the S516 site is conserved in mouse, the critical Arg at P-3 (R513) of the CaMKII motif is not (14). Peptides containing T594 were not recovered by mass spectrometry. Furthermore, PKA and CaMKII regulation of Na_V1.5 both appear to require multiple phosphorylation events on the channel, analogous to the graded regulation that has been observed for some K channels (90).

Collectively, these studies highlight the important role post-translational modifications play in both acute I_Na gating changes and chronic regulation of Na_V1.5 surface expression. Much work remains to fully unravel the complexities of post-translational regulation of Na_V1.5 and its role in cardiac physiology and disease.

GRANTS

This study was supported by the National Institutes of Health grants P01-HL-080101, R01-HL-105242, and training grant T32-GM-099608 and the Fondation Leducq Transatlantic CaMKII Alliance.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

A.W.H. and E.G. conception and design of research; A.W.H. and E.G. interpreted results of experiments; A.W.H. and E.G. prepared figures; A.W.H. and E.G. drafted manuscript; A.W.H., D.M.B., and E.G. approved final version of manuscript; D.M.B. edited and revised manuscript.