Late sodium current is a new therapeutic target to improve contractility and rhythm in failing heart

Abstract

Most cardiac Na⁺ channels open transiently within milliseconds upon membrane depolarization and are responsible for the excitation propagation. However, some channels remain active during hundreds of milliseconds, carrying the so-called persistent or late Na⁺ current (I_NaL) throughout the action potential plateau. I_NaL is produced by special gating modes of the cardiac-specific Na⁺ channel isoform. Experimental data accumulated over the past decade show the emerging importance of this late current component for the function of both normal and especially failing myocardium, where I_NaL is reportedly increased. Na⁺ channels represent a multi-protein complex and its activity is determined not only by the pore-forming α subunit but also by its auxiliary β subunits, cytoskeleton, and by Ca²⁺ signaling and trafficking proteins. Remodeling of this protein complex and intracellular signaling pathways may lead to alterations of I_NaL in pathological conditions. Increased I_NaL and the corresponding Na⁺ influx in failing myocardium contribute to abnormal repolarization and an increased cell Ca²⁺ load. Interventions designed to correct I_NaL rescue normal repolarization and improve Ca²⁺ handling and contractility of the failing cardiomyocytes. New therapeutic strategies to target both arrhythmias and deficient contractility in HF may not be limited to the selective inhibition of I_NaL but also include multiple indirect, modulatory (e.g. Ca²⁺- or cytoskeleton- dependent) mechanisms of I_NaL function.

INTRODUCTION

Chronic HF is associated with profound abnormalities in both cardiac rhythm and contractile function. Despite recent progress in treatment of congestive HF, mortality remains high. Approximately 40% of these patients die suddenly due to the sudden cardiac death syndrome. Ventricular tachycardia and fibrillation have been documented in ∼ 80% of patients with congestive HF in whom ECG Holter recordings were being obtained at the time of sudden death [1, 2].

Despite intensive research, the electrophysiological mechanisms leading to these arrhythmias are not completely clear. It is widely appreciated that perturbation in control of the action potential (AP) duration (APD) and its propagation, is the proximate cases of arrhythmia. Among numerous proteins involved in the cardiac cell remodeling in HF, the voltage-gated Na⁺ channels (NaCh) deserve special consideration, as they seem to be critically involved in abnormal conduction, repolarization, and Ca²⁺ handling [3, 4]. Most NaCh open only transiently and are quickly inactivated resulting in the peak transient current, I_NaT, which determines excitation and conduction. However, some NaCh remain active, carrying so-called persistent or late Na⁺ current (I_NaL) throughout the AP plateau (reviews [5, 6]).

A growing body of evidence shows that I_NaL provides a major contribution to the AP plateau duration in ventricular cardiomyocytes (VCs) in a variety of mammalian species including humans [7-11].

Since late openings of NaCh generate both electric current and Na⁺ influx during the AP plateau, I_NaL is expected to contribute to at least two known HF cellular mechanisms: 1) electrophysiological remodeling and 2) altered cell Na⁺ cycling. The latter mechanism is tightly integrated with Ca²⁺ cycling, as Na⁺ modulates the Na⁺/Ca²⁺ exchanger (NCX) operation [4]. These anticipated I_NaL contributions could be amplified at the state of chronic HF that reportedly increases the whole cell I_NaL [10, 12, 13].

The importance of I_NaL contribution into HF mechanisms has been demonstrated in experiments where “correction” of I_NaL in failing cardiomyocytes resulted in: 1) rescue of normal repolarization, 2) decrease beat-to-beat APD variability, 3) improvement of Ca²⁺ handling and contractility [10, 13-15]. Accordingly I_NaL has emerged as a novel possible target for cardioprotection to treat the failing heart [6, 16, 17].

COMPLEX IONIC MECHANISMS OF IMPAIRED REPOLARIZATION IN CHRONIC HEART FAILURE

Studies with microelectrodes in ventricular myocardial fibers obtained from failing human hearts [18, 19] and patch-clamped ventricular cardiomyocytes (VCs) isolated from failing hearts have demonstrated a prolongation of APD [10, 11, 20-22] indicating impaired repolarization in HF. The prolongation was less prominent at higher rates [18] and varied depending on the etiology of HF [22]. Experimental and clinical studies suggest that dispersion of repolarization and EADs are two major mechanisms underlying torsade de pointes [23, 24]. This type of arrhythmia is induced by a pause or bradycardia [25]. Indeed, the APD prolongation and dispersion of duration, as well as the incidence of EADs, were advanced at lower pacing rates in VCs of humans and dogs with chronic HF [10, 11, 14]. EADs, in turn, can be a substrate for triggered arrhythmias described in patients with HF [23, 26].

A delicate balance between inward and outward currents maintains the cardiac AP plateau, and repolarization occurs as activating outward currents prevail over inactivating inward currents. Accordingly, APD increase can be explained by reduction of hyperpolarizing outward currents and/or by an increase of depolarizing inward currents. The balance of ion currents is substantially altered in HF as a result of the remodeling of ion channels [3, 27]. Decrease of the transient outward potassium current (I_to) has been reported by many investigators and is now accepted as a common feature of ischemia and HF (review [3]). However, some studies also showed that I_to in human VCs shows no dramatic differences between cells derived from failing and non-failing hearts [28]. Remodeling of other potassium currents (I_Kr, I_Ks, and I_K1) is also variable and seems to be related to the HF etiology (ischemic vs. non-ischemic)[27]. While numerous studies tested HF-related changes of L- type Ca²⁺ channel expression and function, the data remain controversial: I_CaL was found decreased, unchanged or increased in HF. In addition, alterations in expression of the electrogenic Na⁺/Ca²⁺ exchanger (NCX) [29] and Ca²⁺ cycling by sarcoplasmic reticulum (SR) [4] contribute to the complex AP remodeling. Numerous pharmacological approaches targeting different players/contributors of abnormal electrical heart function have been tested to treat arrhythmias in HF, however the problem is still far from being solved. A novel perspective target could be I_NaL because this inward current greatly contributes to AP duration, especially in human myocardium [11, 13, 16], and it is reportedly increased by chronic HF [10, 12, 13].

DEFINITION AND MAJOR CHARACTERISTICS OF THE LATE SODIUM CURRENT

Voltage clamp studies have identified several types of single NaCh activity and whole cell Na⁺ currents that could contribute to APD in cardiomyocytes. The variety of NaCh activities identified until the present time was classified (see review [6]) in terms of the late (or persistent) Na⁺ current i.e. I_NaL (or I_pNa), and background Na⁺ currents. Our review is focused on properties and targeting I_NaL rather than background Na⁺ currents. In contrast to I_NaL, background Na⁺ currents have been poorly characterized to date and have no clear molecular identity.

Major biophysical and pharmacological characteristics of the whole-cell I_NaL have been studied in great detail in human VCs by our research group [11, 13, 16] (Fig. (1)) and can be summarized as follows: 1) slow potential-independent inactivation and re-activation (∼0.5 s), 2) steady-state activation and inactivation similar to that for I_NaT, 3) low sensitivity to the specific toxins TTX and STX similar to the cardiac NaCh isoform Na_v1.5. A slowly inactivating I_NaL with aforementioned biophysical characteristics has been identified in VCs of dogs [10, 13, 15, 30], guinea pigs [31-33], rabbits [34] and rats [35]. I_NaL is also produced by heterologously expressed cardiac NaCh isoform main α-subunit Na_v1.5 (Fig. (1F)).

Figure (1).

Biophysical properties of the slowly inactivating, late Na⁺ current (I_NaL) evaluated by whole cell patch clamp in human ventricular cardiomyocytes (A-E) and human cloned Na_v1.5 expressed in tsA201 cells (F). A-B: I_NaL can be carried either by Na⁺ or Li⁺. B: I-V relation for I_NaL. C: examples of steady-state activation and availability curves, G(V_m) and A(V_p), respectively. D: Examples of original traces illustrating voltage-independent I_NaL decay. E: slow reactivation of I_NaL. F: I_NaL produced by Na_v1.5 was assessed as difference current before application of a selective Na⁺ channel blocker tetrodotoxin (TTX, 30 μM) and after TTX. Voltage protocols are shown at the traces. Recording was performed at 24°C. Modified from [11] (A-E) and [65](F), used with permission.

It is important to note, however, that I_NaL is not a “window” current that was suggested long time ago as a theoretical mechanism to explain the persistent Na⁺ current in cardiac cells [36, 37]. According to the Hodgkin-Huxley formalism [38], the “window” current is a non-inactivating component of I_NaT, resulting from the crossover of its steady-state activation and inactivation curves. The overlap of steady-state activation and inactivation curves occurs within a relatively narrow region of voltages close to I_NaT activation threshold (Fig 1C). However, I_NaL exhibit time-dependent inactivation and is present even at positive voltages (Fig.1B, filled circles, Fig. (3C)) [11, 31], where the calculated “window” current is negligible.

Figure (3).

Inactivation of both late scattered mode (A) or burst mode (B) of the late openings of Na⁺ channel was slowest in failing human cardiomyocytes compared with those from normal human hearts or heterologously expressed Na_v1.5. *P<0.05, heart failure vs. normal heart or clone (Mean±SEM). Cell-attached patches, 24°C. (Adapted from [12]). C: recordings of action potentials in failing human cardiomyocytes are shown along with the late scattered mode and burst mode openings occurring at -10 mV, i.e. within the voltages of the action potential plateau. Adapted from [12, 40], used with permission.

I_NAL IS INCREASED IN FAILING HUMAN AND CANINE VENTRICULAR MYOCARDIUM

The first evidence of I_NaL importance in electrophysiological remodeling of cardiomyocytes in HF was found in 1995 in an experimental dog model of chronic HF [39]. Further patch clamp studies in human and canine hearts have conclusively shown that chronic HF increases I_NaL density and significantly slows inactivation kinetics of I_NaL in VCs [10, 13] (Fig. (2A)). Analysis of idealized I_NaL time course (Fig. (2B)) in canine normal and failing VCs shows that 1) absolute I_NaL difference between normal and failing canine VCs at 37°C (top panel in Fig. (2B), gray area) has a maximum at 90 ms after membrane depolarization, i.e. within the time of AP plateau duration; 2) the relative difference between normal and failing cells increases progressively with membrane depolarization, doubling after 330 ms (Fig. (2B), inset)); 3) The integral of I_NaL reflecting Na⁺ influx transferred by I_NaL, is much greater in HF (bottom panel in Fig. (2B), gray area) [13]. Accordingly, I_NaL may disturb cell Na⁺ balance and have different dynamic impacts on the balance of the ionic currents at the different phases of AP plateau in failing heart. The importance of these phenomena is discussed below.

Figure (2).

A: Chronic heart failure slows and increases I_NaL. A: examples of whole cell I_NaL recordings in human and dog ventricular cardiomyocytes. B: Idealized I_NaL and their integrals in normal and failing dog cardiomyocytes of same size (200 pF) calculated using Q₁₀ factors (37°C) and average parameters of I_NaL density and decay time constant measured in normal and HF canine VCs. Larger and slower I_NaL in failing cardiomyocytes results in substantial increase in total charge (or Na⁺ influx) transfer by I_NaL. Gray areas illustrate difference between failing and normal cells. Adapted from [13], used with permission.

THE I_NaL FINE STRUCTURE AND RELATED SODIUM FLUXES: PARADOXES AND THEIR MECHANISMS

In cell-attached patches from heterologously expressed Na_v1.5 and native human cardiomyocytes, the late NaCh activity is arranged in two major gating modes: late scattered mode (LSM) and “burst” mode (BM) (Fig. (3C) inset)). A detailed analysis of the properties of these modes and its contributions to whole cell I_NaL lead to some unexpected, paradoxical conclusions [12, 40]. Paradox #1: The late scattered openings were previously reported in guinea pig and considered to represent “background” activity [41]; however, they actually possess ultra slow (hundreds of ms) inactivation, similar to that of whole-cell current decay in human and dog cardiomyocytes [13, 40]. Paradox #2: While inactivation of I_NaT is voltage-dependent, the inactivation time constant of LSM is potential-independent. Paradox #3: Bursts are often considered as a persistent activity, but they do inactivate and cease well within AP duration (Fig.3B). Paradox #4: While bursts represent abundant NaCh activity, a numerical evaluation of the contributions of BM and LSM to I_NaL based on the Markov model of single channel data unexpectedly pointed to LSM openings, but not the bursts, as a major contributor to whole cell I_NaL. Paradox #5: While I_NaT amplitude is about 3 orders of magnitude larger than that of I_NaL (50 nA in comparison to 50 pA), the total charge (reflecting Na⁺ influx) transferred across the membrane by I_NaT and I_NaL is almost the same! In fact, I_NaT span is about 3 orders of magnitude shorter than that of I_NaL (2 ms in comparison to 2 s), resulting in an almost equal total charge transfer by these currents.

I_NaL transfers significantly more Na⁺ into failing vs. normal VCs. The total charge transferred by I_NaL is predicted to be 28.5 and 45 pC for normal and failing VCs, respectively, or a ∼58% increase. This model estimate is in line with a 53.6% HF-induced increase of Na⁺ influx via I_NaL evaluated from whole cell patch clamp measurements in canine normal and failing VCs (gray area in Fig. (2 B) bottom panel; [13]). Taking into account that I_NaT is reduced by 30-40% in HF VCs [42-44], the role of I_NaL in Na⁺ homeostasis should be even more substantial in the failing cells, in line with the recent finding that [Na]_i is significantly increased in failing paced cardiomyocytes [45].

One possibility for the increased I_NaL in HF could be an additional, HF-specific NaCh activity. However, no qualitative changes in NaCh gating were found in failing human VCs in comparison to normal VCs and Na_v1.5 clone [40]; they exhibit early openings and the two modes of late gating (LSM and BM) with the same single NaCh conductance [40]. The inconspicuous differences turn out to be quantitative: 1) inactivation of LSM (Fig. (3A)) was significantly slower and 2) burst length was significantly larger in failing compared to normal myocytes or Na_v1.5 clone (Fig. 3B)) [13].

MOLECULAR IDENTITY OF I_NAL IN NORMAL AND FAILING VENTRICULAR MYOCARDIUM

The problem of molecular identity of I_NaL has been approached in many different ways including methods of biophysics, pharmacology, and molecular biology. Late NaCh openings show similar gating modes and Na⁺ conductance in normal human cardiomyocytes and in heterologously expressed Na_v1.5. Furthermore, the heterologously expressed Na_v1.5 produces the slowly inactivating whole cell I_NaL in tsA201 cells (Fig. (1F)) similar to that observed in human cardiomyocytes (Fig. (1A-D)). These data indicate the common molecular origin of the late channel openings i.e., they are produced by Na_v1.5 in human cardiomyocytes. Dose-response curves for blockade of I_NaL by TTX and STX reveal only a single-site binding with the half-concentration that is to say typical for the Na_v1.5 in both dog and human failing hearts (IC₅₀ was 1.2 vs. 1.53 μM for TTX and 62 vs. 98 nM for STX comparing dog vs. human cardiomyocytes) [11, 13]. Furthermore, I_NaL is sensitive to Cd²⁺ (IC₅₀=104 μM) [13], what is typical for cardiac but not neuronal Na⁺ channel isoforms [46]. Silencing the SCN5A gene responsible for Na_v1.5 expression with siRNA decreases I_NaL by 75% in wide range of membrane potentials (including AP plateau), resulting in a significant reduction of AP duration and variability in dogs with chronic HF [47]. Thus, while contributions of other NaCh isoforms to I_NaL are still not excluded [17], Na_v1.5 likely provides a major contribution to I_NaL in both normal and failing canine and human VCs.

MECHANISMS FOR I_NaL ALTERATIONS IN PATHOLOGICAL CONDITIONS: POSSIBLE ROLE OF THE CHANNEL MICROENVIRONMENT

Since results of multiple studies (described above) indicate that HF alters I_NaL likely via modulation(s) of Na_v1.5 gating, the question of which specific modulatory mechanisms may underlie I_NaL alterations, deserve special consideration, because these might represent new indirect targets for I_NaL-related therapies.

NaCh and associated modulatory proteins

The function of the NaCh is not fully determined by its protein structure, but also depends on its environment. Na⁺ channels represent a multi-protein complex comprising not only the main pore-forming α subunit and its auxiliary β subunits, but also components of cytoskeleton, Ca²⁺-sensitive protein calmodulin, regulatory kinases and phosphatases, trafficking proteins, and extracellular matrix proteins embedded into lipid bilayer plasma membrane. A diagram of Na_v1.5 and its interacting proteins is shown in Fig. (4 A). For in-depth reviews on this matter see [48-50]. Here we highlight only the components that seem to modulate I_NaL in different pathological conditions, including hypoxia and HF. The III-IV linker (see Fig. (4 B)) is responsible for NaCh inactivation [51], and mutations in this region disrupt Na_v1.5 inactivation causing persistent Na⁺ current linked to inherited LQT3 syndrome [52]. Recently, COOH terminal that has binding sites for abundant regulatory proteins has been implicated in Na_v1.5 inactivation [53, 54].

Figure (4).

Schematic illustration of Na⁺ channel macromolecular complex. A: The pore forming α subunit of the channel interacts with β-subunits, cytoskeleton and the extracellular matrix (Modified after [50], used with permission). B: schematic presentation of the α subunit of the cardiac Na⁺ channel isoform (Na_v1.5) with reported sites of interaction with β subunits (restricted only to β1 and β2) and other regulatory proteins. Reprinted from [17], used with permission.

Modulation of I_NaL by β subunits

Mammalian voltage gated NaCh are associated with auxiliary β subunits. The β-subunit gene family has four members β₁(SCN1B), β₂ (SCN2B), β₃ (SCN3B) β₄ (SCN4B) (see for review [48, 50]). All these β-subunits are expressed in rodent hearts and are differently localized to specific sub cellular domains and cell types. β₁ subunit is non-covalently attached to α subunit, and β₂ subunit is covalently linked to α subunit by a disulfide bond [55]. The protein of these β subunits contains an extracellular amino-terminus, a single transmembrane segment, and an intracellular COOH terminus (C-terminus) (Fig. (4B)). The extracellular N-terminus of all β subunits contain immunoglobulin domain found in cell adhesion molecules. Particularly, immunoglobulin domain for β₂ (and probably for β₄) is similar to contactin, whereas for β1 and its splice variant β₁A it is similar to myelin P_o. This unique property allows interactions with variety of signaling molecules and components of the extracellular matrix. With the regard to the intracellular domain, direct interactions of β₁ subunit C terminus and ankyrin B in rat brain membranes have been demonstrated, indicating a role of this subunit in main α subunit (Na_v) localization. Direct interaction between cytoplasmic C-terminus domain of Na_v1.1 with β₁ and β₃ has been recently demonstrated [56]. β subunits do not form an ion-conducting pore, but modulate NaCh function, NaCh protein expression at the plasma membrane (trafficking), and cell adhesion [48, 50]. More specifically, β₁-subunit: 1) is involved in abnormal NaCh activity associated with the LQT3 mutation [57], 2) aggravates NaCh dysfunction in Brugada syndrome [58], 3) modifies the blockade of NaCh by fatty acids [59] and lidocaine [60], 4) modulates the trafficking of Na_v1.5 [61], and 5) affects the burst mode of the heterologously expressed skeletal muscle NaCh isoform [62].

Very few reports are currently available about modulation by β subunits of late openings of the cardiac Na⁺ channel. Heterologous co-expression of β₁ subunit with Na_v1.5 in HEK293 cells diminishes I_bNa [63], but increases I_NaL amplitude and significantly slows I_NaL decay in tsA201 cells [64]. At the same time, β₂ subunit does not affect I_NaL parameters [64]. The potency of β₁ subunit to modulate I_NaL has been confirmed in preliminary studies in native cell environment. In normal dog VCs, knocking down of SCN1B by antisense nucleotides significantly accelerated I_NaL decay [65]. In HF, Na_v1.5 protein level is down regulated, whereas β₁ remains unchanged, indicating a relatively higher membrane content of β₁ [43]. This suggests potential involvement of β₁ in the reported I_NaL alterations in HF.

Modulation of I_NaL by cytoskeleton

The cytoskeleton proteins form a framework within the cytoplasm with links to the membrane that include attachments to integral membrane proteins. This framework maintains cell shape, plasma membrane integrity, and localization of membrane proteins such as ion exchange carriers and ion channels (Fig 7, see for review [48-50, 66]). Multiple experimental studies demonstrated that the sub-membrane cytoskeleton modulate I_NaL in cardiomyocytes.

Figure (7).

Simplified hypothetical diagram of the intracellular Ca²⁺ signaling pathways modulating late sodium current in normal and failing ventricular cardiac myocytes. Pathways 1, 2 and 3 (marked by black digits) represent Ca²⁺ binding to: the E-F domain, CaM-binding site (IQ motif) on C-terminus of NaCh as well as CaM/CaMKII complex, respectively. In addition, shown are the inhibitory sites of the CaM antagonist peptide P290-309 and KN93 - inhibitor of CaMKII, which were used in our original study [110] to discover the I_NaL modulation depicted in the diagram. Dashed line arrows indicate the regulation pathways which are operational only in heart failure. Reprinted from [110], used with permission.

Ankyrin-B

An adapter protein ankyrin-B, which is expressed in heart, links Na_v1.5 to the cytoskeleton. A knockout of ankyrin B in affects late Na⁺ channel openings in mouse cardiomyocytes [67]. Furthermore, mutations in ankyrin B cause the LQT4 syndrome in one French family [68], indicating the importance of channel environment for the channel function. Accordingly, a disruption of any member of this multi-protein complex by pathological conditions may lead to alterations of I_NaL.

F-actin

Cytochalasin-D, an agent that interferes with F-actin polymerization as well as anti-actin antibody [69], slows inactivation of cardiac Na⁺ channel by inducing bursts of openings. It also affects coupling of steady state activation and availability of Na⁺ current [70, 71].

Fodrin

Fodrin (spectrin)-based cytoskeleton, another element of the NaCh microenvironment in heart, is a dynamic structure, which is altered under a variety of pathological conditions (e.g. ischemia or heart failure [72-74]). The role of the fodrin-based cytoskeleton in I_NaL modulation has been suggested in experiments with the antisense oligonucleotides targeting mRNA’s encoding α- and β-fodrin in dog VCs [75].

The fodrin breakdown that happens in some disease states featuring poor Ca²⁺ handling can be mediated by the Ca²⁺ - activated enzyme calpain and caspase [72, 76, 77]. Therefore, disturbances of the ankyrin and/or fodrin-based cytoskeleton may affect the Na⁺ channel inactivation process. Cytoskeleton elements such as ankyrin may bind directly to α and β subunits (see above). The link to β subunits may thus modulate I_NaL indirectly.

Tubular cytoskeleton

also may be involved in NaCh gating regulation. For example, antitumor agent taxol, that stabilizes microtubules, shifts NaCh activation threshold towards more negative membrane potentials [78] and thus may increase the pool of NaCh that underlies I_NaL.

Modulation of I_NaL by the metabolites of membrane phospholipids

Lysophosphatidylcholine (LPC) is the endogenous amphiphilic lipid metabolite that rapidly accumulates in myocardium during ischemia and represents a major factor causing the electrophysiological alterations that contribute to cardiac arrhythmia [79, 80]. Recently, a significant prolongation of QTc interval has been found during early transmural ischemia in patients undergoing balloon angioplasty [81]. Experimentally, LPC causes membrane depolarization, reduction of the maximal upstroke velocity of AP, sustained abnormal rhythmic activity in Purkinje fibers, and delayed afterpotentials (DADs) in isolated tissue [79, 82]. The cellular mechanisms of the LPC effects include specific modifications of Na⁺ current: significant decrease of I_NaT and an emergence of late NaCh openings that produce a sustained Na⁺ current [35, 83-86]. Interestingly, the late NaCh openings caused by LPC form clusters of the synchronized multiple channel openings [83, 85]. One of the mechanisms underlying these LPC-induced modifications might be an integration of LPC into the lipid membrane, which would increase the membrane fluidity [87]. This, in turn, enhances motility and interaction of proteins within the membrane. On the other hand, LPC can activate neuromodulation signaling via PKA and PKC [88, 89], affecting NaCh slow inactivation [90].

Recently emerged modulators

A novel protein partner of Na_v1.5 has been discovered using a yeast 2-hybrid screen [91]. This protein, called 14-3-3η, interacts with the cytoplasmic I inter-domain of NaCh (see Fig. (4 B)). Although its direct effect on I_NaL properties has not yet been studied, it was shown that this protein influences the inactivation process by delaying recovery from inactivation[91]. Additionally, Src family tyrosine kinase Fyn has the phosphorylation site on Na_v1.5A III-IV linker [92], which is known to be responsible for the inactivation. The most recently emerged modulator is a membrane micro domain protein Caveolin-3. This protein was previously linked to NaCh trafficking, [93] but now is implicated in LQT syndrome [94]. Implementation of these new players in I_NaL modulation in different pathological conditions awaits further studies.

Modulation of I_NaL by the Ca²⁺signaling pathways

Structurally, the carboxyl terminus of NaCh has binding sites for Ca²⁺ itself [95] and for Ca²⁺-binding protein calmodulin (CaM) that acts as a Ca²⁺ sensor translating changes in cytoplasmic Ca²⁺ into cellular responses [96] (Fig. (7)). Discovery of these sites inspired multiple studies in heterelogously expressed NaCh, including a brain, skeletal and cardiac muscle isoforms [97-101]. It was found that some inactivation states of I_NaT of heterologously expressed cardiac and skeletal NaCh isoforms may be modulated directly by Ca²⁺, CaM and/or via Ca²⁺ /CaM/CaM-kinase signaling cascade. There are only a few studies to date performed in cardiomyocytes that addressed the question about I_NaL modulation by Ca²⁺ signaling specifically in NaCh native environment. It was shown that over expression of CaMKIIδ_c enhances I_NaL and increases [Na]_i [102]. Our recent detailed studies in normal and failing dog cardiomyocytes have demonstrated multiple, complex effects of Ca²⁺, CaM, and CaMKII on I_NaL properties (summarized in Fig.7), including the fine structure of I_NaL, described by fast (originated from bursts) and slow (originated from LSM) exponentials. More specifically, I_NaL greatly enhances as [Ca²⁺]_i increase: its maximum density increases, decay of both exponentials describing I_NaL decay slows, and steady-state inactivation curve (SSI) shifts towards more positive potentials. The latter means that more available NaCh generate a larger I_NaL. Testing inhibition of CaMKII and CaM revealed similarities and differences of I_NaL modulation in failing vs. normal dog myocytes. Similarities were as follows: 1) CaMKII slows I_NaL decay and decreases the amplitude of fast exponential; 2) Ca²⁺ shifts SSI rightward. The following differences in failing vs. normal myocytes were found: 1) slowing I_NaL by CaMKII is greater; 2) CaM shifts SSI leftward; 3) Ca²⁺ increases the amplitude of slow exponential. These data suggest that Ca²⁺/CaM/CaMKII signaling increases I_NaL and Na⁺ influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na⁺ influx provides a novel Ca²⁺ positive feedback mechanism (via Na⁺/Ca²⁺ exchanger), enhancing contractions at higher beating rates, but worsening cardiomyocytes contractile and electrical performance in conditions of poor Ca²⁺ handling in heart failure (multiple I_NaL-mediated Na⁺ - Ca²⁺ interactions in HF are discussed in detail below).

EXPERIMENTAL EVIDENCE OF I_NaL IMPORTANCE FOR ABNORMAL REPOLARIZATION, Ca²⁺ HANDLING, AND CONTRACTILITY IN FAILING MYOCARDIUM

Inhibition of I_NaL normalizes AP duration and beat-to-beat variability and eliminates EADs in HF VCs

APD is extremely frequency dependent in failing VCs [10]. At low pacing rates of 0.2-0.5 Hz the mean APD is significantly larger in failing VCs (Fig. (5 A)), and failing cells eventually exhibit EADs (Fig. (3 C)) [10, 11, 13]. In addition to the prolongation, APD also exhibits significant beat-to-beat variability in failing VCs. Although AP prolongation is not evident at physiological frequencies (1Hz and higher), variability of APD in failing cells is substantially larger than in normal cells (compare APD distributions and their SD values in Fig. (5 B)) [10, 13]. Increased and slowed I_NaL (Fig. (2)) greatly contributes to APD prolongation, variability, and EADs. Partial reduction in the magnitude of I_NaL caused by specific NaCh blockers (TTX, 1.5 μM, STX, 100 nM), a new antianginal drug ranolazine (that turned out to be a specific I_NaL blocker), or injection of external current opposite to I_NaL during the AP plateau significantly shorten the prolonged APD, decrease beat-to-beat variability of APD, and abolish EADs [10, 11, 13, 15].

Figure (5).

A: Frequency-dependence of action potential duration in ventricular cardiomyocytes of normal dogs and dogs with chronic heart failure. Note that largest difference occurs at low pacing rates. B: at the low (0.2 Hz) and the physiologic (1 Hz) pacing rates, AP duration in failing myocytes exhibits significant beat-to beat variability (see respective SD values in the APD₉₀ distribution histograms) Adapted from [10] with permission.

Blockade of I_NaL improves Ca²⁺ transient and contractility in HF

The contractile dysfunction in HF is related not only to ongoing loss of functional cardiac units, but also to abnormal function of cardiomyocytes. HF is characterized by systolic dysfunction as a result of depressed Ca²⁺ transients [4] and by abnormal relaxation of cardiac myocytes [103, 104]. At low pacing rates, the prolonged relaxation is associated with the spike-dome configuration of contractile response of cardiomyocytes (Fig. (6 A)). The ratio between amplitudes of the spike and dome phases was suggested to be an index for the severity of HF [103]. Similar to the shape of abnormal contraction, abnormally prolonged Ca²⁺ transients have also been observed in both ventricular muscle strips [103] and cardiomyocytes isolated from failing hearts [14, 105] (Fig. (6 A)). At the higher frequencies, these abnormalities account for the reversal of the force-frequency relationship in failing myocardium, leading to an increase in diastolic [Ca²⁺]_i and diastolic tension (Fig. (6 B) “Control”) [15, 106]. A partial blockade of I_NaL by STX, TTX or ranolazine greatly improves performance of failing VCs; it abolishes the dome phase of both contraction-relaxation cycle and Ca²⁺ transient at low pacing rates and prevents the rising diastolic tension and [Ca²⁺]_i at the higher pacing rates in failing VCs [14, 15] (Fig.(6)).

Figure (6).

A: Examples of effects of a specific Na⁺ channel blocker saxitoxin (STX) on AP duration, contraction and Ca²⁺ transient in ventricular cardiomyocytes of dogs with chronic heart failure at a low pacing rate of 0.2 Hz STX reduces AP duration, abolishes “dome” phase of contraction and of Ca²⁺ transient in failing cells. B: At higher pacing rates a specific I_NaL blockers ranolazine reduces diastolic tension, and a specific Na⁺ channel blocker tetrodotoxin (TTX) reduces Ca²⁺ accumulation (Fluo-4 signals). Adapted from [14, 15, 17], used with permission.

INTEGRATION OF I_NaL INTO ELECTROPHYSIOLOGICAL AND Ca²⁺ MECHANISMS IN HF

Dramatic improvement of cardiomyocyte function by I_NaL inhibition described above provides an evidence for a substantial I_NaL contribution to the functional remodeling in the failing heart. However, the interpretation of these effects needs extreme care because electrophysiology, contraction, and Ca²⁺ dynamics in cardiomyocytes are interrelated via multiple feedback mechanisms. The extent of deterioration of cardiomyocyte function and these feedback mechanisms vary greatly with the progression of HF and etiology. Since late openings of NaCh generate both an electric current and a Na⁺ influx, I_NaL is expected to contribute to at least two established HF mechanisms: 1) electrophysiological remodeling [3], and 2) altered cell Na⁺ and Ca²⁺ cycling [4].

The role of I_NaL in AP prolongation and EADs

Given the high membrane resistance during the AP plateau, I_NaL provides a critical contribution to the altered delicate balance of ion currents and thus to the APD. The relative contribution of I_NaL to the AP plateau in failing VCs is amplified by the reduced K⁺ currents in HF [3]. Since HF simultaneously increases and slows I_NaL it is expected to contribute directly to AP prolongation in HF, thus explaining APD normalization with I_NaL inhibition described above. Prolonged APD allows more time for I_CaL reactivation and thus facilitates EADs [107]. Since I_NaL contributes to AP prolongation, it thus indirectly contributes to EADs. Accordingly, I_NaL inhibition eliminates EADs likely as a result of APD shortening.

I_NaL and elevated [Na⁺]_i increase Ca²⁺ entry via NCX and limit depression of systolic function

A major problem in HF is systolic dysfunction, which is associated with smaller Ca²⁺ transient and sarcoplasmic reticulum (SR) Ca²⁺ content. The extent of deterioration of systolic function is limited by multiple compensatory mechanisms (listed below), which are indirectly linked to I_NaL and elevated Na⁺.

1. NCX function depends on Na⁺ and Ca²⁺ concentrations and membrane voltage. In HF VCs, increased Na⁺ influx (including that via I_NaL) shifts the NCX operation from the predominant forward mode to the reverse mode i.e. from Ca²⁺ efflux to Ca²⁺ entry [108]. Elevated Na⁺ in HF thus limits SR unloading and provides additional Ca²⁺ influx during the AP [4]. Interestingly, in addition to preserving SR Ca²⁺ load, this operational shift in failing human myocardium results in the direct activation of contraction during the terminal phases of the AP via the reverse mode NCX Ca²⁺ influx [109]. Thus, a larger cellular Na⁺ load (contributed by I_NaL) may be also important to drive contractions via this HF-specific mechanism.

2. The vast majority of studies demonstrated that NCX is upregulated in HF [29], therefore the above effects could be amplified by the enhanced NCX function.

3. I_NaL contributes to APD prolongation and thus indirectly prolongs Ca²⁺ influxes via I_CaL and the reverse mode NCX.

4. I_NaL in HF is positively modulated by intracellular Ca²⁺ [110] (Fig.7), which could yield a new possible amplification mechanism of the Ca²⁺ entry. This creates a positive feedback loop from I_NaL via NCX to larger cell Ca²⁺ load, then from the larger Ca²⁺ load back to I_NaL.

Adverse effects of increased [Ca²⁺]_i in HF. Role of I_NaL and NCX in diastolic dysfunction

While the increased I_NaL boosts a cell Ca²⁺ load and thereby limits the depression of systolic function in HF, it also leads to diastolic dysfunction, especially at high rates as described above. Relaxation of cardiac myocytes occurs when [Ca²⁺]_i declines, allowing Ca²⁺ dissociation from the myofilaments. Ca²⁺ is removed from cytosol, mainly via SERCA, which takes Ca²⁺ back into the SR, and, by NCX operating in the forward mode during diastole [111]. It is believed that the diastolic dysfunction in HF is mainly due to a reduced SERCA function in HF. At the same time, increased expression and function of NCX in HF tends to offset the deficiency of Ca²⁺ removal by SERCA (review [4]). The contribution of the increased I_NaL to the Ca²⁺ removal could be twofold. First, as discussed above, I_NaL and related increase of [Na⁺]_i facilitate Ca²⁺ influx. Secondly, higher [Na⁺]_i during diastole partially offsets the function of the forward mode NCX and thus worsens the problems of Ca²⁺ removal from the cytosol and diastolic dysfunction. The improvement of diastolic function by the inhibition of I_NaL (Fig.10B) can be attributed both to a decrease in Ca²⁺ load during the AP plateau and to improve removal of Ca²⁺ by forward mode NCX during diastole. Indeed partial blockade of the NCX improves EC coupling in HF [112] and reduces both EADs and DADs [113, 114].

Ca²⁺ overload and increased diastolic Na⁺: potential importance of I_NaL for DADs

DADs occur as a result of spontaneous Ca²⁺ releases during diastole via the activation of the forward mode of NCX. I_NaL does not occur at low diastolic potentials in humans and dogs (see Fig. 2B, filled circles), so it cannot have a direct contribution to DADs. However, as discussed above, in HF VCs a larger Na⁺ influx via I_NaL increases Ca²⁺ influx via NCX during AP leading to SR Ca²⁺ overload, which, in turn, is critical for initiation of spontaneous Ca²⁺ release (such as Ca²⁺ waves) during diastole. I_NaL involvement in DADs is thus limited to the contribution of the I_NaL to the Ca²⁺ overload. On the other hand, high diastolic [Na⁺]_i in HF [45] decreases the forward mode NCX current and, hence, attenuates the amplitude of DADs. This positive factor of I_NaL can be counterbalanced by down regulation of I_K1 in HF [115], facilitating membrane excitations.

Role of I_NaL in dispersion of repolarization: a feedback from abnormal Ca²⁺ cycling?

The mechanisms of dispersion of repolarization in HF remain unclear. A possible mechanism involves beat-to-beat alternations and/or fluctuations in intracellular Ca²⁺ cycling transduced to abnormal repolarization by electrogenic feedback mechanisms (review [116]). As discussed above, there are at least three possible indirect contributions of I_NaL to the beat-to-beat variability problem.

1. The I_NaL contribution to the abnormal Ca²⁺ cycling.

2. The I_NaL dependence on Ca²⁺/CaM/CaMKII. Thus I_NaL can serve as one of the electrogenic Ca²⁺ feedback mechanisms along with other Ca²⁺ -dependent ion currents, such as I_CaL inactivation, I_Ks, I_to, Ca²⁺-activated Cl^--current, and NCX.

3. The synchronicity of Ca²⁺ release depends on the state of phosphorylation of Ca²⁺ cycling proteins [117] and on the AP shape [118] (including the AP shape of failing VCs, [119]). Thus I_NaL contributes to the fluctuations of Ca²⁺ transients to the extent to which I_NaL contributes to the AP shape. More specifically, the contribution of I_NaL could be important for the abnormal early repolarization phase in HF via its increased burst mode [12] (Fig. (3 B)), especially when I_to is decreased [3]. Further, the activity of LSM of NaCh also increased in human HF VCs [12] (Fig. (3 A)); they persist on the AP plateau (Fig. (3 C)) and, hence, tend to prevent further repolarization.

Summary of I_NaL role in failing myocardium: friend or foe?

I_NaL and its Na⁺ influx can directly and indirectly contribute to several important HF mechanisms related to electrophysiological remodeling and ion homeostasis. These contributions could either improve or worsen performance of HF myocardium, i.e. being “friend” or “foe”, respectively (Fig.1). I_NaL is “friend” as it contributes to 1) APD prolongation as an adaptive and an anti-arrhythmic (anti-re-entry) response [3], and 2) Ca²⁺ entry to limit depression of systolic function. The latter mechanism is an intrinsic, adaptive, digitalis-like effect with all corresponding risks and benefits. Interestingly, a large slow component of the Na⁺ current decay (burst mode) has been identified in post-myocardial infarction-remodeled myocytes [120], i.e. in the transitional period from an infarction to HF. The increased I_NaL may indeed serve as an initial mechanism of adaptation to match an increased contractility demand for the survived VCs.

Although APD prolongation can be beneficial in HF, the temporal and spatial dispersion of repolarization that accompanies AP prolongation is critical for arrhythmia and sudden cardiac death [3]. Additionally, DADs and EADs have critical importance for non-reentrant arrhythmia or triggered activity (recent review [4]). Accordingly, I_NaL is “foe” as it contributes to EADs, DADs, dispersion of repolarization, and diastolic dysfunction.

I_NaL IS A NOVEL TARGET FOR CARDIOPROTECTION

Na⁺ channel: To block or not to block?

After negative outcomes of Cardiac Arrhythmia Suppression Trial (CAST) [121, 122] Class I antiarrhythmic drugs [123] targeting NaCh became close to a “taboo”. The trial tested whether the suppression of ventricular arrhythmias by encainide, flecainide, moricizine after myocardial infarction improves survival. The conclusion was that the “treatment strategies designed solely to suppress these arrhythmias should no longer be followed” [122]. However, the discoveries of inherited mutations in SCN5A gene that lead to an increased I_NaL (LQT3 syndrome, see for review [124]) and of increased and slowed I_NaL in acquired, chronic HF (discussed in this review) give rise to a revival of NaCh as a therapeutic target. However, these studies suggest that not all NaCh must be equally targeted. The emerging paradigm for Na⁺ channels in HF is that I_NaT is decreased [42-44] but simultaneously I_NaL is increased (see above). Blockers of I_NaT are proarrhythmic in HF because they slow conduction, thus worsening conduction problems (review [124]) and facilitating development of re-entry. Accordingly, new strategies for treatment must be considered: the new type of “smart” drugs should preferentially block I_NaL over I_NaT. This requirement calls for a new classification of Class 1 drugs in the future.

As discussed above increased I_NaL likely contributes to both electrical and contractile abnormalities of failing VCs, the potential benefits of a preferential I_NaL blockade in HF could be both an antiarrhythmic effect and an improvement of diastolic function. A preferential blockade of I_NaL over I_NaT in failing human VCs was reported for amiodarone [16], suggesting an explanation of why amiodarone, classified as Class III anti-arrhythmic drug, shows a remarkable efficiency among K⁺-channel blockers. Accordingly a new index of “smart drug” has been recently suggested as a ratio of the drug potencies to block I_NaL over of I_NaT (i.e. (IC_{50_INaT}/IC_{50_INaL})) [15]. Based on this new index three different drugs were compared and fell in the following sequence: ranolazine (37.8) > amiodarone (12.9) > lidocaine (2.7), suggesting that the most promising drug is a new antianginal drug ranolazine [15].

The potential great benefits (preventing arrhythmias and Ca²⁺ overload) of the preferential I_NaL blockade can be expected not only in HF but also in other cardiac diseases, such as hypoxia and ischemia, in which I_NaL increase and Na⁺-Ca²⁺ overload are major features. A hypothesis that Na⁺ initiates Ca²⁺ overload in hypoxia, ischemia and reperfusion was based on the experimental finding that Na⁺ accumulation precedes the Ca²⁺ overload [125-127]. NaCh are critically involved in this process because NaCh blockers or activators reduce or increase Ca²⁺ overload in these pathological conditions, respectively [128-130]. In ischemia, targeting I_NaL is especially encouraged because LPC dramatically increases I_NaL [84, 85]. Indeed, in recent clinical trial ranolazine has been effective to reduce the incidence of arrhythmias in patients with non ST-segment elevation acute coronary syndrome [131]. On the other hand, the therapeutic strategy of a preferential I_NaL blockade should be exercised with care. The balanced and effective therapy targeting I_NaL should take into account that I_NaL might be involved in adaptive response at different states of cardiac disease, making the question whether I_NaL is “friend” or “foe” vitally important.

Alternative targets delineated by multiple mechanisms of I_NaL modulation

Figure 8 summarizes discussed above NaCh modulatory mechanisms, which could be involved in the I_NaL increase in HF. Thus, I_NaL increase in HF can be prevented, at least in part, in different ways: i. Stabilizing membrane phospholipid composition by preventing LPC accumulation, ii. Normalizing β-subunits membrane content, iii. Stabilizing subsarcolemmal cytoskeleton, iv. Normalizing Ca²⁺ homeostasis.

Figure (8).

Simplified diagram of the modulatory mechanisms that lead to the I_NaL increase in HF. These mechanisms may serve as a road map to develop new strategies for HF treatment (see text for detail).

List of abbreviations

AP

action potential

APD

action potential duration

CaM

calmodulin

CaMKII

Ca²⁺-calmodulin activated protein kinase II

BM

burst mode of NaCh gating

DADs

delayed afterdeplarizations

DHP

dihydropiridine

EADs

early afterdepolarizations

HF

heart failure

I-V

current-voltage relationship

I_NaT

fast transient sodium current

I_NaL

late sodium current

LPC

lysophosphatilylcholine, the membrane phosholipid metabolite

LSM

late scattered mode of NaCh gating

NaCh

voltage sensitive sodium channels

NCX

Na⁺/Ca²⁺ exchanger

PKA

protein kinase A

PKC

protein kinase C

SR

sarcoplasmic reticulum

TTX

tetrodotoxin, a specific blocker of NaCh

STX

saxitoxin, a specific blocker of NaCh

VCs

left ventricular cardiomyocytes

(dV/dt)_max

maximal AP upstroke velocity