The Late Na⁺ Current - Origin and Pathophysiological Relevance

In excitable tissues, voltage-dependent Na⁺ current (I_Na) is best known for supporting autoregenerative depolarization and impulse propagation. Its transient component (I_NaT), which is large and terminated within several milliseconds by channel inactivation, fulfils this role. Nevertheless, I_Na also includes a smaller sustained component, i.e. one persisting during prolonged membrane depolarization, which contributes to repolarization course. Sustained I_Na implies slow or incomplete inactivation of a proportion of the Na⁺ channels activated during the action potential upstroke. Several mechanisms may underlie this phenomenon and contribute to arrhythmogenesis in different conditions.

Sustained Na⁺ Currents

Window Currents

Truly steady-state currents may flow through otherwise inactivating channels if membrane potential is maintained in a restricted range (“window”). These currents are commonly referred to as “window currents”. The window corresponds to the region of overlap between “activation” and “inactivation” curves, in which the probabilities of channels to be open and not inactivated are both greater than 0 at steady-state. A window is normally present in cardiac myocytes for I_CaL and, to a lesser extent for I_Na (Fig. 1), but it may be enhanced, or shifted to different potentials, by channel mutations (e.g. the Nav1.5 ΔKPQ mutation) [1].

Fig. 1

Comparison of the membrane potential “window” for I_Na and I_CaL in rat ventricular myocytes. Steady-state activation and inactivation curves for I_Na (left) and I_CaL (right). The “window”, i.e. the membrane potential range in which activation and inactivation overlap, is filled in grey. I_Na window (zoomed in the inset) is smaller and at more negative potentials than I_CaL one. (from Rocchetti et al. unpublished)

When, during repolarization, membrane potential moves into the “window”, a fraction of channels may recover from inactivation and immediately reactivate. The resulting depolarization supports autoregenerative activation which, albeit concerning a small fraction of total channels, may substantially distort membrane potential course (early afterdepolarizations, EADs) and even trigger propagated activity [2, 3]. The I_Na window is narrow and more negative than the plateau phase in normal ventricular myocytes; thus, in the latter I_CaL window more likely accounts for EADs generation [4] (Fig. 1). I_Na window, somewhat more prominent in normal Purkinje myocytes [5, 6], may be widened in all cell types by Na⁺ channel mutations.

A further mechanism of I_Na enhancement during repolarization is provided by an increase in the rate of channel recovery from inactivation relative to deactivation one. This mechanism, observed in some NaV1.5 mutations, is referred to as “reactivation under non-equilibrium conditions” [7]. This is because the excess Na⁺ current can be observed only during dynamic changes of membrane potential (non-equilibrium). This is not a proper steady-state component and, notably, it can escape detection by the standard “voltage step” clamp protocols used to assess the functional phenotype of channel mutations.

Late Na⁺ Current (I_NaL)

Figure 2a shows that sustained I_Na, identified as current sensitive to blockade by tetrodotoxin (TTX), exists also at potentials positive to the I_Na window (−65 to −50 mV; shown in in Fig. 1a for the same cell type). This current is provided by a component named “late” Na⁺ current (I_NaL). I_NaL, much smaller than I_NaT in normal ventricular myocytes, may be enhanced by more than 3-fold under pathological conditions [8]. Single-channel analysis reveals that the channel gating underlying I_NaL is complex and, in addition to late “scattered” openings, it includes a “burst mode” not observed during the transient component of I_Na (I_NaT) [9, 10]. This led to hypothesize that I_NaL might be carried by channel variants other than the one prevailing in cardiac myocytes (NaV1.5), whose expression would be enhanced under pathological conditions (a contribution of NaV1.8 channels to I_NaL in mouse and rabbit cardiomyocytes has been indeed reported [11]). However, I_NaL with all its gating modes, can be detected by expression of Nav1.5 channels in cell lines (Fig. 2b), where no other Na⁺ channel isoforms are present [10, 12]. This proves that I_NaL can be carried by the same molecular entity accounting for I_NaT; according to this view, I_NaL enhancement reflects a gating abnormality. Observations crucial for the development of this interpretation came from single channel analysis of a mutation (ΔKPQ) associated with LQT3 syndrome [13]. The mutant channel had increased probability of burst openings during sustained depolarization, a behaviour interpreted as instability of the inactivated state [1, 13]. The interpretation of I_NaL enhancement as a gating abnormality of Nav1.5 channels may hold true also for acquired conditions, as proven in the case of heart failure [14–16]. Nevertheless, we cannot rule out that changes in isoform expression may contribute in some of the diverse conditions in which I_NaL enhancement occurs.

Fig. 2

TTX-sensitive current (lower panels, largely representative of I_Na) elicited by clamping the membrane with the action potential waveform (upper panel) (AP-clamp); the inset in each panel shows the dynamic I/V relation, obtained by plotting current as a function of membrane potential. TTX-sensitive current flowing during repolarization includes all sustained Na⁺ components. a recording in a rat myocyte, clamped with its own action potential (Rocchetti et al. unpublished); b recording in a cell line (HEK239) transfected with NaV1.5 (α and β subunits) and clamped with a human ventricular action potential (from ref [12], modified). I_NaT is truncated in all recordings

Because of recovery from inactivation upon repolarization takes some time, I_NaT availability depends on the interval between excitations. This is true also for I_NaL, thus conferring to this current “reverse” rate-dependency [17], which is partial at physiological rates (approx 50 % reduction from quiescence to 120 b/min) [18].

Mutations prolonging action potential duration (APD) cause enhancement of plateau Na⁺ current through one or more of the mechanisms described above. For instance, all the above (widened voltage window, inactivation instability and accelerated recovery) may be operative in ΔKPQ mutants [1], but only non-equilibrium reactivation appears to account for the APD prolonging effect observed with the Il768V mutant [7].

Conditions and Mechanisms of I_NaL Enhancement

Although the interest in I_NaL pathophysiological role was mostly triggered by identification of Na⁺ channel mutations leading to arrhythmogenic QT prolongation [1], secondary I_NaL enhancement occurs in association to a surprisingly large number of common disease states (reviewed in [19]), including cardiac hypertrophy/failure and ischemia, not necessarily related to each other in terms of primary pathogenesis. Such a pattern suggests that I_NaL enhancement may be a common response to cell stress/dysfunction. Accordingly, in cardiac myocytes, I_NaL is enhanced by reactive oxygen species (ROS) [20], whose generation is generally associated to cell distress. The question of whether ischemia can enhance I_NaL can be answered only indirectly, because membrane currents can be recorded only in isolated myocytes, which cannot be subjected to ischemia. Nevertheless, I_NaL is enhanced by myocyte exposure to hypoxia [21] or ischemic metabolites [22] and I_NaL blockade effectively prevents ischemia/reperfusion damage in the intact heart [23].

The search of a common mechanism, mediating I_NaL enhancement by cellular stress of heterogeneous etiology, has highlighted the role of Ca²⁺-calmodulin kinase (CaMKIIδ) activation [24]. This is a Ca²⁺- and ROS-activated cytosolic enzyme which may, among other targets, phosphorylates NaV1.5 channels. Converging evidence indicates that CaMKIIδ overexpression (or activation) enhances I_NaL and generates cardiac abnormalities that are reversed by I_NaL blockade [25]. Upstream components of the CaMKII activation pathway, i.e. calmodulin (CaM) and Ca²⁺ itself, have also been shown to enhance I_NaL directly, with differences between normal and failing myocytes [26]. While this does not rule out further modes of I_NaL enhancement (see ref [27]. for review), CaMKIIδ activation is particularly relevant because its relation with I_NaL may set up a vicious feed-back loop (Fig. 3), very likely to contribute to evolution of cell dysfunction and damage in response to stress. Whereas CaMKII inhibition is not available for therapeutic use yet, I_NaL blockade may break such a vicious loop [25].

Fig. 3

Network of events linking I_NaL enhancements to its pleiotropic effects. Notably, many events are linked in positive feed back loops, likely to amplify and sustain the network once it has been initiated. Experimental observations (see text) suggest that I_NaL enhancement and CaMKII activation are key elements in this process. Events in red boxes imply increased ATP consumption, those in blue boxes reduced oxygen supply

To summarize, except for the case of primary (genetic) Na⁺ channel defects, I_NaL enhancement might be viewed as a generic response to cellular stress, that is secondary in origin, but has a pivotal role in mediating functional derangements and disease progression.

I_NaL enhancement can also result from abnormalities in proteins other than the Na⁺ channel itself, but interacting with it to form macromolecular complexes [28, 29]. For instance, mutations of scaffold and adaptor proteins, as ankyrin-B [30] and caveolin-3 [31] respectively, have been recently identified in LQT3 patients. A detailed review of the factors potentially contributing to I_NaL enhancement in heart failure has been provided by Matsev et al. [27].

I_NaL Impact on Electrical Activity and Ionic Homeostasis

I_NaL directly affects electrical activity (it promotes depolarization or counters repolarization) and provides a route of Na⁺ influx. Considering the role of Na⁺ gradient in transmembrane transport, the latter may affect cellular solute homeostasis, thus generating a host of indirect consequences of physiological and pathophysiological relevance.

Electrophysiological Effects

I_NaL flows during repolarization and may directly affect its course. In normal canine ventricles I_NaL is differentially expressed across the wall (M-cells and Purkinje cells > subendocardial cells > subepicardial cells) [17, 32, 33]. Thus, it is conceivably a player in the physiological transmural repolarization gradient and in its rate-dependency in the dog (APD restitution) [34]. In normal ventricular myocytes, I_NaL inhibition by ranolazine (I_Na blocker with selectivity for I_NaL vs I_NaT) causes negligible APD changes. On the other hand, the remarkable effects of I_Kr blockade on APD, on its rate-depencency and, most importantly, on repolarization stability are all reversed by ranolazine [18, 35, 36]. These apparently contrasting findings may be reconciled by considering that ranolazine also partially blocks I_Kr [37]. Under basal conditions, this may offset the effect of I_NaL inhibition on APD; in turn, concomitant I_NaL inhibition limits the effect of I_Kr inhibition, thus preventing repolarization instability. If this interpretation is correct, we can conclude that I_NaL and I_Kr are physiologically in balance during normal repolarization; whenever this balance is altered, by either I_NaL enhancement or I_Kr blockade, repolarization stability is compromised. An extreme example of this condition is advanced heart failure, in which I_NaL enhancement and I_Kr downregulation coexist and are associated with dramatic repolarization instability [15]. Although the concept of I_Kr - I_NaL balance is valid in a broad sense, the effects of I_Kr blockade and I_NaL enhancement on action potential contour are not identical, an observation which may have its counterpart in the differences of clinical presentation between the LQT2 (I_Kr deficiency) and LQT3 (I_NaL enhancement) syndromes [38].

The direct contribution of I_NaL to repolarization course provides a first powerful mechanism linking arrhythmogenesis to I_NaL enhancement. Nevertheless, the latter may also facilitate arrhythmias through Ca²⁺ handling abnormalities (see below) and it is difficult to establish which mechanism prevails in a specific condition. The mutual interplay between Ca²⁺ handling and repolarization course [39] may actually make this distinction pointless.

A role of I_NaL in arrhythmogenesis is often inferred from the antiarrhythmic effect of its selective blocker ranolazine. While this may be considered legitimate in many cases, there are exceptions due to specificities of drug action. The best example is ranolazine efficacy on atrial arrhythmias, to which mechanisms other than I_NaL inhibition may also contribute [40].

Effects on Ionic Homeostasis

I_Na represents the main source of Na⁺ entry during the cardiac cycle. Abeit I_NaL amplitude is normally 1/1000 of that of I_NaT [10], I_NaL persists throughout repolarization; as a result, I_NaT and I_NaL may similarly contribute to Na⁺ influx during a cardiac cycle [41]. Nevertheless, I_NaL inhibition (by TTX or ranolazine) marginally affects Ca²⁺ cycling [42] and contractility [43] in normal hearts, thus suggesting that the attending changes in Na⁺ influx are effectively buffered by matching changes in Na⁺ extrusion. However, cellular homeostasis can be compromised by the marked increase in Na⁺ influx resulting from pathological I_NaL enhancement.

Na⁺ is normally extruded from the cell by the ATP powered Na⁺/K⁺ pump. Therefore excess Na⁺ influx, even when successfully buffered, may increase ATP consumption. Furthermore, if influx exceeds the maximal extrusion rate, Na⁺ accumulates in the cytosol, thus partially dissipating its transmembrane gradient. Because the latter energizes many secondary membrane transport mechanisms, most importantly the Na⁺/Ca²⁺ exchanger (NCX) and the Na⁺/H⁺ exchanger (NHE), a pivotal consequence of I_NaL enhancement is perturbed homeostasis of intracellular Ca²⁺ and H⁺ (Fig. 3).

NCX is the main mechanism of Ca²⁺ extrusion from the cell. Increased cytosolic Na⁺ moves its electrochemical equilibrium potential in the negative direction, thus reducing the driving force for its forward operation during diastole and possibly reversing the direction of transport during systole (i.e. Ca²⁺ entry through NCX). The resulting increase in intracellular Ca²⁺ may re-establish NCX driving force, but the system equilibrium is now moved to higher cytosolic Ca²⁺ levels. Under conditions of I_NaL enhancement (e.g heart failure) NCX expression may be upregulated [44] and, provided that a driving force still exists, this may increase Ca²⁺ transport rate. However, the effect of this change in sustaining Ca²⁺ extrusion can only be partial, because it vanishes as NCX electrochemical equilibrium is approached. Accordingly, unless maximal Na⁺/K⁺ pump transport rate is also incremented, an increase in total cellular Ca²⁺ content is a necessary consequence of I_NaL enhancement. A further aspect of interest is the distribution of such an increment between subcellular compartments. Stimulation of Ca²⁺ uptake by the sarcoplasmic reticulum (SR) (by SERCA2) is a central component of physiological stimuli meant to increase cell Ca²⁺ content (e.g. by β-adrenergic activation). This ensures that most of the gain concerns SR luminal Ca²⁺, which results in larger amplitude of Ca²⁺ transient (larger developed force) and lower diastolic Ca²⁺ (accelerated relaxation). This is not the case for I_NaL enhancement, in which the Ca²⁺ increment concerns primarily the cytosol, which may only secondarily increase Ca²⁺ in the SR. Although the latter may increase maximal developed force, this is expectedly associated with higher diastolic Ca²⁺. The consequences of cytosolic Ca²⁺ being persistently elevated are multiple and of pathophysiological relevance.

Opening of SR Ca²⁺ release channels (RyRs) is directly triggered by cytosolic Ca²⁺, a process facilitated by high Ca²⁺ in the SR lumen [45]. Increased cytosolic Ca²⁺ also activates CaMKII-dependent RyRs phosphorylation, a further mechanism of RyRs facilitation (see below, Fig. 3). It is therefore unsurprising that I_NaL enhancement may lead to facilitation of diastolic “Ca²⁺ waves” and the resulting electrical disturbances (delayed afterdepolarizations, DADs) [46]. This represents an important arrhythmogenic mechanism, probably the one prevailing under conditions of altered Ca²⁺ handling (e.g. heart failure).

With persistently elevated cytosolic Ca²⁺, diastolic function may be hampered by delayed and incomplete sarcomere relaxation. A contribution of I_NaL to diastolic dysfunction has been demonstrated in various experimental models of heart failure [25, 42] and ranolazine improved diastolic relaxation in human ischemic heart disease [47]. In addition to its direct hemodynamic impact, diastolic dysfunction may limit coronary flow by extrinsic compression of intramural vessels. Significance of this effect is indirectly demonstrated by the ability of ranolazine to improve myocardial perfusion in the setting of coronary artery disease (likely I_NaL enhancement) [48]. Indeed, being ranolazine devoid of significant direct vasodilator effects, the perfusion improvement is likely to result from accelerated diastolic relaxation, a well known factor in modulation of coronary flow [49].

Abnormally elevated cytosolic Ca²⁺ may also activate a number of signalling pathways involved in modulation of cell function and, in the long run, structure and fate. CaMKIIδ activation, is at the same time, a cause (see above) and a consequence [50] of I_NaL enhancement. Phosphorylation of SR Ca²⁺ release channels (RyRs) by this kinase increases their open probability [51], ultimately leading to destabilization of the Ca²⁺ store. This accounts for the facilitation of spontaneous Ca²⁺ release events and delayed afterpotentials (DADs, their arrhythmogenic electrical consequence) induced by either I_NaL enhancement or CaMKIIδ overexpression [52] and suppressed in both cases by I_NaL blockade [25]. Calcineurin, a cytosolic Ca²⁺-activated phosphatase, regulates translocation to the nucleus of NFAT, a transcriptional regulator centrally involved in hypertrophic remodelling [53]. While there is still no direct evidence for the involvement of this specific pathway in I_NaL-induced damage, ranolazine has been shown to affect downstream gene transcription and histomorphometric parameters in heart failure induced by coronary microembolization [54].

A further consequence of pathological I_NaL enhancement is a derangement in cell energy balance. The main ATP-consuming mechanism involved in the control of cellular homeostasis (the Na⁺/K⁺ and SERCA pumps) are conceivably short-circuited by enhanced sarcolemmal Na⁺ influx and increased Ca²⁺ leak from the SR (by RyRs facilitation) (Fig. 3). Therefore, I_NaL enhancement is expected to increase ATP consumption by mechanisms not directly involved in force generation, which would translate into decreased mechanical efficiency. This view is supported by the observation that I_NaL blockade prevents the drop in myocardial ATP, but not the positive inotropic effect, of ouabain [55]. In addition to improved coronary perfusion, increased myocardial efficiency might partly account for the ability of I_NaL blockade to improve exercise capacity of ischemic patients without altering systolic mechanical work [56].

It has been reported that increased cytosolic Na⁺ may also jeopardize mitochondrial function (Fig. 4). Concomitant cytosolic Na⁺ loading (to 15 mM) impaired Ca²⁺-triggered mitochondrial energetic adaptation in patch-clamped myocytes, thereby causing an abnormal drop in NADH (i.e metabolic energy) upon catecholamine challenge [57]. On the other hand, ranolazine pre-treatment failed to modify NADH course during global ischemia/reperfusion in isolated hearts [43]. Notably, in the same preparation, ranolazine opposed ischemia-induced rise in cytosolic and mitochondrial Ca²⁺; this was associated, as expected, with diminished ROS generation and delayed opening of mitochondrial permeability transition pore (mPTP) [43]. A plausible network of mechanisms in accord with these observations is illustrated in Fig. 4. Altogether, this preliminary evidence suggests that, during ischemia/reperfusion, I_NaL inhibition may be more effective in preserving integrity of mitochondria than in improving their function. Although ranolazine effects on energy metabolism can be accounted for by I_NaL inhibition, the drug has been also reported to inhibit fatty acid oxidation [58, 59], a further action potentially protecting mitochondria during metabolic stress. However, this action was observed with drug dosage (60–200 mg/Kg/day) far from that required for the anti-ischemic effect in humans (10–15 mg/Kg/day).

Fig. 4

A plausible sequence of events coupling I_NaL enhancement to mitochondrial damage. Functions primarily depressed upon I_NaL enhancement are in purple, those upregulated to compensate in yellow. Elevated cytosolic Ca²⁺ (Ca_cyt) loads the mitochondria through the Ca²⁺ uniporter (MCU) and stimulates NADH production by the tricarboxilic acids (TCA) cycle. Excess mitochondrial Ca²⁺ is partially removed by mitochondrial NCX (mNCX), driven by high cytosolic Na⁺ [57]. This chain of events (in the dotted box) results in positive charge influx, which tends to dissipate the electrical gradient (Ψm) that drives ATP synthesis by the ATP-synthase (F1F0) [64]. The resulting drop in the ATP/ADP ratio is compensated by increasing the electron transport rate (by ETC), fuelled by NADH [57]. In this scheme NADH and ATP levels may be preserved up to a limit [57], but at the cost of increased substrate consumption and ROS production by ETC [43]. Concomitantly elevated mitochondrial Ca²⁺ and ROS facilitate opening of mPTP [43], eventually resulting in loss of membrane selectivity, swelling and disruption of the outer mitochondrial membrane. Release of cyrochrome C and other components of the outer membrane space triggers cell apoptosis [64]

Under normal conditions, cellular H⁺ homeostasis may be less sensitive than Ca²⁺ homeostasis to I_NaL enhancement, because mechanisms other than NHE contribute to H⁺ extrusion. During abrupt reperfusion following acute coronary occlusion, massive H⁺ extrusion through NHE contributes to intracellular Na⁺ loading, which explains why direct NHE inhibition paradoxically improves Ca²⁺ handling and limits myocardial injury [60]. Nevertheless, the situation might be different in the presence of chronic partial ischemia, when reduced H⁺ export by NHE might lead to persistent intracellular acidosis, potentially contributing to impair force development and relaxation. Although awaiting experimental confirmation, this hypothesis is supported by the observation that the increment in intracellular Na⁺ caused by Na⁺/K⁺ pump blockade does produce intracellular acidosis, which can be prevented by concomitant I_NaL blockade [55].

Functional Interaction Between I_NaL and K⁺ Currents

Several observations suggest a complex interplay, with partly unexplained mechanisms, between I_NaL and K⁺ currents during repolarization. Such an interplay may have consequences on electrophysiology and ionic homeostasis.

The impact on repolarization of blocking endogenous I_NaL in the presence of K⁺ current inhibition (I_Kr, I_to or I_K1) [36] may look disproportionately large to the small magnitude of the inward current removed by I_NaL block. Although ranolazine, often used as the I_NaL blocker, shares the channel binding domain on HERG channels (carrying I_Kr) with E-4031, it cannot competitively displace E-4031 [36]. Moreover, ranolazine effects are largely mimicked by TTX, which does not bind to HERG channels. Therefore, this disproportion cannot be attributed to ancillary ranolazine properties and remains largely unexplained.

A further relevant aspect of I_NaL blockade is its ability to interfere with the intrinsic reverse rate-dependency of APD and of its amplification by K⁺ channel blockers [18]. Because I_NaL decreases at faster rates (see above), I_NaL block shortens APD more at slower ones, thus dampening the intrinsic reverse rate-dependency of repolarization [18]. Rate-dependency of I_NaL may also account for the ability of I_NaL block to flatten “APD restitution” curves, an action strongly correlated with antifibrillatory effect [34].

Recent work on olfactory neurons highlights a structurally organized relationship between Na⁺ channels and the Na⁺-activated K⁺ current (I_KNa) [61], whereby the latter is specifically activated by Na⁺ influx through I_NaL. I_KNa activation may oppose I_NaL-induced membrane depolarization but, by doing this, it would increases the driving force for Na⁺ influx. Thus, coupling to I_KNa might partially dissociate the effects of I_NaL enhancement on membrane potential from those on intracellular ionic homeostasis. I_KNa is expressed in (guinea-pig) ventricular myocytes [62] and contributes to regulate APD [63]. However, its structural association with cardiac I_Na channels and functional interaction with I_NaL enhancement has not been investigated thus far.

Conclusions

Although of small magnitude, endogenous I_NaL contributes in setting repolarization course of normal myocytes, being in a delicate balance with K⁺ currents. Disruption of this balance may strongly affect repolarization and its stability. Furthermore, I_NaL enhancement, occurring in many pathological conditions as a general response to cell stress, can profoundly alter intracellular ionic homeostasis with consequences on contractile function, electrical stability and cell fate. Accordingly, I_NaL represents a therapeutic target with an expectedly pleiotropic effect, which is being gradually unveiled by experimental and clinical studies.