Triple Threat: The Na⁺/Ca²⁺ Exchanger in the Pathophysiology of Cardiac Arrhythmia, Ischemia and Heart Failure

Abstract

The Na⁺/Ca²⁺ exchanger (NCX) is the main Ca²⁺ extrusion mechanism of the cardiac myocyte and thus is crucial for maintaining Ca²⁺ homeostasis. It is involved in the regulation of several parameters of cardiac excitation contraction coupling, such as cytosolic Ca²⁺ concentration, repolarization and contractility. Increased NCX activity has been identified as a mechanism promoting heart failure, cardiac ischemia and arrhythmia. Transgenic mice as well as pharmacological interventions have been used to support the idea of using NCX inhibition as a future pharmacological strategy to treat cardiovascular disease.

1. INTRODUCTION

The great majority of cardiac disorders are associated with at least one of three main pathophysiologies: arrhythmia, ischemia and heart failure. In clinical settings these entities often occur in combination sometimes mutually reinforcing and/or depending upon one another. As a clinical example, human heart failure renders the heart more susceptible to arrhythmia [1]. Conversely, ischemia can result in heart failure and increased arrhythmia burden [2]. Both extracellular and cellular mechanisms have been identified in the causal chains leading to either heart failure, arrhythmia or ischemia and some of these have been successfully identified as therapeutic targets [3].

In this issue of Current Drug Targets, cardiac Ca²⁺ regulating proteins such as the ryanodine receptor [4], TRP channels [5] and mitochondrial Ca²⁺ handling proteins [6] are reviewed regarding their role in cardiac pathophysiology and their potential as future pharmacological targets.

Another Ca²⁺ handling protein, the Na⁺/Ca²⁺ exchanger (NCX), also operates in cardiac myocytes. In the physiological setting, NCX serves as the main Ca²⁺ extrusion mechanism of the cardiac myocyte [7]. However, increased activity of NCX, induced experimentally, can promote arrhythmia; exacerbate ischemic necrosis and cause heart failure. Since increased exchanger activity has also been observed in several models of these diseases, increased NCX activity could be interpreted as a common denominator in the mediation of these three pathophysiologies. Several groups [8–11], including ours [12–14] have been able to define experimental conditions under which genetic or pharmacological inhibition of NCX activity can indeed protect from arrhythmia and ischemia. Although the above studies have demonstrated a promising therapeutic potential of NCX inhibition, pharmacological NCX inhibition has not, to our knowledge, been tested on a chronic condition. Some information on the chronic effects of altered NCX activity stems from transgenic murine models. We have investigated adaptive mechanisms and pathophysiological aspects of chronically altered NCX activity in transgenic overexpressors of NCX and in cardiac specific NCX knockout (KO) mice (for review see [15–18]). Mice are viable with both ventricular-specific KO and 3-fold overexpression of NCX. Although homozygous cardiac specific KO of NCX eventually leads to hypertrophy and a heart failure phenotype in later development, this does not seem to be the case in heterozygotes when NCX expression is modestly reduced to about 50% [19]. However, these observations do not speak to the safety of chronic pharmacological NCX inhibition since murine cardiac physiology is drastically different than humans. Furthermore, transgenic manipulation in early development may offer unrecognized but beneficial adaptive mechanisms that may not operate during pharmacological suppression started during later development.

Thus, before pharmacological or genetic NCX antagonism can be tested as treatment for human cardiac disease, a considerable amount of data defining bioavailability, safety, and reliability has yet to be collected. Nevertheless, the animal data currently available is encouraging. The conduct of future studies may in the end make available a new therapeutic concept by which we can defend against the “triple threat” of cardiac arrhythmia, ischemia and heart failure by countering one single molecular target.

It is noteworthy that NCX has also been characterized in extracardiac tissue such as arterial smooth muscle [20] and neuronal tissue [21]. Though not directly involved in cardiac disease, arterial and neuronal NCX may play a significant role in arterial hypertension and brain ischemia. These diseases are also part of the burden of cardiovascular disease, however, the role of NCX in these pathophysiologies has been reviewed elsewhere [22, 23].

Here we will give an overview on the role of NCX in cardiac physiology and pathophysiology. We will furthermore review the current data on experimental therapeutic approaches targeting NCX to treat ischemia, arrhythmia and heart failure.

2. NCX IN CARDIAC EXCITATION CONTRACTION COUPLING

In cardiac myocytes, the contractile cycle is initiated by Ca²⁺ entry via voltage activated L-type Ca²⁺ channels (dihydropyridine receptor (DHPR)). This “trigger Ca²⁺” binds to the ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR) and thus induces further release of Ca²⁺ into the cytosol, which allows contraction of the myofilaments. During diastole, Ca²⁺ is shifted back into the sarcoplasmic reticulum by the Ca²⁺ ATPase of the sarcoplasmic reticulum (SERCA) (Fig. 1, for review, see [24]).

Fig. 1.

Role of NCX in cellular Ca²⁺ cycling during excitation-contraction coupling in cardiac myocytes. During the action potential Ca²⁺ enters the myocyte via the voltage dependent sarcolemmal L-type Ca²⁺ channel, and thus triggers further Ca²⁺ release from the sarcoplasmic reticulum via sarcoplasmic Ca²⁺ release channels (ryanodine receptors). The released Ca²⁺ diffuses towards the myofilaments and induces the contraction. During diastole Ca²⁺ is shifted back into sarcoplasmic reticulum by the Ca²⁺ ATPase of the sarcoplasmic reticulum (SERCA). The Na⁺/Ca²⁺ exchanger extrudes Ca²⁺ from the cytosol into the extracellular space.

To maintain Ca²⁺ homeostasis and to ensure diastolic relaxation of the myocyte, the same amount of Ca²⁺ that enters the cell with each contractile cycle has to be extruded. As the main cardiac Ca²⁺ removal mechanism, the Na⁺/Ca²⁺ exchanger extrudes 1 Ca²⁺ ion in exchange for 3 Na⁺ ions [25–28]. As an additional Ca²⁺ removal mechanism, the plasma membrane Ca²⁺ ATPase (PMCA) also extrudes Ca²⁺ from the cell, however in far smaller quantities than NCX [29].

The RyR and the DHPR are colocalized to the cleft at the t-tubular sarcoplasmic-sarcolemmal junctions [30]. The proximity of the RyR and the DHPR is believed to ensure a high fidelity of excitation-contraction coupling. There is also mounting evidence that a sizeable proportion of NCX is also expressed within the dyadic cleft [31–33] and that NCX could thus modulate cleft Ca²⁺ concentration which again may have important physiological implications [34, 35].

Under certain conditions, NCX transport mode can reverse to mediate influx of Ca²⁺ into the myocyte instead of extruding Ca²⁺ [36–38]. Thus, in the presence of increased intracellular Na⁺, the driving force for Ca²⁺ extrusion is reduced or even reversed, so that Na⁺ leaves the cell and is exchanged for Ca²⁺ entering the cell. Since NCX is electrogenic, membrane depolarization during early systole also favours NCX reverse mode. It has been a matter of debate whether NCX reverse mode may contribute to systolic Ca²⁺ influx and thus add to force development in cardiac myocytes. Indeed, it was demonstrated that NCX reverse mode induced by long depolarizing voltage clamps could evoke contractions [39, 40]. We have recently produced new evidence from our genetic models that this reverse NCX mechanism is a manifest contributor to trigger Ca²⁺ [41]. As will be discussed below, NCX reverse mode is believed to play a significant role in the pathophysiology of ischemia-reperfusion injury.

In 1990, Nicoll et al. [42] first sequenced and published their initial analysis of the transsarcolemmal structure of NCX. It was found that the protein consists of 9 transmembrane segments and a long cytoplasmic loop, which separates the first 5 from the following 4 α helical transmembrane segments. Currently, three isoforms of the Na⁺/Ca²⁺ exchanger have been characterized, that have about 70% amino acid identity. While NCX1 is the predominant isoform of the heart [43, 44], NCX2 and NCX3 are detected in the central nervous system and in skeletal muscle [45, 46]. An as yet not cloned form of NCX is also thought to be present in mitochondria. Interestingly, this form of NCX does not seem to be electrogenic [47].

NCX activity is regulated by a variety of mechanisms among them Ca²⁺ and Na⁺, that besides being substrates for NCX also exert separate regulatory influences (for review, see [7]). At least one study has found that Na⁺/Ca²⁺ exchange current (I_NCX) may respond to adrenergic stimulation [48] but most studies investigating this effect have been negative [49–51].

3. PRINCIPLES OF NCX INHIBITION

Experimental NCX inhibition has been an important tool in defining the role of NCX in cardiac physiology and pathophysiology. Both pharmacological and genetic inhibition of NCX have been used experimentally to evaluate the therapeutic potential of NCX suppression. The potential of a future therapy of cardiovascular disease by inhibition of NCX will depend directly on the reliability, specificity and safety of the means available to suppress NCX activity in vivo. We will therefore give a brief review on the tools that are currently available to suppress NCX activity.

Pharmacological Inhibition

Synthetic NCX inhibitors have been available since the mid-1990s and have since been used in numerous studies investigating the physiology and pathophysiology of Na⁺/Ca²⁺ exchange and Ca²⁺ cycling. A common reservation about the use of pharmacologic NCX inhibition vs. genetic ablation is the potential lack of specificity. Indeed, KB-R 7943, the first synthetic NCX inhibitor widely used experimentally shows interactions with several extracardiac [52, 53] and cardiac ion channels and functional proteins. Among the latter are L-type-Ca²⁺, K⁺ and Na⁺ channels [54], the RyR [55] and mitochondrial uniporters [56]. SEA0400, a synthetic inhibitor which became available in 2001 [57] appears to offer a higher specificity [54] though there is still evidence that it may also modify cardiac function via a mechanism independent of NCX [58].

Further synthetics with NCX inhibitory potential are under development [59, 60].

Genetic Knockout (KO) of NCX

Global KO of NCX is embryonically lethal in mice [61, 62], while mice with inducible cardiac specific knockout survive into adulthood. Mice with modest (=heterozygous KO) [19, 63] and complete (=homozygous KO) [64] genetic ablation of NCX have been investigated. Cardiac myocytes from NCX KO mice with complete ablation of NCX do not exhibit significant alterations of resting or systolic Ca²⁺ concentration, or sarcoplasmic reticular (SR) Ca²⁺ load when compared to WT littermates. NCX inward current is absent and the decrease of the Ca²⁺ transient is drastically slowed during caffeine exposure, indicating that no alternative Ca²⁺ extrusion mechanism is upregulated to compensate for the absence of NCX. Instead, peak L-type Ca²⁺ current (I_Ca) is decreased [32] and AP duration is reduced [65] resulting in a further reduction of net Ca²⁺ entry into the myocyte to 20% [35]. Thus, in the absence of NCX, transsarcolemmal Ca²⁺ traffic is drastically reduced. The plasma membrane Ca²⁺ ATPase (PMCA) has been estimated to provide 10–25% of myocyte Ca²⁺ efflux from the cytosol into the extracellular space depending on species variation [29, 66, 67]. Thus, a Ca²⁺ influx reduced to 20% as observed in NCX KO myocytes is well within the range of capacity of the PMCA (Fig. 2a). Despite these alterations, there is no difference in the cellular Ca²⁺ transients implying an increase in the gain of excitation-contraction coupling in KO cells. The converse has been observed in NCX overexpressor mice [68] (Fig. 2b). Though there are considerable differences between murine cardiac physiology and that of higher mammals, these studies provide information on the cellular adaptations that may be active during future therapeutic NCX manipulation.

Fig. 2.

Concept of cellular mechanisms of adaptation to reduced and increased NCX activity as modelled from studies in NCX knockout (a, [32, 35]) and overexpressor mice (b, [68]).

Gene-transfection methods to acutely inhibit NCX in isolated cardiac myocytes are currently available [69] and may be investigated as therapeutic tools in future studies.

4. NCX AND CARDIAC ARRHYTHMIA

NCX activity and Action Potential (AP) Kinetics

To understand the involvement of NCX in the generation of cardiac arrhythmia, it is necessary to define the role that NCX plays in the repolarization of the cardiac AP. During systole, when cytosolic Ca²⁺ concentration is high, NCX extrudes Ca²⁺ from the cytosol into the extracellular space. Due to the electrogenic nature of this process, NCX generates an inwardly directed membrane current (I_NCX) during SR Ca²⁺ release. It has been assumed that via this mechanism NCX would contribute to maintain the plateau of the cardiac action potential. Indeed, computer modelling revealed that increased I_NCX activity significantly delays repolarization of the cardiac AP [70]. Sham and Spencer [71] also demonstrated in guinea pig cardiomyocytes that an inhibition of NCX caused an abbreviation of the AP while a prolongation was observed when NCX activity was maximal. Many groups including ours have used transgenic models with cardiac specific alteration of NCX expression to further define the role of NCX in AP repolarization. A prolonged AP plateau was observed in NCX overexpressor mice, and – conversely – a shortened AP in NCX knockout mice [35] (Fig. 3), although in knockout mice, additional mechanisms contribute to shortening AP duration [65].

Fig. 3.

NCX activity regulates AP repolarization in adult cardiac myocytes. Middle column: typical “pyramid”-like mouse AP recorded in a myocyte isolated from a WT animal. Left column: A lack of the plateau phase due to a faster repolarization of the AP is observed in myocytes isolated from NCX KO mice. Right column: In myocytes isolated from mice that homozygously overexpress NCX (HOM), a slowed repolarization with a pronounced plateau phase can be observed (modified from [35, 68]).

In addition to a direct influence of NCX inward current on AP repolarization, an alternative mechanism may explain NCX-mediated repolarization changes: we have proposed a functional interplay between NCX and L-type Ca²⁺ channels. One of the strongest regulators of I_Ca is cytosolic Ca²⁺ itself. Thus, at high intracellular Ca²⁺ concentrations, Ca²⁺ binds to calmodulin, thereby inactivating I_Ca. At low cytosolic Ca²⁺ concentrations, when no Ca²⁺ is bound, L-type Ca²⁺ current is considered to be maximal [72, 73]. The DHPR is primarily located at sarcolemmal SR junctions in close vicinity to the RyR and NCX [30]. It has been accepted that Ca²⁺ release from the SR is the primary mechanism to influence and terminate L-type Ca²⁺ current [74, 75]. However, due to its close vicinity, NCX activity may also influence I_Ca. We have reported that blockade of NCX may increase cleft Ca²⁺ concentration [32–34]. This may have important implications for I_Ca regulation via Ca²⁺ induced inactivation: In the absence of NCX activity elevated cleft Ca²⁺ decreases I_Ca open probability and accelerates I_Ca inactivation. Conversely, when NCX activity is high, this may increase I_Ca open probability and delay I_Ca inactivation. Indeed, we could demonstrate that in NCX knockout mice, I_Ca is reduced via a Ca²⁺ dependent mechanism [32], while – conversely - I_Ca in NCX overexpressor mice is increased, a mechanism that also appears Ca²⁺ dependent [68]. Via this functional interplay with I_Ca, NCX may – indirectly - influence AP repolarization in the same direction as it does directly via reduced or increased NCX inward current as explained above. Thus, increased I_NCX would also increase net I_Ca thereby further slowing repolarization. Conversely, reduced I_NCX would also reduce I_Ca thus reducing AP duration.

NCX and Afterdepolarizations: Pathophysiological Concept

It has been hypothesized that increased NCX activity could induce afterdepolarizations of the ventricular AP that could then trigger tachyarrhythmias [76–78]. Afterdepolarizations are defined as oscillations of the electric membrane potential during (early afterdepolarization [EAD]) or after (delayed afterdepolarization [DAD]; Fig. 4) the cardiac action potential. Afterdepolarizations can induce reentry and are commonly regarded as a major trigger of cardiac tachyarrhythmias.

Fig. 4.

Pathophysiological concept on the role of NCX in the mediation of cardiac arrhythmia (modified from [76]).

EADs have been shown to occur in animal models with slowed AP repolarization [79]. The prolonged AP duration prolongs the period of time the myocytes spend in a depolarized state which again will broaden the “time window” for voltage dependent I_Na or I_Ca to reactivate and induce an EAD (Fig. 4). This mechanism has been evoked to explain proarrhythmia in animal models of hypertrophy and heart failure where a reduction of K⁺ carried outward currents and an increased AP duration have been observed (for review see [80]). Likewise, specific mutations of K⁺ channel subunits resulting in loss of function, or Na⁺ channel gain-of-function mutations [81], can both slow AP repolarization and cause EADs resulting in ventricular tachyarrhythmia [81–83]. Since NCX also slows repolarization it may be a further candidate for the promotion of EADs.

NCX is also essential for the development of DADs. The initiating event of a DAD consists of a spontaneous release of Ca²⁺ from the SR, as occurs in the setting of SR Ca²⁺ overload. This sudden increase of cytosolic Ca²⁺ then leads to an activation of NCX inward current which depolarizes the sarcolemmal membrane. If this reaches the threshold for Na⁺ channel activation, a new and premature AP is triggered [76, 84] (Fig. 4).

Initial studies in isolated cardiac myocytes from several different species have indeed demonstrated that DADs can be triggered via Ca²⁺ overload of the SR [85–88]. An increased occurrence of afterdepolarizations of the cardiac AP has also been observed in animal models of hypertrophy and heart failure [89–94], where NCX is known to be overexpressed [95, 96].

Studies testing the proarrhythmic potential of increased NCX expression have mostly been limited to hypertrophied and failing myocardium. Therefore, their informations on the role of NCX in the generation of cardiac arrhythmia are somewhat limited because a multitude of structural, expressional and functional changes other than the upregulation of NCX have been observed in human and animal failing and hypertrophic myocardium (for review, see [97]) and any one or a combination of these could be potential promoters of cardiac arrhythmia. The NCX overexpressing mouse on the other hand has increased NCX activity in the absence of cellular hypertrophy or reduced K⁺ channel activity. Before 14 weeks, these animals have normal whole heart and cellular function and structure and preserved K⁺ outward currents. We have found that these animals are prone to whole heart ventricular tachyarrhythmia. In isolated cardiac myocytes, APs are prololonged and there is an increased occurrence of EADs as well as DADs [14]. These observations indicate that NCX may be a promoter for arrhythmia independent of structural or ionic remodelling associated with heart failure.

Experimental Inhibition of NCX to Counter Proarrhythmia

Early studies investigating the antiarrhythmic potential of the NCX inhibitor SEA0400 yielded inconsistent results. While Nagy et al. [8] observed that the NCX inhibitor SEA0400 inhibited EADs induced by dofelitide and barium in guinea pig cardiac muscle strips, Amran et al. did not observe a protective effect of SEA0400 in either in vivo studies or isolated cardiac myocytes when monitoring aconitine-induced arrhythmia in the same species [98]. Two independent studies investigated the effects of SEA0400 on arrhythmias induced by ouabain. Tanaka et al. [9] showed an antiarrhythmic effect in guinea pig right ventricular muscle strips. In an in vivo dog model, Nagasawa and coworkers [99] observed that pharmacological inhibition of NCX protected against ouabain-induced arrhythmia but not against arrhythmia induced by acute ischemia. A recent study has specifically investigated the effect of SEA0400 on cardiac myocytes isolated from the pulmonary vein regions of guinea pig left atria [100]. Spontaneous electrical activity generated by this cell population is regarded to be the trigger for atrial fibrillation, one of the most frequent tachyarrhythmias.

We have also attempted to characterize the antiarrhythmic effect of pharmacological NCX inhibition. To produce proarrhythmic experimental conditions in which afterdepolarizations occur, we utilized a recently developed intact heart model of the rabbit heart mimicking the electrophysiological alterations of long QT 2 and 3 syndromes (LQT 2 and 3) [101–103]. Under these experimental conditions, SEA0400 significantly reduced AP duration and suppressed EADs and the Torsade-de-Pointes form of ventricular tachycardia [13].

The discrepancies evident in studies investigating the antiarrhythmic potential of acute NCX inhibition may be due to the following factors: 1. Proarrhythmic interventions of a different nature have been applied (i.e. aconitine-, vs. ouabain-, vs. dofetilide- vs. ischemia-induced arrhythmia), 2. Different species (guinea pig vs. dog vs. rabbit) have been investigated and species differences in the composition of repolarizing ion channels are well known [104]. 3. Finally, different preparations of cardiac tissue ranging from in vivo models over trabecular preparations to patch clamp experiments in isolated cardiac myocytes have been used.

5. NCX AND ISCHEMIA

Role of NCX in the Pathophysiology of Ischemia Reperfusion Injury

During cardiac ischemia, a rise in intracellular Na⁺ occurs due to a reduction of Na⁺/K⁺ ATPase activity. This increase in Na⁺ concentration is followed by a rise in intracellular Ca²⁺. This accumulation of cytosolic Ca²⁺ concentration is dependent on the rise in cytosolic Na⁺ concentration and it has therefore been suggested that the process of Ca²⁺ accumulation during ischemia is mediated via NCX working in reverse mode [105–110]. As mentioned above, depending on the transsarcolemmal Na⁺ and Ca²⁺ gradients and the membrane potential, NCX can switch into reverse mode and transport Ca²⁺ into the cell instead of extruding Ca²⁺ [7]. In reverse mode, NCX extrudes 3 Na⁺ in exchange for 1 Ca²⁺ ion entering the cell. NCX reverse mode is thus favored by the high cytosolic Na⁺ concentration characteristic for ischemic conditions. Consistent with this hypothesis, mice with heterozygous overexpression of NCX exhibit increased susceptibility to ischemia-reperfusion injury [10, 111].

However, during ischemia, there is also a rise in cytosolic H⁺ concentration that contributes to the rise in cytosolic Na⁺ concentration via activation of the Na⁺/H⁺ exchanger (NHE). This increased Na⁺ favors NCX reverse mode and cytosolic Ca²⁺ accumulation. Inhibition of NHE blocks the rise in Na⁺ and Ca²⁺ and is cardioprotective during ischemia [110, 112]. Some studies have suggested that preventing the rise in cytosolic Na⁺ concentration via NHE inhibition may be cardioprotective, independent of the effect on Ca²⁺ accumulation [113, 114]. Both cytosolic accumulation of Ca²⁺ and Na⁺ are inhibited by NHE inhibitors, and it is therefore difficult to distinguish between the relative protective effects of preventing the increase in Na⁺ vs. Ca²⁺ concentration.

The development of transgenic mice and genetic transfection techniques has provided a unique tool to further investigate this question. We have demonstrated that NCX KO mice are significantly less susceptible to ischemic injury [12]. Cellular studies have demonstrated that there is no detectible Ca²⁺ entry during experimental lowering of the transsarcolemmal Na⁺ gradient in NCX KO myocytes while WT myocytes exhibited significant cytosolic Ca²⁺ accumulation under these conditions (Fig. 5). During ischemia-reperfusion, hearts from NCX KO mice exhibited less necrosis, better post-ischemic recovery of cardiac performance, higher levels of high-energy phosphates, and improved recovery of Na⁺ homeostasis when compared to WT animals [12]. Maddaford et al. [115] have demonstrated that acute targeted genetic suppression of NCX from isolated cardiac myocytes can also protect from ischemic injury. Similarly, it was shown that introduction of specific splice variants of NCX into neonatal cardiac myocytes via transfection prevented ischemia induced cytosolic Ca²⁺ accumulation [116]. These observations add further support to the hypothesis that NCX is a direct mediator of ischemic injury. Moreover, these data indicate that inhibition of NCX seems to be protective independent of a direct inhibition of H⁺ accumulation via NHE.

Fig. 5.

Ca²⁺ entry induced by Na⁺ depletion is absent in NCX KO myocytes: Recording of cytosolic Ca²⁺ concentration in patch clamped cardiac myocytes isolated from NCX KO and WT mice. Ca²⁺ entry via NCX reverse mode was induced in WT myocytes by hyperpolarization and simultaneous rapid removal of external Na⁺. Under these conditions, no increase in cytosolic Ca²⁺ concentration was observed in NCX KO cells. (Modified from [12]).

NCX Inhibition in Models of Ischemia-Reperfusion Injury

Pharmacological studies in animal models have also been conducted to investigate the therapeutic potential of NCX inhibitors as a means to protect against ischemia-reperfusion injury.

The most frequently used agents to test the effect of NCX inhibition on ischemia reperfusion injury are KB-R7943 and more recently SEA0400. KB-R7943 has been demonstrated to reduce ischemic injury under a variety of experimental conditions ranging form cellular to in vivo models. Thus, it has been shown to inhibit ischemia-reperfusion induced cytolsolic Ca²⁺ accumulation in isolated cardiac myocytes [117, 118] and to improve post-ischemic contractility in trabecular preparations [119, 120] and whole heart models [121–123].

In a similar range of experimental settings, SEA0400 has as well been demonstrated to reduce cellular Ca²⁺ entry upon ischemia-reperfusion [124], limit myocardial infarction size [125] and improve post-ischemic contractility [124, 125] even several days after the ischemia-reperfusion event [126]. A new NCX inhibitor, NCC-135, has also been shown to inhibit Ca²⁺ overload and reduce ischemia-reperfusion injury [127]. One comparative study using a whole heart rabbit model to investigate the protective effect of SEA0400 and KB-R7943 under identical experimental conditions has observed a similar efficiency of both drugs in reducing infarct size. However in this model only SEA0400 improved post-ischemic cardiac contractility while KB-R7943 significantly reduced heart rate and cardiac output [125]. This may be a consequence of the previously reported lack of KB-R7943’s specificity.

6. NCX AND CHRONIC HEART FAILURE

NCX and Chronic Heart Failure: Pathophysiological Concept

In the failing human heart, contractile force is reduced and relaxation is retarded. These alterations are caused in part by defective intracellular Ca²⁺-handling (for review see [128]). In failing myocytes, there is an increase in diastolic and a decrease of systolic Ca²⁺ [129] resulting in a reduced Ca²⁺ transient and contractile force, especially at increased cardiac frequencies [130]. These alterations have been attributed to decreased sarcoplasmic Ca²⁺-load, which could be caused by insufficient Ca²⁺-reuptake in diastole due to decreased activity of the SERCA [131, 132] or to excessive Ca²⁺ leak through hyperphosphorylated ryanodine receptors [133]. NCX is upregulated in the failing and hypertrophic heart in both activity and expression [93, 96]. It is unknown whether increased NCX activity is a cause or a consequence of chronic heart failure. Evidence suggests that increased NCX activity may provide an adaptive mechanism which protects against diastolic Ca²⁺ overload [95]. Yet, it may also be possible that the increased NCX current in the failing heart competes with the reduced SERCA of failing myocardium for cytosolic Ca²⁺, a maladaptive mechanism contributing to sarcoplasmic reticular Ca²⁺ loss and reduced contractility.

Cardiac glycosides such as digoxin have been used for over a 100 years to manipulate this relationship between SERCA and NCX in order to restore contractility in failing myocardium. Digoxin inhibits the Na⁺/K⁺ ATPase, resulting in increased cytoplasmic Na⁺. This increased Na⁺ concentration alters the transmembrane gradient for Na⁺, which reduces Ca²⁺ efflux via NCX and allows SERCA to accumulate Ca²⁺. In heart failure, the restored SR Ca²⁺ load provided by this indirect manipulation of NCX improves contractility. As noted above, this can unfortunately result in SR Ca²⁺ overload, DADs and triggered arrhythmias, which likely explains why digoxin does not provide a mortality benefit [128]. If intracellular Na⁺ could be more carefully regulated, it might reduce the arrhythmias associated with digoxin. The approach of reducing digoxin target levels may help in this regard.

Therapeutic Approaches and Lessons from Transgenic Mice

As mentioned above, there is evidence, that in acute heart failure induced by ischemia NCX inhibition can be beneficial (for review see [134]). However, experimental testing of the therapeutic potential of NCX inhibition in chronic heart failure is difficult. In an ideal experimental setup, the development of chronic heart failure would be monitored in the absence and presence of pharmacological NCX inhibition. However, depending on the model, the development from a non-failing phenotype to overt heart failure may take up to several weeks. Alternatively, one could choose an approach in which NCX inhibitors are administered in a phenotype that already exhibits heart failure to test whether a reduction of NCX activity may cause reverse remodelling. However, to detect macro- or microstructural remodelling in chronic heart failure, one would require an observation period of at least weeks. To our knowledge there is no study that has administered pharmacological NCX inhibitors over such a long period of time. This may in part be due to the fact that there is no published information available on the bioavailability or plasma removal rates of pharmacological NCX inhibitors.

Some information on the effect of altered NCX activity on the development of heart failure has been gathered in transgenic mice. In accordance with the hypothesis that increased NCX activity can promote chronic heart failure, transgenic mice with 3-fold overexpression develop clinically overt heart failure under experimental conditions under which their WT littermates do not [135].

In mice with complete ablation of NCX using cre/lox technology, cellular excitation-contraction coupling and Ca²⁺ handling can adapt so that cellular contractility is preserved [16, 35, 64]. Nevertheless, with progressing age, these animals also exhibit overt clinical heart failure and premature death [19]. This does not necessarily contradict the hypothesis that reduction of NCX activity may protect from heart failure since a beneficial effect may only be evident with modest NCX inhibition. Accordingly, heterozygous NCX KO mice show normal cardiac function and structure with normal lifespan [19]. Thus the heterozygous model may be better suited to further investigate the potential of reduced NCX activity in the therapy of chronic heart failure.

7. FUTURE DIRECTIONS: NCX INHIBITION AS A NEW THERAPEUTIC TOOL?

A wide variety of therapeutic tools exist to treat cardiovascular disease. These consist of catheter-based, surgical and pharmacological approaches. Nevertheless, cardiovascular disease remains the leading cause of death in western industrial nations. Since the 1980s, several innovations have been made in the field of interventional therapies such as catheter based coronary revascularization and arrhythmia ablation, implantable cardioverter/defibrillators and cardiac resynchronization therapy to name only a few. Innovations have also been made in the pharmacological field. Since the advent of angiotensin converting enzyme blockers (ACE inhibitors), novel promising drugs such as ranolazine or ivabradine have emerged. The overwhelming experimental data reviewed here point towards a benefit of using NCX inhibition as a novel therapy as well. However, research on NCX inhibition – may it be pharmacological or genetical – has to be taken to the next level. Long term animal feeding studies will have to be conducted to give some information on the safety of pharmacological NCX inhibitors. This will be a precondition of eventually testing NCX inhibitors on their safety in humans.

Contemplating the broad spectrum of pathophysiologies that depend on NCX activity (arrhythmia, ischemia, heart failure, arterial hypertension) the authors imagine, that once established in terms of safety and benefit, inhibition of NCX for the treatment of cardiovascular disease may have a similar impact on the field as the advent of ACE inhibitors in the 1980s.