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1 Introduction

Heart disease, including heart failure (HF), myocardial infarction (MI), and their complications, is a global problem accounting for more than 12% of all deaths worldwide in 2011 according to the World Health Organization (2011). In the United States, 5.8 million people carry a diagnosis of heart failure; 1.1 million are hospitalized with HF each year as a primary diagnosis, and 3.39 million patients visit an outpatient clinic annually because of HF (Roger et al. 2011). The CDC estimates the US cost of HF in 2010 to be $39.2 billion (2011). This is an enormous financial expenditure as well as disease burden. There are also one million myocardial infarctions annually in the United States. Fifty percent of patients with MI will die of arrhythmia before hospitalization. Another 5% develop cardiogenic shock, and half of these patients die as well (Roger et al. 2011). Thus, the severity and the prevalence of heart disease in the world are astounding. In this chapter, we will briefly review the pathogenesis of HF and then discuss how new insights into the role of NCX in excitation-contraction (EC) coupling may offer opportunities to improve the treatment of this debilitating disease.

2 Pathogenesis of Heart Failure

The pathogenesis of HF has been an intense area of investigation. Although several lines of evidence suggest that NCX activity is increased in HF and contributes to contractile dysfunction by depleting sarcoplasmic reticulum (SR) Ca2+ content (Studer et al. 1994; Flesch et al. 1996; Hobai and O’Rourke 2000; Hasenfuss and Pieske 2002; Armoundas et al. 2007), recent clinical advances have ignored the exchanger and instead target abnormal activation of neuroendocrine signals. Neuroendocrine activation has multiple deleterious effects but with respect to EC coupling, it is thought to lead to hyperphosphorylation of ryanodine receptors (RyRs) by kinases (PKA and/or CaMKII), leading to SR Ca2+ leak (Marks 2000). Neuroendocrine activation also promotes beta-adrenergic receptor downregulation and associated abnormal G protein signaling, which likewise blunts the response of LCCs and SR Ca2+ loading to adrenergic signals (Koch et al. 2000). Other factors contribute to contractile dysfunction: these include defective SR Ca2+-ATPase activity, leading to reduced SR Ca2+ content (Schmidt et al. 1998); myofilament dysfunction, which decreases the contractile response to released Ca2+ (Hajjar and Gwathmey 1990); mitochondrial dysfunction, which leads to energy starvation; and fibrosis, which replaces myocytes with noncontracting cells (Ingwall and Weiss 2004).

3 Manipulating Contractility in Heart Failure

Although targeting the neuroendocrine system through the use of beta blockers, angiotensin-converting enzyme inhibitors, and aldosterone antagonists has been a relatively effective strategy to manage HF (Fonarow et al. 2011), patients still complain of fatigue, shortness of breath, and limited exercise tolerance. Ultimately, their disease progresses and hospitalizations for decompensation become more frequent as resting blood flow to vital organs decreases. Thus, another approach is necessary. One such approach is to employ inotropic agents to directly stimulate contractile function. These agents most commonly operate by further stimulating beta-adrenergic receptors, which in turn trigger a signaling cascade that results in (1) increased Ca2+ influx via Ca2+ current (ICa), (2) increased SR Ca2+ uptake rate (via phospholamban (PLB) phosphorylation), and (3) increased myofilament Ca2+ responsiveness. However, several seminal studies have established that inotropes increase mortality and morbidity in the HF population (Felker et al. 2003) despite improved pump function. For example, the ADHERE registry of >10,000 patients showed significantly higher in-hospital mortality (adjusted by propensity score) for HF patients treated with the beta agonist dobutamine or the phosphodiesterase inhibitor milrinone instead of vasodilators (Abraham et al. 2005). The ESCAPE trial of severe HF patients undergoing evaluation for heart transplantation found that those who were “electively” treated with inotropes had a 1.8-fold increase in 6-month mortality (Elkayam et al. 2007). Thus inotropes, while sometimes unavoidable in the short run, are dangerous in the long run. The problem appears to be the very thing that improves contractility: increased cellular Ca2+ load leading to SR Ca2+ overload, which has a variety of deleterious consequences including arrhythmia and cell death.

4 A Modern View of Excitation-Contraction Coupling in Health and Disease

Recent developments in understanding of the role of NCX in EC coupling may help reveal new and safer strategies to improve contractility than the current generation of inotropes. We have long known that a Ca2+-induced Ca2+ release (CICR) mechanism controls EC coupling in cardiac cells (Fabiato 1983). Ca2+ entering through sarcolemmal L-type Ca2+ channels (LCCs) triggers release of Ca2+ by RyRs on the SR surface (London and Krueger 1986). This reaction occurs throughout the ventricular cell within functional units known as couplons (Stern et al. 1997; Franzini-Armstrong et al. 1999). These units, which are located primarily along transverse (t) tubules, permit sarcolemmal LCCs to admit Ca2+ into a restricted junctional region (the diadic cleft), leading to a significant rise in Ca2+ concentration. This Ca2+ gates a cluster of RyRs on the apposing membrane of the junctional SR, allowing Ca2+ release from the SR to generate a Ca2+ spark (Cheng et al. 1993). The spatial separation between couplons is sufficient to permit their local control (Stern 1992), which explains the voltage dependence of Ca2+ transients. However, we now know that action potentials in healthy cells trigger each couplon simultaneously in a coordinated and synchronous manner (Inoue and Bridge 2003). This synchronous activity appears to be critical for optimum contractility.

Failing cardiac muscle is characterized by the loss of synchronized Ca2+ release upon depolarization, as exemplified by postinfarct remodeling in the rabbit (Litwin et al. 2000). We have found similar loss of synchronization of Ca2+ release in rabbit cells exposed to metabolic inhibitors (Fig. 30.1), an experimental condition that recapitulates the metabolic stress of HF (Chantawansri et al. 2008). The loss of synchronization can in large part be explained by changes in the single-channel characteristics of LCCs. For example, the Ca2+ spark probability and distribution of spark latencies are predicted by LCC latency, open time, and opening probability (Po). Primary changes in RyR behavior (Meissner 1994) and cellular structure (Gomez et al. 2001) may also contribute to loss of synchronization.

Fig. 30.1
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Effect of metabolic inhibition on LCCs and triggered Ca2+ sparks in isolated adult rabbit ventricular myocytes. (a) Frequency distribution of single L-type Ca2+ channel latency under control conditions (red), and after 4 (green) and 6.5 (blue) min of oxidative and glycolytic metabolic inhibition (MI) with FCCP (50 nM) and 2-deoxyglucose (10 mM). Normalized frequency distribution is shown in Panel b. Note the increase in the proportion of delayed latencies during MI. (c) Shows high-speed line-scan images of Ca2+ sparks in response to depolarization by action potentials (shown above each image) under control conditions and during MI. Spark latency increases during MI while spark probability declines, in parallel with the increased latency and reduced open probability of LCCs as shown in (a) and in the inset of original single Ca2+ channel records. In this example, spark probability and latency returned to normal as the action potential duration became very short, the result of increased Ca2+ influx caused by rapid early repolarization. The ability to trigger Ca2+ release with a short action potential indicates that RyRs can still respond to Ca2+ even during advanced MI and that changes in Ca2+ channel behavior are the primary reason for reduced spark probability and increased latency (From Chantawansri et al. 2008 with permission)

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5 Excitation-Contraction Coupling in NCX KO Mice

We wondered whether NCX might alter EC coupling independent of changes in SR Ca2+ stores and Ca2+ channel activity. To explore this possibility, we took advantage of our ventricular-specific NCX knockout mice. These mice live into adulthood with normal cardiac function. Isolated cells from these mice exhibit normal resting Ca2+, preserved SR Ca2+ stores, and normal Ca2+ transients in response to electrical stimulation (Henderson et al. 2004). Because NCX is absent and no other Ca2+ efflux mechanism increases to compensate, Ca2+ removal in response to caffeine-induced SR Ca2+ release is dramatically reduced. A major adaptation in this model appears to be a reduction in Ca2+ influx through LCCs and an associated increase in EC coupling gain (Pott et al. 2005). The reduced Ca2+ current (ICa) is caused by an increase in subsarcolemmal/diadic cleft Ca2+ concentration and the resulting Ca2+-dependent inactivation (Pott et al. 2007a). Action potential shortening caused by upregulation of the transient outward current (ITO) also limits Ca2+ entry during depolarization (Pott et al. 2007b). Resting Ca2+ sparks, the elementary events of EC coupling that reflect CICR activity at the single-couplon level, are reduced in frequency compared to wild-type cells. However, the sparks that do occur are larger and last longer (Neco et al. 2010). The frequency reduction is consistent with reduced diastolic triggering of sparks by the smaller KO ICa, and the difference in spark size is caused by the lack of NCX-mediated Ca2+ removal from the diadic cleft in KO cells. Spark activity and size equalize when cells from WT and KO mice are permeabilized to eliminate the influence of NCX, ICa, and differences in cleft Ca2+ (Neco et al. 2010). This indicates that RyR function is not responsible for differences in spark frequency and directly implicates ICa and NCX as the responsible elements.

6 Reverse NCX and SR Ca Release Triggering

How then does NCX affect cleft Ca2+ and microscopic EC coupling during depolarization? In the cardiac-specific NCX knockout (KO) mouse, effective EC coupling is dependent upon elevated diadic cleft Ca2+ throughout the cardiac cycle. This is made clear by experiments buffering Ca2+ in the cytoplasm using EGTA. Under strong Ca2+ buffering conditions, KO mice exhibit reduced coupling efficiency (exemplified by decreased spark number and increased spark latency), whereas wild-type (WT) mice display normal coupling (Fig. 30.2, from Neco et al. 2010). Keep in mind that under these highly buffered conditions, we expect ICa to be as large in the KO as it is in the WT (Pott et al. 2007b). The best explanation for preserved EC coupling in buffered WT cells is that NCX helps maintain coupling during depolarization. We have hypothesized that reverse NCX primes the diadic cleft with a subthreshold amount of Ca2+ during the initial upstroke of the action potential in response to Na+ entry via INa into the subsarcolemmal space. Only a small amount of additional Ca2+ brought in by LCCs is needed to trigger release in all couplons. A similar argument was proposed by LeBlanc and Hume in 1990 when they showed that blocking INa reduced Ca2+ release (LeBlanc and Hume 1990). However, these authors argued that reverse NCX was a direct trigger. Although subsequent reports from several other groups supported LeBlanc and Hume’s findings (Haworth and Goknur 1991; Nuss and Houser 1992; Kohmoto et al. 1994; Wasserstrom and Vites 1996; Lines et al. 2006), others refuted NCX ability to trigger SR Ca2+ release in any fashion that was remotely close to what could be triggered by ICa (Bers et al. 1990; Sham et al. 1992; Lipp and Niggli 1994; Lopez-Lopez et al. 1995; Sipido et al. 1995, 1997). Furthermore, many of the experiments supportive of Leblanc and Hume were criticized on technical grounds: poor voltage control, inadvertent activation or inactivation of Ca2+ channels by voltage protocols, instability in SR Ca2+ content, incomplete blockade of ICa by voltage-dependent blockers, and nonphysiologic intracellular Na+ concentrations.

Fig. 30.2
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Buffering Ca2+ in the diadic cleft reduces spark probability in NCX knockouts, but not in wild type. (a) Representative action potentials stimulated by current commands, and (b) corresponding high-speed (0.24 ms/line) line-scan images recorded simultaneously in representative WT and NCX KO myocytes. Cells were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer Ca2+ in the diadic cleft. Action potentials and images are also shown on a higher-resolution temporal scale (scale bar, 10 ms). Dashed line indicates the time when the earliest Ca spark was activated. Arrowheads mark the positions where couplons failed to activate. Fluorescence intensities are reported in self-ratioed ∆F/F magnitude as indicated in the adjoining palette. (c) Ca2+ spark latency histograms (15-ms bins) constructed from line-scan images recorded in WT (left, n  =  8 cells from four mice) and NCX KO (right, n  =  8 cells from four mice) myocytes. Note the increased spark latency in the KO compared to WT, a consequence of buffering the diadic cleft with EGTA. These data show that KO mice require elevated cleft Ca for efficient EC coupling, whereas WT mice are able to prime the diadic cleft with Ca2+ via reverse NCX (From Neco et al. 2010, with permission).

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To address these criticisms, we once again took advantage of the NCX KO mouse and also carefully constructed voltage clamp protocols and waveforms in the shape of an action potential so as to minimize voltage errors and inactivation of ICa that might confound interpretation. In order to trigger Ca2+ release in the absence of INa, the action potential clamp was preceded by a linear ramp depolarization from −70 to −40 mV over a period of 1.3 s. This prepulse strategy was designed to inactivate INa without first generating the large Na+ influx that typifies square-wave prepulses. It also prevented unwanted activation of LCCs by voltage errors produced by saturating Na+ currents (INa) activated during the prepulse. This was verified in control experiments. Thus, we were able to expeditiously eliminate INa without the use of tetrodotoxin (TTX) and without introducing voltage errors or unplanned changes in Ca2+ channel activity. Using this protocol, we found that eliminating INa selectively decreases (but does not eliminate) Ca2+ release in WT but has no effect in NCX KO (Fig. 30.3). The absence of an effect of inactivating INa in NCX KO confirms that reverse NCX in response to rapid influx of Na+ via INa makes an important contribution to the triggering process. To confirm this finding using a different approach, we applied the Na+ channel blocker TTX (5 μM) using a rapid solution exchange device 1 s prior to depolarization by the action potential voltage clamp. TTX rapidly and reversibly reduced the Ca2+ transient without reducing SR Ca2+ load, confirming the effect of Na-induced reverse NCX on CICR.

Fig. 30.3
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Reverse NCX is an essential component of the Ca2+-induced Ca2+ release mechanism of cardiac EC coupling. We used a ramp prepulse to inactivate INa immediately prior to application of an action potential voltage clamp waveform in WT (left) and NCX KO (right) myocytes. In this representative example of Ca transients recorded with Fura-2 in patch clamped myocytes, we found that in WT the prepulse markedly reduced the Ca2+ transient, whereas in KO there was no effect. This shows that reverse NCX driven by Na+ entry during the upstroke of the action potential increases coupling fidelity, i.e., the probability of triggering Ca2+ release from the SR (From Larbig et al. 2010, with permission)

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7 Importance of Na Channel Isoforms Concentrated in Transverse Tubules

Ventricular myocytes contain numerous isoforms of Na+ channels in addition to the cardiac isoform Nav 1.5. One group of isoforms (NaV 1.1, 1.2, 1.3, and 1.6), often referred to collectively as “neuronal Na+ channels,” appears to be concentrated in transverse tubules (t-tubules) (Gershome et al. 2011). Blocking these channels in rats apparently has no effect on EC coupling (Brette and Orchard 2006). However, some other groups have suggested that these channels do have an effect on contractility (Maier et al. 2002). We reasoned that since the process of EC coupling in ventricular myocytes is mainly concentrated in couplons located in t-tubules, then selective inhibition of “neuronal” Na+ channels should be sufficient to eliminate the contribution of reverse NCX to the trigger for SR Ca2+ release. We tested this hypothesis in rabbit, a species which is more dependent on Ca2+ influx from LCCs for triggering than mouse (i.e., less EC coupling gain). When we exposed rabbit cells to 100-nM TTX, a low concentration that specifically inhibits “neuronal” Na+ channels (Goldin 2001; Catterall et al. 2005), we found reduced SR Ca2+ release similar to the reduction in Ca2+ release observed during a slow ramp prepulse and similar to that described above for mouse (Torres et al. 2010).

8 Essential Role of NCX in Priming the Diadic Cleft

Our results suggest that NCX plays an essential role in the process of Ca2+-induced Ca2+ release, not simply by direct triggering of RyRs (which seems unlikely based on the relative inefficiency of NCX as demonstrated by Sham et al. (1992) and Sipido et al. (1997)), but through the following sequence of events: in response to t-tubular “neuronal” Na+ channel activation upon depolarization, the rise in junctional Na+ concentration activates reverse NCX which primes the diadic cleft with Ca2+. We know that the relationship between RyR Po and activating Ca2+ is sigmoid (Copello et al. 1997). The NCX-mediated priming of cleft Ca2+ moves Ca2+ concentration along the flat part of this sigmoid curve without increasing RyR Po appreciably. However, the Ca2+ concentration reaches all the way to the inflection point for the steep portion of the sigmoid curve. We propose that this priming takes place during the 4 ms of the action potential that precedes activation of ICa. Subsequent Ca2+ entry upon activation of ICa will further raise Ca2+ in a concentration range where it is related steeply to RyR Po, so that the NCX and ICa effectively sum their activities in a nonlinear fashion (Torres et al. 2010). Without this priming effect, the entry of Ca2+ via ICa may still be sufficient to trigger but with less efficiency than when the system is first primed by NCX. Thus, it seems that NCX is necessary to increase the coupling efficiency (Polakova et al. 2008) of CICR. In NCX KO myocytes, the cleft Ca2+ is elevated throughout the cardiac cycle, so further priming by INa and NCX is not required (Larbig et al. 2010).

9 Conclusion

These findings raise the intriguing possibility of manipulating NCX as a therapeutic tool in HF, not simply to alter Ca2+ efflux and SR Ca2+ load like a cardiac glycoside (e.g., digitalis), but rather as a way to prime the diadic cleft and maximize coupling efficiency. The goal is to provide maximum inotropic support without provoking SR Ca2+ overload and the consequent arrhythmias and cellular damage. The increase in Ca2+ entry via reverse NCX required to accomplish this increase in coupling efficiency is unknown but should be minimal (Torres et al. 2010). On the other hand, we have shown evidence that ablation of NCX substantially reduces ischemia/reperfusion injury (Imahashi et al. 2005) and may also reduce triggered arrhythmias (Nagy et al. 2004). Thus, we are faced with two opposing strategies for involving NCX in the protection and improvement of cardiac function: enhancing reverse NCX to optimize CICR and blocking NCX during acute ischemia/reperfusion to prevent Ca2+ overload. Unfortunately, pharmacological agonists and antagonists of the exchanger lack the specificity for these purposes and will require further development. Hopefully, new work involving structure/function of NCX (John et al. 2011) will soon lead to a new family of pharmacological agents.

In summary, we have found that knocking out NCX in the ventricle reduces LCC activity through Ca2+-dependent inactivation, independent of SR Ca2+ load and global cytoplasmic Ca2+ levels, which are unchanged. The reduction in LCC activity also reduces the frequency of resting Ca2+ sparks. Nevertheless, the size of Ca2+ sparks is increased, supporting the concept that NCX resides within or at least very near couplons and thereby locally regulates the removal of diadic cleft Ca2+. Conversely, we have found that effective EC coupling in mouse and rabbit requires activation of TTX-sensitive Na+ channels in order to promote reverse NCX, which primes the diadic cleft with Ca2+ and increase coupling fidelity. We conclude that cardiac NCX is a key transporter responsible for normal contractility in addition to its classic function as a regulator of cellular Ca2+ by facilitating Ca2+ efflux. NCX is therefore a potentially major therapeutic target with a higher safety margin than current agents.