Regulation of Cardiac L-Type Ca²⁺ Current in Na⁺-Ca²⁺ Exchanger Knockout Mice: Functional Coupling of the Ca²⁺ Channel and the Na⁺-Ca²⁺ Exchanger

Abstract

L-type Ca²⁺ current (I_Ca) is reduced in myocytes from cardiac-specific Na⁺-Ca²⁺ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca²⁺ overload in the absence of NCX. However, Ca²⁺ channel expression is unchanged, suggesting that regulatory processes reduce I_Ca. We tested the hypothesis that an elevation in local Ca²⁺ reduces I_Ca in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I_Ca was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca²⁺ concentration on I_Ca, we used Ba²⁺ instead of Ca²⁺ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca²⁺ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba²⁺ current between WT and KO myocytes. Also, dialysis with the fast Ca²⁺ chelator BAPTA eliminated differences in both I_Ca amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca²⁺-dependent inactivation of I_Ca reduces I_Ca amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca²⁺ that results from the absence of NCX activity inactivates some L-type Ca²⁺ channels. Modulation of subsarcolemmal Ca²⁺ by the Na⁺-Ca²⁺ exchanger may be an important regulator of excitation-contraction coupling.

INTRODUCTION

The Na⁺-Ca²⁺ exchanger (NCX) is the main Ca²⁺ extrusion mechanism of the cardiac myocyte, maintaining Ca²⁺ homeostasis by removing Ca²⁺ that enters the myocyte during each cardiac cycle (1–3). Although functional excitation-contraction coupling would seem impossible without NCX, we have produced a murine cardiac-specific knockout (KO) of NCX. Strikingly, these animals survive into adulthood and exhibit only minor cardiac dysfunction (4,5). One mechanism that enables KO myocytes to maintain Ca²⁺ homeostasis in the absence of NCX is a reduction of Ca²⁺ entry via the L-type Ca²⁺ current (I_Ca) (5), which is accentuated by a shortening of the action potential (6).

The mechanism underlying the reduction in I_Ca is unknown. Because dihydropyridine receptor (DHPR) expression is unaltered in KO myocytes (7), regulatory processes must be responsible. We have demonstrated previously that the voltage dependence of I_Ca is similar in WT and KO, and I_Ca is reduced by a similar amount at all voltages in KO compared to WT (5,7). Thus, it seems unlikely that the mechanisms involved in the reduction of I_Ca in KO are primarily voltage dependent.

Intracellular Ca²⁺ itself potently inactivates I_Ca by a process known as Ca²⁺-dependent inactivation (8,9). Changes in local subsarcolemmal Ca²⁺ caused by the absence of normal Ca²⁺ extrusion via NCX could explain the reduction of I_Ca in NCX KO mice. Such a functional relationship between NCX and I_Ca has indeed been demonstrated in a noncardiac mammalian cellular expression system (10). However, the experimental investigation of this relationship in intact cardiac myocytes remains challenging: Pharmacological manipulation of NCX activity is difficult because of a lack of sufficiently specific inhibitors (11). Furthermore, when NCX is acutely blocked by removal of external Na⁺, there is an almost immediate increase in the sarcoplasmic reticular (SR) Ca²⁺ load and the Ca²⁺ transient (12). Thus, it is difficult to directly link changes in I_Ca to altered NCX activity and to establish a functional relationship between the two proteins.

Cardiac-specific NCX KO mice exhibit unaltered SR Ca²⁺ load, and their global systolic and diastolic Ca²⁺ concentrations measured with the Ca²⁺-sensitive indicator fura-2 are indistinguishable from those in wild-type (WT) littermates (5,7). Thus, these animals present a unique tool for investigating the effects of NCX on the behavior of the L-type Ca²⁺ current. An increase in subsarcolemmal Ca²⁺ might increase the degree of I_Ca inactivation and reduce current amplitude yet be undetectable by conventional fluorescent indicator techniques. We report here that Ca²⁺-dependent inactivation is indeed responsible for the reduction of I_Ca in NCX KO mice. We attribute this to elevated subsarcolemmal Ca²⁺ in the absence of NCX activity.

METHODS

Generation of transgenic mice

NCX cardiac-specific KO mice were generated using cre/lox technology as previously described (7). Animals used in this study were between 8 and 12 weeks of age, did not display any gross pathology, had good cardiac function, and exhibited no evidence of heart failure.

Isolation of ventricular myocytes from adult mouse hearts

Before (20 min) explantation of the hearts, mice were injected with 200 μl heparin (10,000 units/ml) i.p. Animals were then anesthetized with 200 μl nembutal (50 mg/ml) i.p., and hearts were quickly removed via thoracotomy. Isolation of ventricular myocytes by collagenase/protease digestion was performed as reported previously (5,7). All procedures were in accordance with the guidelines of the UCLA Office for Protection of Research Subjects. Isolated cardiomyocytes were stored for up to 6 h at room temperature in modified Tyrode solution containing (in mM) 136 NaCl, 5.4 KCl, 10 Hepes, 1.0 MgCl₂, 0.33 NaH₂PO₄, 1.0 CaCl₂, 10 glucose, pH 7.4 with NaOH. To prevent Ca²⁺ activated Cl⁻ current, 0.1 mM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate) was present when SR Ca²⁺ release was not depleted by thapsigargin and ryanodine. DIDS was prepared as a 100 mM stock solution by heating in 0.1 M KHCO_3. The above solution was also used, with modifications described below, as the standard bath for electrophysiological recordings.

Electrophysiology

Whole-cell membrane currents were recorded as described previously (5,7). The pipette solution contained (in mM) 120 CsCl, 10 TEA-Cl, 10 NaCl, 20 Hepes, 5 MgATP, 0.05 cAMP, pH 7.2 with CsOH, with modifications described below. We recorded whole-cell membrane current using an Axopatch 200 or 200B patch-clamp amplifier (Axon Instruments, Union City, CA) in voltage-clamp mode and a Digidata 1322A (Axon Instruments) data acquisition system controlled by pCLAMP 9 software (Axon Instruments). We applied series resistance compensation to all recordings. To measure differences in decay kinetics of I_Ca, single exponential curves were fitted, and the time constant (τ) was calculated.

Rapid solution exchange

Miniature solenoid valves (The Lee Co., Westbrook, CT) controlled by PCLAMP digital outputs controlled the bath solution flow through a micromanifold (ALA Scientific Instruments, Westbury, NY). This enabled precise timing of solution exchanges in relation to the voltage-clamp protocol. The solution surrounding a patched cell exchanged with a half-time of <150 ms. The procedure has been described previously (5,7).

Statistical analysis

Data are expressed as means ± SE. Student's unpaired t-test was used for direct comparisons of WT versus KO.

RESULTS

Determination of cellular phenotype

It has been a common finding that cardiac-specific gene knockout using cre/lox technology does not occur with 100% efficiency (13,14). In NCX KO mice, those cells in which gene excision occurs have complete absence of NCX, and the remaining cells have normal (WT) expression levels. Immunofluorescence and functional data indicate that 80–90% of myocytes from NCX KO mice have no detectable NCX (5).

To identify the 10–20% of myocytes isolated from KO animals that express NCX, each myocyte was tested for NCX inward current (I_NCX) before exposure to thapsigargin and ryanodine. Cells were held at a voltage of −40 mV. After six 100-ms conditioning pulses from −40 to 0 mV (at 1 Hz), cells were depolarized from −40 to 0 mV for 50 ms to induce I_Ca and SR Ca²⁺ release. In WT myocytes, a slowly inactivating inward current is detected on repolarization to −40 mV, which is generated by the forward mode of NCX to remove the elevated cytosolic Ca²⁺ (15). This “tail” NCX current is absent in myocytes with a KO phenotype (Fig. 1). Because NCX KO myocytes have the same SR Ca²⁺ content and Ca²⁺ release on depolarization as WT, the absence of inward current is indicative of the absence of NCX. As expected, only 10–20% of myocytes isolated from NCX KO mice expressed I_NCX. This result is consistent with findings from our previous studies (5,7) and the published efficiency of the cre recombinase system (13,14). Cells with detectable I_NCX were excluded from the KO group.

FIGURE 1.

Identification of cellular phenotype. (Top panel) Cells were held at −40 mV and then depolarized to 0 mV for 50 ms. (Middle panel) Depolarization induces I_Ca. In cells with a WT phenotype, this led to a second inward current on repolarization generated by NCX. This NCX “tail current” is absent in myocytes with a KO phenotype. Insets from the middle panel are magnified in the bottom panel to visualize I_NCX on repolarization.

I_Ca amplitude and kinetics in NCX KO myocytes

We recorded I_Ca in WT and KO cells. After six prepulses to 0 mV (1 Hz; 100 ms) to ensure steady-state SR Ca²⁺ load, cells were depolarized from −75 to −40 mV for 100 ms to inactivate Na⁺ current and then further depolarized to 0 mV (200 ms) to elicit I_Ca. Consistent with our previous studies (5,7), we observed a reduced amplitude of I_Ca in KO myocytes as compared to WT myocytes (KO, −6.2 ± 0.8 pA/pF, n = 9; WT, −12.3 ± 0.8 pA/pF, n = 15; p < 0.01) (Fig. 2 A, Fig. 3). We also found that the decay rate of I_Ca was accelerated in KO compared to WT (KO, τ = 17.8 ± 2.5 ms; WT, τ = 27.6 ± 1.0 ms; p < 0.01) (Fig. 2 B, Fig. 3). These changes in amplitude and decay rate are consistent with increased Ca²⁺-dependent inactivation of the Ca²⁺ current, which we hypothesize is caused by increased subsarcolemmal Ca²⁺ resulting from the elimination of NCX.

FIGURE 2.

Using Ba²⁺ as a charge carrier while depleting SR Ca²⁺ eliminates differences in I_Ca. (A) Representative traces of I_Ca (left), I_Ca after depletion of SR Ca²⁺ with Tg and Ry (middle), and I_Ba in the presence of Tg and Ry (right) for KO and WT myocytes. (B) Recordings shown in A were normalized to peak to emphasize the equivalent rates of inactivation after eliminating SR Ca²⁺ release (I_Ca in the presence of Tg and Ry, middle) and Ca²⁺ entry into the diadic cleft (I_Ba in the presence of Tg and Ry, right).

FIGURE 3.

Summary data comparing amplitude of I_Ca in KO and WT (KO, n = 9; WT, n = 15) under three conditions: 1), using Ca²⁺ as the charge carrier with intact SR, 2), using Ca²⁺ as the charge carrier with SR Ca²⁺ eliminated by Tg/Ry (KO, n = 5; WT, n = 4), and 3), using Ba²⁺ as the charge carrier (I_Ba) in the presence of Tg/Ry (KO, n = 6; WT, n = 4). Amplitude is shown on the left, and decay kinetics on the right. *p < 0.01 for WT versus KO.

Effect of SR Ca²⁺ on I_Ca in NCX KO myocytes

To assess the effect of SR Ca²⁺ on I_Ca in NCX KO cells, we recorded I_Ca as described above after exposing the cells to thapsigargin (Tg; 0.2 μM) and ryanodine (Ry; 10 μM) for 10–15 min to deplete SR Ca²⁺ stores. Tg/Ry eliminated the difference in decay kinetics between KO and WT (KO, τ = 26.5 ± 2.0 ms; WT, τ = 29.1 ± 0.9 ms; p > 0.05; Fig. 2 B, Fig. 3) but did not affect the amplitude of I_Ca in either KO or WT (KO, −6.0 ± 0.9 pA/pF, n = 5; WT, −12.0 ± 1.5 pA/pF, n = 4; p < 0.01) (Fig. 2 B, Fig. 3). Thus, although SR Ca²⁺ has an important influence on I_Ca kinetics in KO cells, this source of Ca²⁺ does not account for all differences in I_Ca between WT and KO. Interestingly, elimination of SR Ca²⁺ had a greater effect on I_Ca decay kinetics in KO myocytes than in WT. We discuss the significance of this finding in more detail below.

Effect of Ca²⁺ entry on I_Ca in NCX KO myocytes

We next tested the contribution of extracellular Ca²⁺ entry through L-type Ca²⁺ channels on I_Ca kinetics in KO myocytes. To do this, we replaced bath Ca²⁺ (1 mM) with Ba²⁺ (3 mM). These experiments were conducted in the presence of Tg and Ry to deplete SR stores so that no Ca²⁺ would be available to influence I_Ca. Ba²⁺ is readily conducted by L-type Ca²⁺ channels. However, unlike Ca²⁺, Ba²⁺ has no direct effect on I_Ca inactivation. Thus, under these conditions, inactivation of I_Ca is predominantly voltage dependent (9). When Ba²⁺ was used as the charge carrier, the differences in I_Ca amplitude between KO and WT were completely eliminated (KO, −15.9 ± 2.9 pA/pF, n = 6; WT, −17.8 ± 3.7 pA/pF, n = 4; p > 0.05) (Fig. 2 A, Fig. 3), and decay kinetics were further slowed (KO, τ = 57.8 ± 4.3 ms; WT, τ = 52.0 ± 6.9 ms; p > 0.05) (Fig. 2 B, Fig. 3). These data indicate that both intact SR Ca²⁺ stores and also Ca²⁺ entry through L-type Ca²⁺ channels are required to produce the differences in I_Ca amplitude and kinetics observed between WT and KO cells. Because global cytosolic Ca²⁺ concentration measured with fura-2 is the same in both cell types (5,7), this finding suggests that differences in subsarcolemmal Ca²⁺ are responsible for the smaller and more rapidly inactivating Ca²⁺ current we observe in NCX KO mice.

Effect of intracellular Ca²⁺ buffering on I_Ca in NCX KO myocytes

To further investigate the influence of intracellular Ca²⁺ on I_Ca in NCX KO myocytes, we dialyzed myocytes with the fast Ca²⁺ chelator BAPTA (10 mM) via the patch pipette to buffer cytosolic Ca²⁺ (Fig. 4). With Ca²⁺ as the charge carrier, BAPTA eliminated differences in I_Ca amplitude (KO, −11.8 ± 1.1 pA/pF, n = 9; WT, −13.9 ± 1.3 pA/pF, n = 11; p > 0.05) (Fig. 4, A and B) and decay kinetics (KO, τ = 44.7 ± 4.1 ms; WT, τ = 44.7 ± 3.0 ms; p > 0.05) (Fig. 4, A and B) between KO and WT myocytes. Thus, buffering cytosolic Ca²⁺ with BAPTA negates the effect of knocking out NCX on I_Ca amplitude and inactivation.

FIGURE 4.

Magnitude and decay kinetics of I_Ca are equal when cytosolic Ca²⁺ is buffered with BAPTA. (A) Representative tracings of a KO and a WT myocyte under control conditions (left) and from a separate set of KO and WT myocytes that had been dialyzed with BAPTA (right). (B) Summary data comparing I_Ca in the presence and absence of BAPTA (KO, n = 9; WT, n = 11). Amplitude is shown on the left-hand graph, and decay kinetics on the right. *p < 0.01 for WT versus KO.

Abrupt block of NCX

In cardiac myocytes, blockade of NCX leads to an almost immediate increase in SR Ca²⁺ load and the Ca²⁺ transient (12). To evaluate the effects of NCX blockade on I_Ca before loading of the SR can occur, we used the following protocol: I_Ca was measured during constant pulsing from a holding potential of −40 mV to 0 mV at 1 Hz. After seven pulses, external Na⁺ was rapidly replaced with Li⁺ using the rapid solution exchanger described in the Methods. To prevent reverse Na⁺-Ca²⁺ exchange, pipette Na⁺ was replaced with Cs⁺ for these experiments. I_Ca during the final pulse in control solution was then compared with I_Ca elicited during the first pulse in Li⁺ solution, i.e., before an increase in SR Ca²⁺ load and the Ca²⁺ transient could occur. An example is shown in Fig. 5. In WT cells, acute blockade of NCX reduced I_Ca amplitude to 86.8 ± 1.3% of control (n = 14; p < 0.05), whereas in KO cells, rapid removal of external Na⁺ had no effect on I_Ca (98.0 ± 6.5%; n = 10; p > 0.05). These results suggest that abrupt blockade of NCX in WT cells can raise subsarcolemmal Ca²⁺ and thereby inactivate Ca²⁺ channels sufficiently to reduce I_Ca. This is consistent with the report by Goldhaber et al. (16), who found that acute blockade of NCX (without reversal) rapidly raises subsarcolemmal Ca²⁺ and activates Ca²⁺ sparks in rat ventricular myocytes. In KO cells, removing Na⁺ has no effect on subsarcolemmal Ca²⁺ or I_Ca because NCX is already blocked.

FIGURE 5.

Effect of abrupt blockade of NCX on I_Ca. (A) Representative traces showing effect on I_Ca of rapidly replacing external Na⁺ with Li⁺ before loading of SR Ca²⁺ can occur. The left-hand traces show the seventh and final Ca²⁺ current elicited in Na⁺-containing solution, whereas the right hand traces show the first Ca²⁺ current elicited in Li⁺-containing solution. Note that Li⁺ replacement causes a small but significant reduction in I_Ca amplitude in the WT cell (upper panel) but no change in the KO cell (lower panel) because of the lack of NCX. (B) Summary results (*p < 0.05, n.s. = difference not significant).

Experimental limitations

As mentioned earlier, some myocytes (10–20%) isolated from KO hearts have a WT phenotype. To identify and exclude these cells from the study, we tested for the presence of Na⁺-Ca²⁺ exchange current as described above. However, testing for I_NCX depends on the presence of cytosolic Ca²⁺. It was not feasible, then, to test for I_NCX when cells were dialyzed with BAPTA. Thus, for this subset of experiments (Fig. 4), we cannot exclude the possibility that some cells in the KO group actually have a WT phenotype. However, no more than 20% of KO cells are likely to be of WT phenotype (5,7), which would not alter our conclusions.

DISCUSSION

The Na⁺-Ca²⁺ exchanger is the dominant Ca²⁺ efflux mechanism in cardiac myocytes. Surprisingly, knocking out the exchanger in a cardiac-specific manner is not lethal. NCX KO mice live into adulthood with only modest hemodynamic abnormalities (5,7). NCX KO mice do not up-regulate alternative Ca²⁺ extrusion mechanisms and therefore have severely impaired Ca²⁺ removal capacity (7). To compensate for the absence of NCX, KO mice limit Ca²⁺ entry via I_Ca, and the net effect is a decrease in transsarcolemmal Ca²⁺ flux without compromising contractility (5,7). Although I_Ca is reduced in NCX KO mice, DHPR expression is unaltered (5). Thus, regulatory mechanisms must underlie the reduction of I_Ca in NCX KO mice. How does this occur?

Knockout of NCX promotes Ca²⁺-dependent inactivation of I_Ca

Ca²⁺-dependent inactivation is one of the strongest modulators of the cardiac L-type Ca²⁺ channel and can regulate Ca²⁺ influx on a beat-to-beat basis. Ca²⁺-dependent inactivation of I_Ca occurs when Ca²⁺ binds to DHPR-bound calmodulin (17). Although the amount of Ca²⁺ released from the SR is severalfold higher than that entering the cell via the L-type Ca²⁺ channel, both sources of Ca²⁺ are major contributors to Ca²⁺-dependent inhibition of I_Ca (18–21), and both sources may thereby influence I_Ca amplitude (10).

In this study, we show that either buffering intracellular Ca²⁺ (Fig. 4), or eliminating both SR Ca²⁺ release and Ca²⁺ entry (Fig. 3) eliminates the differences in I_Ca between NCX KO and WT mice. Thus, we conclude that the mechanism underlying the reduced I_Ca in NCX KO mice is most likely Ca²⁺-dependent inactivation. Because global cytosolic Ca²⁺ levels are unchanged in NCX KO cells, we conclude that I_Ca is inactivated by elevated subsarcolemmal Ca²⁺.

Regulation of I_Ca by subsarcolemmal Ca²⁺

Suppression of SR Ca²⁺ release is not sufficient to normalize I_Ca amplitude in NCX KO myocytes (Figs. 2 and 3). Only the additional inhibition of Ca²⁺ entry (Figs. 2 and 3) or heavily buffering cytosolic Ca²⁺ (Fig. 4) leads to normalization of I_Ca amplitude and decay kinetics. These observations suggest that in NCX KO myocytes, the absence of a robust Ca²⁺ efflux mechanism keeps subsarcolemmal Ca²⁺ elevated, even when SR Ca²⁺ is eliminated. We do not completely understand the source of the elevated subsarcolemmal Ca²⁺ in the absence of SR Ca²⁺. One possibility is that stochastic openings of L-type Ca²⁺ channels are sufficient to raise subsarcolemmal Ca²⁺ (even before depolarization) in the absence of NCX. Also, we cannot exclude the possibility of other Ca²⁺ sources or adaptations of which we are not yet aware. For example, ablation of NCX may lead to an increase in the sensitivity of L-type Ca²⁺ channels to inactivating Ca²⁺, although we have no direct evidence for this.

I_Ca amplitude in WT myocytes is also regulated by subsarcolemmal Ca²⁺. However, in WT cells, with an active efflux of Ca²⁺ by NCX, subsarcolemmal Ca²⁺ is lower at rest than in KO myocytes. Thus, I_Ca amplitude is greater and inactivation slower in WT compared with KO even at baseline. Furthermore, because subsarcolemmal Ca²⁺ is relatively low in WT myocytes to begin with, elimination of SR Ca²⁺ using thapsigargin and ryanodine has a relatively small effect on subsarcolemmal Ca²⁺ (and therefore inactivation of I_Ca; see Fig. 3) compared to the effect in KO.

Conventional fluorescent indicator methods are incapable of measuring the local Ca²⁺ concentration in the diadic cleft or the subsarcolemmal space. Indeed, resting and systolic cytoplasmic Ca²⁺ levels measured by fura-2 and fluo-3 are similar in WT and KO myocytes (5,7). Nevertheless, as shown in this study, a Ca²⁺-dependent mechanism underlies the reduced I_Ca in NCX KO mice. The Ca²⁺ current thereby serves as a reporter of subsarcolemmal and diadic cleft Ca²⁺. Elevated Ca²⁺ in these restricted spaces caused by a lack of normal Ca²⁺ extrusion by the exchanger results in reduced I_Ca amplitude and accelerated inactivation kinetics.

Effect of acute NCX blockade on I_Ca

Unlike KO of NCX, rapid blockade of NCX by Li⁺ substitution in WT myocytes had only a small effect on the I_Ca recorded during the first pulse after blockade (Fig. 5). An explanation for the mild effect of acute NCX blockade on I_Ca could be that several cardiac cycles in the absence of NCX activity are necessary to sufficiently raise subsarcolemmal Ca²⁺ in WT cells to levels required to inactivate I_Ca. This is difficult to investigate in cardiac myocytes because the blockade of NCX leads to a rapid increase of SR Ca²⁺ load and Ca²⁺ transients during continued pulses (12,22). Isaev et al. (10) addressed this problem by investigating the effects of NCX on I_Ca in a noncardiac mammalian expression system in which DHPR and NCX were coexpressed. Cytosolic Ca²⁺ was raised to levels expected in the subsarcolemmal space of cardiomyocytes during systole. Under these conditions, they found that suppression of NCX leads to a reduction of I_Ca similar to what we have described here in KO myocytes (Fig. 2).

We cannot exclude the possibility that there are longer-term adaptive responses participating in the survival of the NCX KO mice. Adaptations could, for example, sensitize KO myocytes to Ca²⁺-induced inactivation of I_Ca; we do not presently have evidence for such adaptations.

Ca²⁺-dependent versus voltage-dependent inhibition of I_Ca

We have reported previously that there is no difference in the relative voltage dependence of I_Ca in WT versus KO myocytes (5,7). In this study, we demonstrate a complete reversal of the reduction in I_Ca in KO myocytes when regulatory Ca²⁺ is eliminated. We therefore conclude that the underlying mechanism is primarily Ca²⁺-dependent rather than voltage-dependent.

However, evidence has been presented recently that subsarcolemmal Ca²⁺ can alter the voltage sensor of the DHPR (10), and thus, voltage-dependent and Ca²⁺-dependent regulatory mechanisms may be interconnected. Therefore, we cannot exclude the possibility that differences in subsarcolemmal Ca²⁺ may differentially influence the voltage-dependent availability of I_Ca in KO versus WT. Future experimental work will be necessary to investigate this issue.

Functional implications

The reduction of I_Ca in NCX KO mice via a Ca²⁺-dependent mechanism suggests a functional coupling between NCX and the L-type Ca²⁺ channel (Fig. 6): Reduced Ca²⁺ extrusion capacity leads to an accumulation of subsarcolemmal Ca²⁺. This promotes Ca²⁺-dependent inactivation of I_Ca, leading to a reduction of I_Ca. The reduction of Ca²⁺ influx balances Ca²⁺ fluxes. There is then an overall reduction in transsarcolemmal Ca²⁺ traffic.

FIGURE 6.

Schematic diagram of functional coupling between NCX and the L-type Ca²⁺ channel (LTCC). (A) Under physiological conditions, Ca²⁺ influx via I_Ca is in balance with Ca²⁺ efflux via NCX. (B) In situations of reduced Ca²⁺ extrusion capacity, Ca²⁺ accumulates in the subsarcolemmal space. (C) The increased subsarcolemmal Ca²⁺ concentration promotes Ca²⁺-dependent inactivation of I_Ca. This reduces Ca²⁺ influx so that a new balance between influx and efflux is established. ec, extracellular; ic, intracellular.

This feedback mechanism may enable the cardiomyocyte to adapt to impaired Ca²⁺ extrusion and may stabilize the balance of Ca²⁺ fluxes. The interplay between the Na⁺-Ca²⁺ exchanger and the L-type Ca²⁺ channel may be essential for maintenance of Ca²⁺ homeostasis in both healthy and diseased heart.