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(Circulation Research. 2004;95:604.)
© 2004 American Heart Association, Inc.

Cellular Biology

Functional Adult Myocardium in the Absence of Na⁺-Ca²⁺ Exchange

Cardiac-Specific Knockout of NCX1

Scott A. Henderson^*, Joshua I. Goldhaber^*, Jessica M. So, Tieyan Han, Christi Motter, An Ngo, Chana Chantawansri, Matthew R. Ritter, Martin Friedlander, Debora A. Nicoll, Joy S. Frank, Maria C. Jordan, Kenneth P. Roos, Robert S. Ross, Kenneth D. Philipson

From the Departments of Physiology and Medicine and the Cardiovascular Research Laboratories (S.A.H., J.I.G., J.M.S., T.H., C.M., A.N., C.C., D.A.N., M.C.J., J.S.F., K.P.R., R.S.R., K.D.P.), David Geffen School of Medicine at the University of California, Los Angeles; and the Department of Cell Biology (M.R.R., M.F.), The Scripps Research Institute, La Jolla, Calif. Present affiliation for R.S.R. is the Department of Medicine, University of California, San Diego School of Medicine.

Correspondence to Kenneth D. Philipson, Cardiovascular Research Laboratory, MRL 3-645, David Geffen School of Medicine, Los Angeles, CA 90095-1760. E-mail kphilipson{at}mednet.ucla.edu

	Abstract

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The excitation–contraction coupling cycle in cardiac muscle is initiated by an influx of Ca²⁺ through voltage-dependent Ca²⁺ channels. Ca²⁺ influx induces a release of Ca²⁺ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca²⁺ homeostasis, Ca²⁺ entry is balanced by efflux mediated by the sarcolemmal Na⁺-Ca²⁺ exchanger. In the absence of Na⁺-Ca²⁺ exchange, it would be expected that cardiac myocytes would overload with Ca²⁺. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na⁺-Ca²⁺ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na⁺-Ca²⁺ exchanger as measured by both immunoblots and microarray analysis. Ca²⁺ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca²⁺ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca²⁺ influx. Rather than upregulate other Ca²⁺ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na⁺-Ca²⁺ exchanger by limiting Ca²⁺ influx. The magnitude of Ca²⁺ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca²⁺ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.

Key Words: Na⁺-Ca²⁺ exchange • excitation–contraction coupling • genetically altered mice

	Introduction

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Cardiac excitation–contraction coupling is initiated by the influx of Ca²⁺ through voltage-dependent Ca²⁺ channels that triggers release of Ca²⁺ from the sarcoplasmic reticulum (SR). Ca²⁺ influx must, of course, be balanced by Ca²⁺ efflux, and the sarcolemmal Na⁺-Ca²⁺ exchanger and ATP-dependent Ca²⁺ pump are the two mechanisms that mediate myocardial Ca²⁺ efflux. A voluminous literature has documented the dominant role of Na⁺-Ca²⁺ exchange in the Ca²⁺ efflux process.^1,2 The sarcolemmal Ca²⁺ pump appears to have little role in excitation–contraction coupling. By regulating intracellular Ca²⁺ levels, Na⁺-Ca²⁺ exchange is a determinant of cardiac contractility, and acute alterations in exchange activity have major effects on contractile strength. For example, small changes in intracellular Na⁺ in response to digitalis produce positive inotropy.

With our current understanding of excitation–contraction coupling, it is almost inconceivable that myocardium could survive in the absence of Na⁺-Ca²⁺ exchange activity. Without a vigorous Ca²⁺ efflux mechanism, cardiac myocytes should Ca²⁺ overload, leading to a nonfunctional myocardium. Four laboratories, including ours, have reported that global knockout of the Na⁺-Ca²⁺ exchanger, NCX1, is embryonic lethal.^3–6 The result is not surprising and the lethality has been attributed to a cardiac phenotype.^3,5 One study, however, indicated that the lethality had an extracardiac origin.⁷

Unexpectedly, heart tubes from NCX knockout embryos, dissected at day 9.5, had almost normal Ca²⁺ transients in response to electrical stimulation.^5,8 Excitation–contraction coupling was still dependent on Ca²⁺ influx and, apparently, the sarcolemmal Ca²⁺ pump was sufficient to maintain Ca²⁺ homeostasis. The heart tubes were operating under low stress conditions (1 Hz stimulation at 26°C), however, and did not tolerate interventions (eg, increased stimulation rate) that increased the need for Ca²⁺ efflux. Additionally, day 9.5 NCX knockout embryos are within 1 day of death and other adaptations of the myocardium have occurred.⁸ Thus, embryonic heart tubes are a difficult preparation and not ideal for analysis of NCX ablation.

We have now generated, using Cre/loxP technology, mice with a cardiac-specific knockout of NCX1. The exchanger is completely ablated in at least 80% of the myocytes. Strikingly, these mice survive to adulthood with diminished, yet surprisingly good, cardiac function. We report our initial characterization of these mice.

	Methods

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Cardiac-Specific Knockout of NCX1
To ablate NCX1 in the ventricular myocardium, a mouse line was produced such that exon 11 was flanked by loxP sites (floxed). Typically, male and female breeders were homozygous for the NCX1 floxed allele (NCX1^fx/fx), and male breeders were additionally hemizygous for Cre expression under transcriptional control of the endogenous ventricular myosin light chain-2 (MLC2v) promoter.⁹ Offspring were genotyped by polymerase chain reaction (primer sequences provided on request) of DNA from tail biopsy specimens. All experiments were performed under approved institutional recombinant DNA and animal protocols.

For a description of other methods, see the expanded Methods available in the online data supplement at http://circres.ahajournals.org.

	Results

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Knockout of NCX1
We generated mice with a loxP site inserted into each of the two introns flanking exon 11 of the Na⁺-Ca²⁺ exchanger, NCX1. These mice were mated with mice expressing Cre recombinase under the control of the cardiac-specific MLC2v promoter. Cre recombinase excises DNA between loxP sites from the chromosomes of ventricular myocytes beginning during embryonic development.⁹ No other Na⁺-Ca²⁺ exchange activity besides NCX1 is known to be present in myocardium.

We chose to excise exon 11 because we had previous evidence that amino acids encoded by this exon are critical for exchange activity. Single site mutations within this region eliminate exchange activity.¹⁰ Exon 11 codes for amino acids 722 to 813 (Figure 1A), encompassing two transmembrane segments including part of the -2 region essential for Na⁺-Ca²⁺ exchange activity.¹¹ Excision of exon 11 leaves the reading frame intact. To rigorously test the assumption that an exchanger with amino acids 722 to 813 deleted is inactive, we constructed NCX1 cDNA with this deletion (722–813). Function of 722–813 was tested in three different expression systems: after transient transfection of BHK and HEK cells and after cRNA injection into Xenopus oocytes. Using a Na⁺-gradient–dependent ⁴⁵Ca²⁺ uptake assay, the deletion mutant displayed no detectable exchange activity in any expression system (Figure 1B). Expression of 722–813 protein was confirmed by immunoblot (not shown).

View larger version (38K): [in this window] [in a new window]   Figure 1. Deletion of amino acids 722 to 813 from NCX1. Excision of exon 11 results in a Na+-Ca2+ exchanger with amino acids 722 to 813 deleted (722–813). A, Topological model of the Na+-Ca2+ exchanger, NCX1. The 9 cylinders represent transmembrane segments with the extracellular surface at the top. The -1 and -2 regions (shaded) are homologous and are essential for exchange function. The approximate locations of amino acids 722 and 813 are indicated (arrows). The region between the arrows is deleted in 722–813. B, Na+-Ca2+ exchange activity of wild-type (WT) and 722–813 () exchangers expressed in BHK or HEK cells or in Xenopus oocytes. Exchange activity is indicated as the difference of 45Ca2+ uptake of Na+-loaded cells in K+ and Na+ media. No difference in uptake in K+ and Na+ media, and hence no exchange activity, is detected for the deletion mutant. See Methods for details.

Homozygous NCX1 knockout mice survive to adulthood, and all experiments described used homozygous knockout mice. We performed no experiments with heterozygous knockout mice. In initial experiments, we compared hearts from wild-type mice and mice with exon 11 of NCX1 flanked by loxP sites (floxed), but not crossed with Cre recombinase-expressing mice, so no excision had occurred. There were no differences in NCX1 protein levels or cardiac function as assessed by echocardiography. That is, floxing per se had no effects. In all experiments reported here, control animals for the knockout mice were floxed.

NCX1 Expression
We directly assessed the level of Na⁺-Ca²⁺ exchanger protein in the hearts of homozygous NCX1 knockout mice by immunoblot (Figure 2A). As shown, the level of NCX1 protein is greatly reduced. A typical pattern of bands at 120 and 160 kDa is seen with homogenate of control hearts. With the knockout hearts, an additional band arises at 110 kDa (arrow in Figure 2A), representing exchangers with the 722–813 deletion. The intensity of this band is diminished, suggesting that the abnormal and nonfunctional protein gets degraded more rapidly. The level of decrease in functional exchanger protein is 88% (Figure 2A and Table 1).

View larger version (66K): [in this window] [in a new window]   Figure 2. Protein levels of Ca2+-handling proteins in NCX1 knockout mice. A, Immunoblot of mouse heart homogenate proteins (20 µg) using an antibody to the Na+-Ca2+ exchanger. Homogenate from a separate heart was loaded in each lane from a mouse not expressing or expressing cardiac Cre recombinase. Only in the presence of Cre recombinase does NCX1 knockout occur. Arrow indicates the new band representing nonfunctional exchanger that arises in the knockout hearts after excision of exon 11. B, Protein levels of other Ca2+-handling proteins in NCX1 knockout hearts. Shown are immunoblots developed with antibodies to the plasma membrane Ca2+ pump (PMCA), the sarcoplasmic reticular Ca2+ pump (SERCA2), the 2 subunit of the dihydropyridine receptor (DHPR2), and calsequestrin. Molecular weight markers (kDa) are shown on the right. Quantification of all data is shown in Table 1. C, Immunofluorescence of isolated myocytes from a NCX1 knockout heart. Myocytes were immunolabeled using the monoclonal antibody R3F1 to the cardiac Na+-Ca2+ exchanger. In this representative experiment, 90% (35/39) of the knockout myocytes displayed only a diffuse background staining (left panel). The other 10% (4/39) of the myocytes showed a staining pattern indistinguishable from that of wild-type myocytes (right panel).

View this table: [in this window] [in a new window]   Table 1. Lack of Adaptation to the Absence of the Na+-Ca2+ Exchanger

Why does any NCX1 protein remain in knockout mice? The extent of ablation depends on the efficacy of the Cre recombinase, and it has been a common finding that cardiac-specific knockouts do not occur with 100% efficiency.^12,13 Those cells in which excision occurs will have a complete absence of NCX1 and the remaining cells will have a normal expression level; however, there is also the possibility that in some cells excision will occur on only one allele. The immunoblot data indicate that 88% of the myocytes have no NCX1. We further addressed this issue by using immunofluorescence with isolated myocytes.

Figure 2C shows the immunofluorescence of myocytes isolated from a NCX1 knockout heart. When stained using a monoclonal antibody to NCX1, a majority of myocytes showed only a weak diffuse background staining (left panel of Figure 2C). This staining is equivalent to that obtained when primary antibody is omitted (not shown). A minority of myocytes from a knockout heart showed staining similar to that observed in myocytes from control hearts (right panel in Figure 2C). In this representative experiment, 35 of 39 cells showed the weak background staining pattern, whereas 4 of 39 cells displayed a staining pattern similar to that of wild-type cells. One hundred percent of control cells had a staining pattern similar to that displayed in the right panel of Figure 2C (not shown). The results are consistent with the immunoblot data. The ultrastructure of knockout hearts was normal as assessed by thin-section electron microscopy (not shown). Myofibrils showed normal alignment, and structures involved in excitation–contraction coupling (transverse tubules; subsarcolemmal and junctional SR) also appeared normal.

Expression of Other Myocardial Proteins
It might be expected that the myocardium would respond to the absence of NCX1 by altering the expression of other proteins involved in excitation–contraction coupling. We could find no evidence for any adaptations at either the protein or the transcript level. By immunoblot, there were no changes in the levels of the plasma membrane Ca²⁺ pump, the dihydropyridine receptor, SERCA, or calsequestrin (Figure 2B and Table 1). Lack of adaptations, as detected by microarray analysis, was striking. The expression of no transcripts stood out as having been altered by the ablation of the Na⁺-Ca²⁺ exchanger. Table 1 shows the unchanged transcript levels of several proteins involved in the handling of Ca²⁺. Notably, the transcript levels of other known Na⁺-Ca²⁺ exchangers (NCX2 and NCX3) remained at nondetectable levels after knockout of NCX1. NCX2 and NCX3 were also nondetectable by immunoblot (not shown). Transcript levels of NCX1 were unchanged in the myocardium of NCX1 knockout mice (Table 1). This is compatible with normal levels of the abnormal transcript being synthesized from NCX1 alleles lacking exon 11. Two K⁺-dependent Na⁺-Ca²⁺ exchangers were represented on the microarray (NCKX1 and NCKX3) but were not present at significant levels even after ablation of NCX1. The hearts used in the immunoblot and microarray analyses were from young mice (5 to 6 weeks of age) to eliminate changes secondary to the development of pathology as the animals became older.

Cardiac Function
We assessed the cardiac function of the NCX1 knockout mice by echocardiography (Table 2). At 7 to 8 weeks of age, a relatively modest diminution of cardiac function is apparent. Fractional shortening, velocity of circumferential fiber shortening, and ejection fraction (three measures of contractility) are decreased by 30%, 25%, and 21%, respectively. Wall thicknesses and left ventricular chamber size are unchanged, demonstrating absence of hypertrophy. The increase in end systolic dimension reflects the decrease in contractility.

View this table: [in this window] [in a new window]   Table 2. Cardiac Function in the Absence of the Na+-Ca2+ Exchanger from Echocardiography

Although at 7 to 8 weeks of age the NCX1 knockout mice have myocardium without a large decrease of function (Table 2), the animals are not normal and cannot withstand stress. Thus, homozygous knockout females could not survive the stress of breeding. Of 6 knockout females that gave birth, all died within 9±2 days of delivery. Another 4 knockout females died after mating but before delivery. Cardiac function displayed further decline with age as assessed by echocardiography (not shown), and lifespan was reduced. Nineteen knockout animals died at 103±7 days, representing 10% of the animals. No animals in the control group died within this time span. Postmortem inspection indicated enlarged hearts, and death was probably associated with heart failure. Mice beyond this age were either euthanized or used for experiments, so average life span was not determined. In this report, we focus on younger knockout mice (6 to 8 weeks of age) with minimal pathology.

Excitation–Contraction Coupling
We were unable to detect any difference in the appearance or yield of enzymatically isolated ventricular myocytes from control and knockout hearts. We assessed Na⁺-Ca²⁺ exchange activity in patch-clamped myocytes by rapidly applying 5 mmol/L caffeine to the bath solution while recording membrane current at a constant holding potential of –40 mV. To ensure constant SR loading, cells were prepulsed from –80 to 0 mV (100 ms duration) at 1 Hz. Caffeine elicited Ca²⁺ transients of similar amplitude in control and knockout myocytes (Figure 3), suggesting similar SR Ca²⁺ contents. The increases in the [Ca²⁺] induced by caffeine were 368±34 (n=7) and 329±22 nmol/L (n=5) for control and knockout cells, respectively (P=0.4). The Ca²⁺ transient declined in control cells, even in the presence of caffeine, as Ca²⁺ was extruded by Na⁺-Ca²⁺ exchange. The decline in the Ca²⁺ transient in knockout cells in the presence of caffeine was always slower. Based on the initial decline in the Ca²⁺ transient in the presence of caffeine, we estimated the half-time for the decline in the transient to baseline. These values were 1.2±0.1 (n=7) and 6.3±2.2 s (n=5) for control and knockout cells, respectively (P<0.02).

View larger version (12K): [in this window] [in a new window]   Figure 3. Absence of caffeine-induced Na+-Ca2+ exchange currents in NCX1 knockout myocytes. Patch-clamped myocytes were held at a constant holding potential of –40 mV during rapid application of caffeine (5 mmol/L) to the bath to release SR Ca2+ stores. In control myocytes, this procedure always elicited an inward Na+-Ca2+ exchange current (middle trace), whereas 11 of 12 myocytes from knockout animals exhibited no detectable Na+-Ca2+ exchange current.

In control cells, application of caffeine always induced an inward Na⁺-Ca²⁺ exchange current. However, in 11 of 12 knockout cells, caffeine-induced Na⁺-Ca²⁺ exchange current was completely absent (Figure 3, middle trace). Thus, the wild-type phenotype was present in <10% of randomly chosen knockout myocytes. These functional data are consistent with the immunoblot and immunofluorescence data. We estimate that we would detect any residual Na⁺-Ca²⁺ exchange current in knockout myocytes that was >1% of that in control myocytes.

We examined Ca²⁺ transients in externally paced isolated ventricular myocytes loaded with fluo-3 acetoxymethyl ester (fluo-3 AM) (Figure 4). Ca²⁺ transients from NCX1 knockout myocytes were of similar magnitude to those in wild-type myocytes and had no relaxation deficit. Diastolic Ca²⁺ levels were similar. Both the wild-type and knockout myocytes displayed a negative staircase typical of mouse myocytes. In an attempt to expose consequences of NCX1 ablation, we stimulated isolated myocytes with the ß agonist, isoproterenol (1 µmol/L for 3 minutes). Robust and identical inotropic responses to isoproterenol were induced in both wild-type and knockout myocytes (Figure 5). Remarkably, we could detect no effects of the NCX1 knockout on cellular Ca²⁺ dynamics. Cells from knockout myocardium retained dependence on external Ca²⁺; on removal of external Ca²⁺, transients were rapidly eliminated (not shown).

View larger version (40K): [in this window] [in a new window]   Figure 4. Effects of pacing rate on Ca2+ transients from control and NCX1 knockout myocytes. Ca2+ transients were recorded from externally paced control and knockout myocytes loaded with fluo-3 AM. Both control and knockout myocytes exhibited similar rate-dependent decreases in the amplitude of the Ca2+ transient. There were no differences in the kinetics of the relaxation phase () of the Ca2+ transient. Summary data are shown in the bar graphs; n=18 and 16 for control and knockout myocytes, respectively.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effects of isoproterenol on Ca2+ transients from control and NCX1 knockout myocytes. Ca2+ transients from externally paced control and knockout myocytes loaded with fluo-3 AM. Both control and knockout myocytes exhibited similar increases in the amplitude and relaxation kinetics of the Ca2+ transient in response to isoproterenol (1 µmol/L for 3 minutes). Summary data are shown in the bar graphs; n=25 and 21 for control and knockout myocytes, respectively. *P<0.05 as compared with values in the absence of isoproterenol by paired t test.

A difference between control and NCX1 knockout myocytes did become manifest in measurements of voltage-dependent L-type Ca²⁺ currents (I_Ca) under whole-cell clamp conditions. Current–voltage (I-V) relationships are shown in Figure 6 A and 6B. I_Ca in knockout cells was substantially decreased compared with control cells. The percentage decreases were 48%, 49%, 47%, and 32% at –10, 0, +10, and +20 mV, respectively. Both I-V curves peaked at 0 mV. The rates of inactivation of I_Ca were similar in the two cases. It is notable that all four measures of L-type Ca²⁺ channel expression shown in Table 1 show a modest reduction in the knockout hearts, although in none of the cases is statistical significance achieved. We have no other data on the mechanism of decreased Ca²⁺ current, although perhaps posttranslational modification is involved.

View larger version (25K): [in this window] [in a new window]   Figure 6. L-type Ca2+ currents and action potentials in NCX1 knockout myocytes. A, Representative Ca2+ currents from patch-clamped control and knockout myocytes. Cells were depolarized from a holding potential of –40 mV to a family of test potentials from –30 to +40 mV. Note the reduced ICa at all potentials in the knockout. B, Ca2+ current summary data; n=4. C, Representative action potentials recorded in control and knockout myocytes using the perforated patch clamp technique. The distinct action potential plateau of wild-type myocytes is absent in knockouts, consistent with the absence of Na+-Ca2+ exchange. *P<0.05 by 2-way ANOVA with Tukey–Fisher least significant difference post hoc testing.

There was also a substantial difference in the morphology of action potentials between wild-type and NCX1 knockout myocytes. Whereas action potentials from control myocytes always exhibited a distinct plateau after the upstroke and rapid repolarization, action potentials from NCX1 knockout myocytes completely lacked a plateau in 6 of 8 myocytes (Figure 6C). At 85 ms after onset of the stimulus, the plateau level in control myocytes was –48±3 mv, whereas the voltage in the 6 knockout myocytes was –67±2 mv (P<0.001). These data are consistent with the absence of NCX1 in the majority of isolated myocytes as expected. Resting membrane potentials were indistinguishable in control and knockout myocytes (–68.1±1.0 [n=10] and –68.0±1.7 mV [n=5], respectively). At 0 mV, the width of the action potential spike was 3.6±0.5 ms for control and 1.5±0.3 ms for knockout myocytes, respectively (P<0.01).

	Discussion

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We generated mice in which the Na⁺-Ca²⁺ exchanger, NCX1, was completely removed from 80% to 90% of the cardiac myocytes as assessed by immunoblot, immunofluorescence, and function. In vivo, mouse myocardium contracts 600 times per minute, and Ca²⁺ influx accompanies each excitation. The expectation was that excitation–contraction coupling would fail in the absence of the dominant Ca²⁺ efflux mechanism. Whole animal knockout of NCX1 is embryonic lethal,^3–6 although the cause may not be cardiac in origin.⁷ Also, in our cardiac-specific knockout, disruption of the NCX1 gene will begin during embryonic development and is limited to ventricular myocytes.⁹ The presence of NCX1 in the atria and early ventricular cells may prevent the lethality that occurs in the global knockout. In any case, the survival of the cardiac-specific knockout mice to adulthood is surprising.

There are several issues to consider. Mice, like rats, are known to have quantitatively distinct Ca²⁺ flux pathways. Ca²⁺ influx is required to initiate excitation–contraction coupling in all myocardium, but the contribution of transsarcolemmal pathways is smaller in mice and rats than in other mammalian species. For example, 25% of activator Ca²⁺ crosses the sarcolemma during each contraction–relaxation cycle in rabbit cardiac myocytes.^14–16 The remaining 75% of the Ca²⁺ is released from and then sequestered by the SR. In contrast, mice and rats are much more SR-dependent. Only 8% of activator Ca²⁺ traverses the sarcolemma.^14,16 Thus, the role of Ca²⁺ efflux mechanisms, although still essential, is reduced in magnitude in adult mouse myocardium. Nevertheless, the mouse heart undergoes excitation 600 times per minute. Ca²⁺ enters myocytes with each excitation, and over time the magnitude of Ca²⁺ that must be extruded may equal that of other species.

Although the Na⁺-Ca²⁺ exchanger is absent in most myocytes from knockout myocardium, Ca²⁺ efflux must occur after each excitation. An alternative efflux mechanism must function, and the only known viable candidate is the sarcolemmal Ca²⁺ pump. The Ca²⁺ pump is thought to have minor significance in excitation–contraction coupling, but this contention may need to be reexamined. Some studies using rat and mouse myocytes find that the capacity of the Ca²⁺ pump is only a small fraction of that of the Na⁺-Ca²⁺ exchanger,^14,17 although other studies using rat myocytes indicate that the Ca²⁺ pump can remove Ca²⁺ at a rate of 30% of that of the exchanger.^18–20 We see no increase in the expression level of the sarcolemmal Ca²⁺ pump (Table 1) in response to knockout of NCX1. Nevertheless, the sarcolemmal Ca²⁺ pump is highly regulated and may be activated by phosphorylation or calmodulin. This possibility is difficult to assess. The only direct measure of Ca²⁺ efflux in our experiments is the decline of the Ca²⁺ transient in the presence of caffeine (Figure 3). The relationship between the diminished Ca²⁺ efflux of the knockout myocytes in caffeine experiments and the need for Ca²⁺ balance during excitation–contraction coupling is not quantitatively clear. The sarcolemmal Ca²⁺ pump may seem an unlikely candidate to completely compensate for the absence of NCX1 but, nevertheless, appears to permit survival of mouse myocytes.

The myocardium is a syncitium and the 20% of myocytes with normal NCX1 levels may compensate for the absence of exchanger in adjoining cells as Ca²⁺ diffuses from cell to cell. We expect that this mechanism is unlikely to make a substantial contribution to Ca²⁺ homeostasis for two reasons. First, diffusion is sufficiently slow that cells with no exchanger would experience elevated Ca²⁺ levels for prolonged times, leading to contractile abnormalities and altered gene expression. Second, and more cogently, we see unaltered Ca²⁺ transients in isolated myocytes. In this case, the syncitial nature of myocardium has been eliminated, and NCX1 knockout cells cannot rely on adjoining cells for Ca²⁺ extrusion.

The expression in heart of a member of the K⁺-dependent Na⁺-Ca²⁺ exchanger family (NCKX6) has recently been described.²¹ These exchangers countertransport 4 Na⁺ for 1 Ca²⁺ plus 1 K⁺. It is unlikely that this exchanger, or other members of the NCKX family, contribute significantly to Ca²⁺ efflux in myocardium. First, no Na⁺-Ca²⁺ exchange activity with a high-affinity K⁺-dependence has ever been observed in cardiac sarcolemma.²² Second, by immunofluorescence, this exchanger was absent from T tubules.²¹ Third, exchangers of the NCKX family are electrogenic, and we can detect no exchanger-associated currents in NCX1 knockout myocytes. In short, no other known Na⁺-Ca²⁺ exchangers have been shown to be active in cardiac myocytes other than NCX1.

As mentioned, cardiac pathology develops in the knockout mice as they age, but it is quite remarkable that isolated myocytes from younger knockout mice have Ca²⁺ transients indistinguishable from those in wild-type myocytes. In addition, responses to changes in stimulation frequency or isoproteronol were normal. These results are completely consistent with our inability to detect any adaptation of the myocardium to the absence of NCX1, as indicated by immunoblots and microarray analysis. Ca²⁺ regulates transcription as well as contraction; chronic abnormalities in Ca²⁺ handling would induce alterations in protein expression. We conclude that mouse myocytes lacking the Na⁺-Ca²⁺ exchanger display normal Ca²⁺ dynamics.

How is Ca²⁺ homeostasis maintained in the absence of Na⁺-Ca²⁺ exchange? Rather than upregulate other Ca²⁺ efflux mechanisms, it appears that the cell limits Ca²⁺ influx. Thus, the need for robust Ca²⁺ efflux is lessened and the sarcolemmal ATP-dependent Ca²⁺ pump suffices. Ca²⁺ influx through the L-type Ca²⁺ channel is substantially diminished (Figure 6), supporting this hypothesis, although the mechanism of decreased I_Ca is unknown. Interestingly, we have recently reported that overexpression of NCX1 results in an increased I_Ca, again by an unknown mechanism.²³ In addition, the action potential of knockout myocytes is markedly abbreviated. The plateau phase is eliminated and the duration of the initial spike of depolarization is also shortened. The more rapid repolarization of the cell in the absence of the exchanger may induce a more rapid closing of Ca²⁺ channels, further limiting Ca²⁺ influx beyond that seen under voltage clamp conditions. Inhibition of Na⁺-Ca²⁺ exchange has previously been observed to abbreviate the action potential,^24,25 whereas overexpression of the exchanger prolongs action potential duration.²⁶ Shortening the action potential, especially at positive potentials, can limit Ca²⁺ influx without compromising subsequent Ca²⁺-induced Ca²⁺ release.²⁷ That is, the initial phase of I_Ca appears to be most important as the trigger for SR Ca²⁺ release.

If Ca²⁺ influx into NCX1 knockout myocytes is diminished, then the presence of normal Ca²⁺ transients must be explained. We hypothesize that the gain of Ca²⁺ release is increased. That is, the trigger Ca²⁺ (I_Ca) more efficiently induces SR Ca²⁺ release than in wild-type mice. Thus, an equivalent Ca²⁺ transient could be evoked with a reduced I_Ca. Our observations of decreased I_Ca and normal Ca²⁺ transients directly imply that gain in the knockout myocytes is increased, although we have not directly assessed these two properties in the same myocytes. We have recently described that overexpression of the cardiac Na⁺-Ca²⁺ exchanger results in a decreased gain of Ca²⁺ release.²³ It would be an interesting parallel if underexpression of the exchanger produces an increase in gain. In any case, the cardiac-specific NCX1 knockout mice provide a remarkable example of the ability of the myocardium to functionally adapt to altered Ca²⁺ handling.

	Acknowledgments

 
This research was supported by National Institutes of Health grants HL48509 (K.D.P.), HL70828 (J.I.G.), and the Laubisch Foundation.

	Footnotes

 
*Both authors contributed equally to this work.

Original received April 23, 2004; revision received August 2, 2004; accepted August 3, 2004.

	References

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