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J. Biol. Chem., Vol. 276, Issue 21, 17706-17711, May 25, 2001

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Na⁺-Ca²⁺ Exchanger Remodeling in Pressure Overload Cardiac Hypertrophy^*

Zhengyi Wang^§, Bridgid Nolan^§¶, William Kutschke, and Joseph A. Hill^¶**

From the Departments of  Internal Medicine and  Pharmacology, the ^** Interdisciplinary Graduate Program in Molecular Biology, University of Iowa College of Medicine, and the ^¶ Department of Veterans Affairs, Iowa City, Iowa 52242

Received for publication, January 19, 2001, and in revised form, March 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Perturbations of Ca²⁺ metabolism are central to the pathogenesis of cardiac hypertrophy. The electrogenic Na⁺-Ca²⁺ exchanger mediates a substantial component of transmembrane Ca²⁺ movement in cardiac myocytes and is up-regulated in heart failure. However, the role of the exchanger in the pathogenesis of cardiac hypertrophy is poorly understood. Thoracic aortic banding in mice induced 50-60% increases in heart mass and cardiomyocyte size. Despite the absence of myocardial dysfunction, steady-state NCX1 transcript and protein levels were increased to an extent similar to that reported in heart failure. As recent studies indicate that calcineurin is critical to the expression of Na⁺-Ca²⁺ exchanger genes, we inhibited calcineurin with cyclosporin. Calcineurin inhibition blunted the increases in NCX1 transcript and protein levels and eliminated the increases in heart mass and cell volume normally associated with pressure overload. To examine the functional significance of these changes, we measured Na⁺-Ca²⁺ exchanger current in two independent ways. Surprisingly, exchanger current density was decreased in hypertrophied myocytes, and this down-regulation was eliminated by calcineurin inhibition. Together, these data reveal a role for Na⁺-Ca²⁺ exchanger current in the electrical remodeling of hypertrophy and implicate calcineurin signaling therein. In addition, these data suggest the Na⁺-Ca²⁺ exchanger is functionally regulated in hypertrophy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hypertrophic transformation is important in many forms of heart disease including ischemia, hypertension, heart failure, and valvular disease. In each of these types of cardiac pathology, hypertrophy has a crucial compensatory function normalizing wall stress and oxygen consumption. At the same time, hypertrophy may be an early phase in a pathogenic process that culminates in heart failure. The molecular signaling pathways involved in the pathogenesis of hypertrophy and transition to heart failure are the subject of intense investigation (reviewed recently in Refs. 1 and 2).

Electrical remodeling, culminating in action potential prolongation, is a fundamental aspect of both ventricular hypertrophy (3) and heart failure (4-6). Delayed repolarization leads to increased dispersion of refractoriness within the diseased ventricle thereby predisposing to arrhythmia, syncope, and sudden death. Indeed, the clinical impact of cardiac hypertrophy stems largely from disordered electrical currents that predispose patients to devastating arrhythmias. Patients with echocardiographically documented cardiac hypertrophy are at significantly increased risk of malignant cardiac arrhythmia, which accounts for a substantial component of the morbidity and mortality associated with cardiac hypertrophy (7). The molecular mechanisms underlying these arrhythmias are poorly understood and the means of treating them disappointingly ineffective.

Intracellular calcium is increasingly viewed as a central point of regulation in the pathogenesis of hypertrophy (1, 8) and disease-related electrical remodeling (3, 9). It has been known for some time that intracellular Ca²⁺ homeostasis is impaired in heart failure with elevated diastolic Ca²⁺ concentrations and diminished systolic Ca²⁺ transient amplitude (10, 11). A number of Ca²⁺-sensitive signaling pathways have been implicated in cardiac hypertrophy including activation of MAP kinases (12), protein kinase C (13, 14), Ca²⁺/calmodulin-dependent protein kinase (15), and calcineurin (16, 17). Evidence suggests that Ca²⁺ signaling in hypertrophy may be different from heart failure with increased Ca²⁺ transients (3, 5, 9) and increased inward Ca²⁺ current (I_Ca,L) (5).¹

Calcium transport by the Na⁺-Ca²⁺ exchanger (NCX)¹ is a major mechanism of Ca²⁺ elimination during diastole (18). As NCX catalyzes the bidirectional exchange of three Na⁺ ions for a single Ca²⁺ ion, one net charge moves per reaction cycle generating a transmembrane current that approaches one-half the magnitude of the L-type Ca²⁺ current. Indirect inhibition of NCX by diminution of the transmembrane Na⁺ gradient from Na⁺-K⁺ ATPase blockade is thought to underlie the positive inotropic effects of digitalis glycosides. NCX expression is maximal near the time of birth and declines postnatally (19), and is required for normal cardiac development (20). In several models of heart failure, NCX activity and protein levels are increased (21-26), which has been proposed to be an adaptive mechanism that preserves Ca²⁺ metabolism and hence myocardial function (27-29). On the other hand, increased NCX may contribute to anoxia-induced cytosolic Ca²⁺ overload (30) and has potentially proarrhythmic actions (24, 31, 32).

Given the central role of Ca²⁺ metabolism in the pathogenesis of hypertrophy and the major role played by NCX in controlling intracellular Ca²⁺ levels, we hypothesized that NCX may be altered in hypertrophy. To test this hypothesis, we measured NCX1 transcript and protein levels in a model of compensated hypertrophy where systolic dysfunction or heart failure do not occur. To examine the functional significance of changes in NCX expression to the action potential prolongation of hypertrophy, we measured NCX activity by recording NCX current (I_NCX) using 2 independent means.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pressure Overload Hypertrophy Model-- Male mice (C57BL6, 6-8 weeks old) were subjected to pressure overload by thoracic aortic banding (TAB) (33). Some mice were subjected to a sham operation in which the aortic arch was visualized but not banded. Perioperative (24 h) mortality was less than 5%. On the morning of post-operative day 1, TAB (or sham-operated) mice were randomized to receive 25 mg/kg CsA (or an equal volume of vehicle) subcutaneously twice daily. Sham-operated mice were similar in every aspect to unoperated controls, and vehicle injections were similarly without effect.

Myocyte Isolation and Electrophysiological Recordings-- Left ventricular cardiomyocytes were isolated by retrograde enzymatic perfusion and superfused at 1-2 ml/min (21-23 °C) with Tyrode's solution containing (mmol/liter) 137 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.0 CaCl₂, 10.0 dextrose, 10.0 HEPES (pH 7.4). Calcium-tolerant, quiescent myocytes with typical rod-shaped appearance and clear cross-striations were chosen for experimentation. Borosilicate glass capillaries (7052 glass, 1.65-mm OD, A-M Systems) were prepared with tip resistances of 1.5-3.0 M when filled with solution containing (mmol/liter): 130.0 KCl, 1.0 MgCl₂, 0.5 CaCl₂, 10.0 HEPES, 5.0 EGTA, 5.0 Mg₂ATP, 5.0 Na-creatine phosphate, 0.5 GTP-Tris (pH 7.2). Tip potentials were compensated before the pipette touched the cell. After obtaining whole cell voltage clamp with a gigaohm seal, whole cell membrane capacitance was calculated as the time integral of the capacitive response to a 10 mV hyperpolarizing step. Cells with significant leak current (100 pA) were rejected (20%). When measuring whole cell currents (depolarized every 15 s), series resistance and membrane capacitance were compensated electronically 85%.

Inward I_NCX Induced by Caffeine-mediated SR Ca²⁺ Release-- Myocytes were held at 80 mV with a pipette filled with a solution containing (mmol/liter) 15 NaCl, 100 CsCl, 30 tetraethylammonium-Cl, 5 MgATP, 10 HEPES, and 5.5 dextrose (pH 7.1 adjusted with CsOH). The voltage-clamped cell was superfused in a microstream containing (mM) 138 NaCl, 1.0 MgCl₂, 4.4 KCl, 1.08 CaCl₂, 2 CsCl, 0.1 BaCl₂, 11 dextrose, and 24 HEPES (pH 7.4 adjusted with NaOH to give a final extracellular [Na⁺] ([Na⁺]_o) of 145 mM). After a train of steady-state conditioning pulses (eight 200-ms pulses to 0 mV, 0.25 Hz), the cell was rapidly switched to a solution containing 10 mmol/liter caffeine and superfused for 6 s.

I_NCX Measured as Ni²⁺-sensitive Current-- Myocytes were depolarized to 45 mV from a holding potential of 90 mV to inactivate sodium current. The voltage was then stepped to +80 mV and ramped down to 140 mV to induce remaining currents. After currents reached steady state, the protocol was repeated in the presence of 5 mM NiCl₂. In this way, I_NCX is defined as the nickel-sensitive current induced during the ramped potential (24). Pipette solutions contained (mmol/liter) 45 CsCl, 55 Cs-methanesulfonic acid, 10 ATP-Tris, 0.3 GTP-Tris, 20 HEPES, 5 BAPTA, 5 DiBr-BAPTA, 10.8 MgCl₂, 2.21 CaCl₂, and 14 NaCl (pH 7.3 with CsOH). Bath solution contained (mM) 137 NaCl, 1.0 MgCl₂, 5.4 CsCl, 1.0 CaCl₂, 10 dextrose, and 10 HEPES (pH 7.4 with NaOH) plus 10 µM nifedipine.

Western Blots-- Purified membrane proteins were prepared from 3 left ventricles harvested from mice subjected to the 4 treatments conditions listed (sham-operated controls, TAB, TAB + CsA, CsA). These proteins were subjected to polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and subjected to immunoblot analysis with ECL detection.

Northern Blots-- Using standard methods, total RNA (50 µg) was fractionated by electrophoresis in a denaturing formaldehyde/agarose gel, transferred to a positively charged nylon membrane, and hybridized with a riboprobe specific to NCX (34) or glyceraldehyde-3-phosphate dehydrogenase.

Ribonuclease Protection Assay-- An NCX-specific riboprobe was synthesized in vitro (MaxiScript, Ambion) using a cDNA corresponding to 31 to +118 base pairs relative to the AUG codon of NCX1 (cDNA kindly provided by Dr. Muthu Periasamy (34)), followed by gel purification. 2-8 × 10⁴ cpm of riboprobe were incubated overnight at 42 °C with total left ventricle RNA (RNeasy, Qiagen) followed by incubation with ribonucleases A and T1 for 30 min at 37 °C. The complex was subjected to ethanol precipitation followed by separation by polyacrylamide gel electrophoresis. The products were then visualized by exposure to autoradiographic film.

Statistical Methods-- Averaged data are reported as mean ± S.E. Sample sizes are listed as n = x/y to denote x cells from y mice. Statistical significance was analyzed using a Student's unpaired t test or one-way ANOVA followed by Bonferroni's method for post-hoc pairwise multiple comparisons.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ventricular hypertrophy was induced by banding the thoracic aorta in mice, which leads to significant hypertrophy at 3 weeks, measured either as heart weight normalized to body weight (HW/BW) (increased 50%, p < 0.05), myocyte two-dimensional surface area (increased 85%, p < 0.05), or myocyte whole cell capacitance (increased 60%, p < 0.05) (Table I, Fig. 1). Echocardiography of randomly selected mice confirmed our previous report (33) of absence of left ventricular dilatation or systolic dysfunction in this model (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Pressure overload-induced increases in heart mass and myocyte size, and effects of calcineurin inhibition. Two-dimensional cell surface area, whole cell capacitance (× 20 for comparison in a single graphic), and heart weight normalized to body weight (HW/BW) from hearts treated as follows: sham-operated controls (gray), TAB (black), TAB + CsA (white), or CsA alone (stippled black). Sample numbers (n) are provided in Table I. Asterisk (*) denotes p < 0.05 or better.

NCX1 Transcript and Protein Levels Are Up-regulated with Hypertrophy-- NCX1 transcript levels are increased in experimental models of heart failure (23, 24) and in patients with heart failure (21, 22). To examine NCX1 expression in compensated hypertrophy, we measured steady-state levels of NCX1 transcript following induction of hypertrophy by pressure overload. Northern blot analysis of NCX1 mRNA in LV myocardium demonstrated a 7-kb hybridization band, corresponding to the expected position for NCX1 (18) (Fig. 2). Steady-state transcript levels increased significantly in hearts hypertrophied by pressure overload (26 ± 4%, n = 2, p < 0.05). To quantify changes in NCX1 transcript levels, a ribonuclease protection assay was employed. Using a probe specific for the N-terminal region of the NCX1 cDNA, a 118-base pair band was protected (Fig. 2C). Hypertrophy was associated with a 75 ± 17% (n = 3, p < 0.05) increase in NCX1 transcript levels.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Steady-state NCX1 transcript levels. Panel A, Northern blot analysis, duplicate samples, of total RNA hybridized with a probe specific for NCX1 from hearts treated as listed. This blot is representative of two independent experiments. Panel B, densitometric quantification of NCX1 transcript bands from hearts treated as listed. Panel C, ribonuclease protection bands from NCX1 transcript, quantified in Panel D after normalization to -actin (asterisk (*) denotes p < 0.05 or better). This pattern of ribonuclease protection assay bands is representative of three independent experiments.

Calcineurin Inhibition Attenuates Up-regulation of NCX Levels-- Recent work has shown that calcineurin is critical to the expression of NCX genes (35). To test the role of calcineurin in hypertrophy-associated increases in NCX1 transcript levels, we exposed mice to cyclosporin (CsA), a specific inhibitor of calcineurin (36, 37). Mice were randomized on the morning following TAB (or sham operation) to receive either CsA (25 mg/kg subcutaneously twice daily) or vehicle.

As previously reported¹ (33), pressure-overloaded hearts exposed to CsA (TAB + CsA) failed to develop significant hypertrophy assayed as either heart mass (HW/BW), myocyte surface area, or myocyte cell capacitance (Fig. 1). Mice exposed to CsA grew, gained weight, and behaved normally. Interestingly, we found that the pressure overload-induced increase in NCX1 transcript levels was partially blocked by CsA (Fig. 2). This suggests that calcineurin is involved in the transcription of the NCX1 gene and/or in the stability of NCX1 mRNA. CsA treatment alone had no effect on NCX transcript levels.

To determine the effects of pressure overload on expression of NCX protein, we measured NCX protein levels by semiquantitative immunoblot analysis (Fig. 3). Left ventricular membrane proteins were prepared from mice subjected to TAB, TAB + CsA, and CsA only, and compared with sham-operated controls. As expected (18), the mature exchanger protein (120 kDa) co-purifies with an active fragment of 70 kDa. Quantification of the 70-kDa protein bands by densitometry revealed a statistically significant increase (71 ± 17%, n = 5, p < 0.05) in NCX steady-state protein levels in hypertrophied myocytes. This increase is similar to the degree of NCX up-regulation reported in models of heart failure (23, 24). Correlating with changes in steady-state transcript levels (Fig. 2), we found that NCX protein levels in cells isolated from hearts treated with CsA were intermediate between non-hypertrophied cells (control or CsA only) and cells with significant hypertrophy (Fig. 3).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Immunoblot analysis of NCX protein. Panel A, immunoblot demonstrating steady-state NCX protein levels in the four treatment conditions listed. This blot is representative of five blots performed from three independent protein preparations. Panel B, immunoblot analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) demonstrating equal protein loading/transfer in each lane. Panel C, densitometric quantification of the 70-kDa bands arbitrarily normalized to sham-operated control. Asterisk (*) denotes p < 0.05 or better.

I_NCX Induced by SR Release of Ca²⁺ Is Diminished in Hypertrophy-- NCX mediates a substantial component of membrane current and is the major mechanism of Ca²⁺ extrusion during diastole. To examine the effects of pressure overload on NCX activity, inward NCX current (I_NCX) was induced by sudden release of Ca²⁺ from SR stores using caffeine (Fig. 4). Whole cell currents were measured in dissociated ventricular myocytes, and I_NCX density was calculated by normalizing total current to cell membrane capacitance. In order to minimize the effects of differential SR Ca²⁺ loading, myocytes were subjected to a train of conditioning impulses prior to caffeine application.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Inward INCX elicited by rapid application of caffeine. Following a train of conditioning voltage pulses to 0 mV, 10 mM caffeine is applied in a microstream to elicit INCX in a control ventricular myocyte (Panel A). Inward current is not elicited by superfusion with bath solution (Panel B) or superfusion with caffeine in the absence of extracellular Na+ (Panel C). Panel D, INCX induced by applying 10 mM caffeine to a hypertrophied myocyte. Panel E, INCX quantified as peak current density (double asterisk (**) denotes p < 0.01) and integrated current density (p = NS).

Given that NCX transcript and protein levels are increased in hypertrophy (Figs. 2 and 3) and heart failure (21-24), our working hypothesis was that I_NCX would be similarly increased. Surprisingly, in myocytes hypertrophied by surgical pressure overload, the caffeine-induced peak inward current transient (0.7 ± 0.1 pA/pF, n = 8/5) was significantly diminished (32%, p < 0.01) compared with control (1.0 ± 0.1 pA/pF, n = 10/5) (Fig. 4). As the caffeine-induced inward current is a complex function of several factors including SR Ca²⁺ stores (see Refs. 38 and 39 for discussions of the interpretation of these measurements), we also estimated the magnitude of SR Ca²⁺ stores by integrating the caffeine-induced inward current. SR Ca²⁺ stores were not statistically significantly different in TAB (0.9 ± 0.2 pA/pF, n = 8/5) versus control myocytes (1.0 ± 0.1 pA/pF, n = 10/5) (Fig. 4E). To confirm our preliminary measurements of diminished I_NCX in hypertrophy, additional independent measures of NCX activity were employed.

I_NCX Measured as Nickel-sensitive Current Is Diminished in Hypertrophy-- Under physiological conditions, NCX mediates inward current during diastole and during the terminal phase of the action potential. During the upstroke of the action potential, however, I_NCX is outward ("reverse mode Ca²⁺ entry"), mediating Ca²⁺ influx at the onset of systole (although the physiological significance of this latter mode is debated (40, 41)). For this reason, we measured inward and outward I_NCX using a voltage-clamp protocol (24), comparing currents in the presence and absence of 5 mM Ni²⁺. Whole cell currents were measured in dissociated ventricular myocytes, and I_NCX density was calculated by normalizing total current to cell membrane capacitance. I_NCX density measured in control myocytes was 1.6 ± 0.1 pA/pF (n = 30/11) at +80 mV and 0.24 ± 0.07 pA/pF (n = 30/11) at 80 mV. Confirming our preliminary findings with caffeine (Fig. 4), myocytes hypertrophied by surgical pressure overload (TAB) exhibited statistically significantly less I_NCX (0.6 ± 0.1 pA/pF at +80 mV, n = 20/8, decreased 61%, p < 0.05) (Fig. 5).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   INCX measured as nickel-sensitive currents. Panel A, voltage protocol used to elicit currents. Membrane currents (Panel B) elicited in the absence and presence of 5 mM NiCl2 with subtracted nickel-sensitive current illustrated in Panel C. I-V relationships (Panel D) for INCX recorded in myocytes from each treatment group listed. Panel E, INCX at +80 mV and 80 mV in myocytes from treatment groups listed. (Double asterisk (**) denotes p < 0.01.)

Exposure to CsA blocked the increase in heart mass and myocyte size (Fig. 1) and blunted the increases in NCX1 transcript (Fig. 2) and protein levels (Fig. 3). To examine the effects of calcineurin inhibition on NCX activity, I_NCX was measured in myocytes isolated from hearts exposed to CsA. I_NCX density in hearts subjected to TAB + CsA (1.25 ± 0.08 pA/pF at +80 mV, n = 12/4) was not significantly different from control mice (Fig. 5). Exposure to CsA alone was similarly not associated with significant changes in I_NCX density (1.35 ± 0.08 pA/pF at +80 mV, n = 18/4) (Fig. 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In recent years, the importance of calcium-regulated pathways controlling transcription in hypertrophy has emerged (reviewed in Ref. 1). In heart failure, control of diastolic Ca²⁺ levels by up-regulated NCX has been proposed to have either adaptive (29, 42) or detrimental (43) effects on cardiac lusitropy (42). This increase in NCX activity shifts the clearance of diastolic Ca²⁺ from uptake into the SR toward trans-sarcolemmal extrusion into the extracellular space. In the long run, this homeostatic response may lead to depletion of intracellular Ca²⁺ stores (44), compromised systolic function (45), and arrhythmogenesis (24, 31, 32) (reviewed in Barry (40)).

As hypertrophy may be a milestone in the pathophysiologic progression of heart failure, we examined NCX expression and function in a model of compensated hypertrophy. Interestingly, even though this model does not exhibit systolic dysfunction or clinical heart failure, we found that NCX1 transcript and protein levels were increased to an extent similar to that reported in heart failure (21-24). We were surprised, however, to find that NCX function, assayed as I_NCX, is decreased in hypertrophy. This contrasts with heart failure where several (21-23, 26, 46), although not all (42) studies, have documented modest increases in exchanger activity (18) and often decreased expression of SR Ca²⁺-ATPase (21, 47-51), reflecting a "fetal" pattern of calcium cycling protein expression (19, 34, 52). I_NCX was recently reported to be down-regulated in a genetic model of modest hypertrophy (53).

The reversal potentials we observe for I_NCX (E_Na-Ca  40 mV) were the same in all 4 treatment groups suggesting similarity of intracellular ionic milieu. This reversal potential is similar to empirically determined values reported by others (26, 38, 54) (although see Ref. 24). Calculations that assume complete dialysis of intracellular ionic conditions with the patch pipette filling solution, however, predict E_Na-Ca = 64 mV. As suggested by others (26, 55, 56), we speculate that NCX, a mediator of substantial ionic flux, alters ionic conditions in subsarcolemmal domains in the vicinity of the exchanger complex. Thus, E_Na-Ca may not be accurately predicted based on measurements of bulk Na⁺ and Ca²⁺ concentrations. In any event, the major point of these electrophysiological experiments stands, viz. I_NCX is diminished in hypertrophy via a mechanism involving calcineurin.

In cerebellar neurons, transcription of NCX1 increases with depolarization whereas that of NCX2 decreases, and depolarization-induced decreases in NCX2 transcription are mediated by calcineurin (35). We report that calcineurin inhibition blunted the increase in NCX1 transcript and protein levels associated with pressure overload, implicating calcineurin in the regulation of transcript synthesis and/or degradation. Together, these data reveal a potential feedback circuit where NCX-mediated Ca²⁺ handling regulates expression of NCX genes (57). In addition, these data implicate the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin in this feedback.

We observed decreased NCX activity (measured as I_NCX) but increased NCX protein and transcript levels, suggesting that NCX activity is regulated in hypertrophy. A substantial fraction of the protein measured by immunoblot may be inactive or localized within the cellular biosynthetic or degradation pathways. Furthermore, NCX activity is controlled by cytosolic concentrations of Ca²⁺, Na⁺, and ATP, intracellular pH (18), and phosphorylation of a large central cytoplasmic domain of the exchanger (58). These data highlight the importance of measuring NCX activity rather than simple steady-state protein or transcript levels (as discussed in Ref. 26). It will be important in future studies to identify post-translational or regulatory mechanisms that control NCX activity in heart disease. Recently, Boateng et al. (59) reported a similar dissociation between NCX protein and activity levels in hypertrophy.

Hearts (and myocytes) exposed to CsA alone were typically 10% smaller than control (Fig. 1) suggesting that basal calcineurin activity exerts tonic control over cell size. As calcineurin acts on several proteins involved in Ca²⁺ handling including the ryanodine and inositol 3-phosphate receptors (60), neuronal NCX (35), the L-type Ca²⁺ channel,¹ and cardiac NCX (this study), it is possible that calcineurin blockade alters intracellular Ca²⁺ metabolism (61-63), resulting in smaller cells.

Cytoplasmic Ca²⁺ is cleared during diastole via 4 mechanisms; two transporters extrude Ca²⁺ into the extracellular space, viz. NCX and a sarcolemmal Ca²⁺-ATPase; two transporters sequester Ca²⁺ within intracellular stores (the SR Ca²⁺-ATPase and a mitochondrial Ca²⁺ uniporter). In rodents, NCX contributes less to diastolic relaxation compared with rabbits and larger mammals because of relatively greater SR Ca²⁺-ATPase activity (64). In heart failure, NCX is often increased (21-26) and SR calcium pump activity decreased (42, 51), the latter reflecting decreased expression of SERCA2A (21, 47-51) and increased inhibition by phospholamban (49, 51, 65, 66). As a result, there is a relative shift toward Ca²⁺ elimination from failing myocytes, which may account for a blunted force-frequency relation (although see Ref. 29). In contrast, decreased I_NCX in hypertrophy may favor Ca²⁺ sequestration in SR stores, thereby augmenting the systolic performance of the hypertrophic heart. This Ca²⁺ overload, however, may be important in the ultimate progression to heart failure.

Concomitant alterations in expression of several Ca²⁺ handling proteins in heart disease make it difficult to predict the functional significance of altered NCX activity in hypertrophy (40, 67). While NCX is the dominant mechanism for sarcolemmal Ca²⁺ extrusion, recent work has revealed a significant role for the Ca²⁺-ATPase and further suggested that the sarcolemmal Ca²⁺-ATPase may be remodeled in response to changes in the expression of NCX (20). Decreased I_NCX is expected to have complex effects on action potential duration (68) as changes in intracellular Ca²⁺ concentration and altered loading of intracellular Ca²⁺ stores will affect NCX activity in vivo. Changes in the expression of NCX result in modifications of SR Ca²⁺ regulation in cardiac muscle (26, 38) and have been implicated in the prolonged action potential duration of failing human myocardium (27, 69). Future work will be required to dissect the complex interplay among these adaptive and maladaptive responses to stress in heart disease.

	ACKNOWLEDGEMENTS

We sincerely thank Kenneth Richardson for technical assistance and Michael Welsh for helpful comments on the manuscript.

	FOOTNOTES

^* This work was supported by grants from the Donald W. Reynolds Cardiovascular Clinical Research Center, Roy J. Carver Charitable Trust, Departent of Veterans Affairs, American Heart Association-Heartland Affiliate, and National Institutes of Health Grant HL-03908.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Contributed equally to the results of this work.

To whom correspondence should be addressed: Cardiovascular Div., University of Iowa College of Medicine, E318GH, UIHC, 200 Hawkins Dr., Iowa City, IA 52242-1081. Tel.: 319-384-9829; Fax: 319-353-6343; E-mail: joseph-hill@uiowa.edu.

Published, JBC Papers in Press, March, 13, 2001, DOI 10.1074/jbc.M100544200

¹ Z. Wang, W. Kutschke, K. E. Richardson, M. Karimi, and J. A. Hill, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: NCX, Na⁺-Ca²⁺ exchanger; TAB, thoracic aortic banding; CsA, cyclosporin ; M, megaohm; SR, sarcoplasmic reticulum; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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