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
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ABSTRACT |
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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.
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 (ICa,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 (INCX) using 2 independent means.
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 Inward INCX Induced by Caffeine-mediated SR
Ca2+ Release--
Myocytes were held at INCX Measured as Ni2+-sensitive
Current--
Myocytes were depolarized to 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 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.
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).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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%.
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.
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, INCX 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.
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Heart mass, myocyte surface area, and whole cell capacitance for each
treatment group in this study
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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.
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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).
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INCX 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 (INCX) was
induced by sudden release of Ca2+ from SR stores using
caffeine (Fig. 4). Whole cell currents
were measured in dissociated ventricular myocytes, and
INCX 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.
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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 INCX 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 INCX in hypertrophy, additional independent measures of NCX activity were employed.
INCX 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,
INCX 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 INCX 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 INCX
density was calculated by normalizing total current to cell membrane
capacitance. INCX 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 INCX (0.6 ± 0.1 pA/pF at +80 mV, n = 20/8, decreased 61%, p < 0.05) (Fig. 5).
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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, INCX was measured in
myocytes isolated from hearts exposed to CsA.
INCX 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
INCX density (1.35 ± 0.08 pA/pF at +80 mV,
n = 18/4) (Fig. 5).
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DISCUSSION |
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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 INCX, 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). INCX was recently reported to be down-regulated in a genetic model of modest hypertrophy (53).
The reversal potentials we observe for INCX
(ENa-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
ENa-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, ENa-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.
INCX 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 INCX) 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 INCX 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
INCX 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.
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ACKNOWLEDGEMENTS |
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We sincerely thank Kenneth Richardson for technical assistance and Michael Welsh for helpful comments on the manuscript.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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