Impaired Cardiac Performance in Heterozygous Mice with a Null
Mutation in the Sarco(endo)plasmic Reticulum
Ca2+-ATPase Isoform 2 (SERCA2) Gene*
Muthu
Periasamy
,
Thomas D.
Reed
,
Lynne H.
Liu§,
Yong
Ji
,
Evgeny
Loukianov
,
Richard J.
Paul¶,
Michelle L.
Nieman¶,
Tara
Riddle§,
John J.
Duffy§,
Thomas
Doetschman§,
John N.
Lorenz¶, and
Gary E.
Shull§
From the § Departments of Molecular Genetics,
Biochemistry and Microbiology,
Medicine, and
¶ Molecular and Cellular Physiology, University of Cincinnati
College of Medicine, Cincinnati, Ohio 45267-0524
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ABSTRACT |
The sarco(endo)plasmic reticulum
Ca2+-ATPase isoform 2 (SERCA2) gene encodes both
SERCA2a, the cardiac sarcoplasmic reticulum Ca2+ pump, and
SERCA2b, which is expressed in all tissues. To gain a better
understanding of the physiological functions of SERCA2, we used gene
targeting to develop a mouse in which the promoter and 5' end of the
gene were eliminated. Mating of heterozygous mutant mice yielded
wild-type and heterozygous offspring; homozygous mutants were not
observed. RNase protection, Western blotting, and biochemical analysis
of heart samples showed that SERCA2 mRNA was reduced by ~45% in
heterozygous mutant hearts and that SERCA2 protein and maximal velocity
of Ca2+ uptake into the sarcoplasmic reticulum were reduced
by ~35%. Measurements of cardiovascular performance via transducers
in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and
negative dP/dt in heterozygous mutants. These
results demonstrate that two functional copies of the SERCA2 gene are
required to maintain normal levels of SERCA2 mRNA, protein, and
Ca2+ sequestering activity, and that the deficit in
Ca2+ sequestering activity due to the loss of one copy of
the SERCA2 gene impairs cardiac contractility and relaxation.
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INTRODUCTION |
The SERCA21 gene encodes
two Ca2+-transporting ATPases, SERCA2a and SERCA2b, which
differ in their C-terminal sequences as a result of alternative
splicing (1-3). SERCA2a is expressed at highest levels in heart, where
it plays a central role in cardiomyocyte Ca2+ handling
required for excitation/contraction coupling (reviewed in Ref. 4).
Contraction is initiated by an increase in Ca2+
concentrations around the myofibrils, which occurs as Ca2+
is released from the SR or enters the cell via channels in the sarcolemma. SERCA2a pumps Ca2+ out of the cytosol and into
the SR, thereby contributing to the low diastolic Ca2+
levels required for relaxation and replenishing Ca2+ stores
needed for the next contraction. SERCA2a serves a similar function in
slow twitch skeletal muscle and is also expressed in some smooth
muscles (5). In contrast to the limited tissue distribution and
organ-specific function of SERCA2a, SERCA2b is expressed in all tissues
and it has been suggested that it plays an essential housekeeping role
(1), although it undoubtedly serves some organ-specific functions as well.
An important role for SERCA2a in cardiac function is well established.
Recent studies have shown that the levels of SERCA2a are decreased in
several animal models of cardiac hypertrophy and human heart failure
(reviewed in Refs. 6 and 7). However, it is currently unknown whether,
and to what extent, the reductions in SERCA2a levels contribute to
altered contractile function. In addition, it is unclear whether there
are homeostatic mechanisms within the cardiac myocyte that are capable
of sensing perturbations in the levels of pump activity and adjusting
its expression accordingly. These are important issues, given the
observed reductions in SERCA2a mRNA and protein expression in the
failing heart, which correlate well with decreased myocardial
Ca2+ transport and contractile function. However, it is
unclear whether the reduction in the level of SERCA2 expression and
activity are a cause or a consequence of impaired cardiac function.
To obtain an animal model that could be used to examine the
physiological functions of SERCA2 in cardiovascular and other tissues,
we used gene targeting to prepare a mouse carrying a null mutation in
the SERCA2 gene. Because homozygous SERCA2 null mutants were not
observed, we examined cardiovascular function and SERCA2a expression
and activity in hearts of heterozygous mutants, which display no
outward manifestations of a disease phenotype. These studies show that
SERCA2 mRNA, protein, and activity levels are reduced in
heterozygous mutants carrying a single functional copy of the SERCA2
gene, and that these reductions have a significant effect on cardiac performance.
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EXPERIMENTAL PROCEDURES |
Preparation of Targeting Construct and Generation of Mutant
Animals--
A genomic clone containing the 5' end of the mouse SERCA2
gene was isolated from a strain 129/SvJ phage library and characterized by restriction mapping and sequence analysis. The targeting vector, pMJKO, was the same as that used previously to prepare a SERCA3 targeting construct (8). Two restriction fragments, a
BglII-SacI fragment containing 2.5 kb of 5'
flanking sequence and terminating 53 base pairs 5' of the transcription
start site and a 2.8-kb SpeI restriction fragment beginning
in exon 3 and terminating in intron 3, were inserted on either side of
the neo gene. The orientation of the neo gene was opposite that of the
SERCA2 gene. Electroporation of ES cells, selection of targeted ES
cells, blastocyst-mediated transgenesis, and mating of chimeric mice
with Black Swiss mice was carried out as described previously (8). DNA
from targeted ES cells and tail biopsies were analyzed by Southern
hybridization using probes from outside the region used to prepare the
construct. The 3' probe was a 2.4-kb EcoRV-SpeI
fragment from intron 3 that began 3.3 kb downstream from exon 3. The 5'
probe was a 360-bp PstI-EcoRI fragment beginning
~3.6 kb upstream of the transcription initiation site.
Genotype Analysis--
Genotyping was performed by Southern blot
analysis of tail DNA using the 3' outside probe or by PCR analysis
using a combination of three primers that amplify both wild-type and
mutant alleles in the same reaction. For PCR analysis the following
primers were used: primer 1 (5'-CGGCCTTCTAGAATTGCCGGCTG-3'),
corresponding to genomic sequences near the 3' end of the
SacI-SpeI fragment that was deleted in the
targeted allele; primer 2 (5'-CTTACTAAAGATATACATGCTGCCAGCAG-3'), complementary to sequences just 3' to the SpeI site in exon
3 that was used to prepare the construct; and primer 3 (5'-CTGACTAGGGGAGGAGTAGAAGG-3'), corresponding to sequences in the
promoter region of the neo cassette. PCR conditions for simultaneous
amplification of wild-type (221-bp product) and mutant (112-bp product)
alleles were: denaturation at 94 °C for 3 min, annealing for 45 s at 52 °C, and extension for 45 s at 72 °C.
Ribonuclease Protection Assays--
DNA fragments used for
generation of riboprobes for mouse SERCA2, phospholamban, and the L32
ribosomal subunit were subcloned into pBluescript. The subcloned
fragments were: for SERCA2, a 451-bp NotI-XhoI
fragment from the mouse SERCA2 gene, which contains 278 bp of exon 1 and 173 bp of intron 1; for phospholamban (9), a 70-bp fragment
corresponding to nucleotides
181 to
111, relative to the initiation
methionine codon; and for L32 (10), a 204-bp fragment corresponding to
nucleotides
18 to +186, relative to the initiation methionine codon.
The pBluescript subclones were linearized, and 32P-labeled
riboprobes were synthesized using T7 RNA polymerase and the
MAXIscriptTM in vitro transcription kit (Ambion,
Inc., Austin, TX). 5 µg of total heart RNA (from 12-14-week-old
mice, n = 7 for each genotype) were analyzed using the
RPA IITM ribonuclease protection assay kit (Ambion, Inc.),
with all three probes included in each reaction. The protected
fragments were separated by electrophoresis in a 5% denaturing
polyacrylamide gel, analyzed by autoradiography, and quantitated by
PhosphorImager analysis (Molecular Dynamics, Wayzata, MN). Levels of
SERCA2 and phospholamban mRNAs were determined relative to L32 mRNA.
Western Blot Analysis--
SERCA2a levels in cardiac homogenates
from 15-16-week-old mice (n = 3 males and 3 females
for each genotype) were determined by Western blot analysis,
essentially as described previously (11, 12). Proteins were separated
in SDS-polyacrylamide gels, transferred to nitrocellulose membranes,
and probed with a polyclonal anti-SERCA2a antibody (13) or a monoclonal
anti-
-actin antibody (Sigma). Binding of the primary antibody was
detected by peroxidase-conjugated secondary antibody and enhanced
chemiluminescence (Kirkegaard and Perry Laboratories, Gaithersburg,
MD). The signal obtained for each heterozygous mutant sample (5 µg of
protein; duplicate lanes for each sample) was compared with a standard
curve prepared by plotting the signal intensities obtained with
increasing amounts of wild-type cardiac homogenates (1-5 µg of
protein from pooled samples of 6 wild-type mice). Quantitation of the
signals was performed by densitometry using a UMAX Astra 1200 s
scanner and analyzed using NIH Image version 6.1 software.
Ca2+ Uptake Assays--
Hearts were removed from
15-16-week-old wild-type and heterozygous mutant mice, rinsed in
phosphate-buffered saline, and stored in liquid nitrogen. Preparation
of whole heart homogenates and analysis of both the maximum rate of
Ca2+ uptake and the Ca2+ dependence of
Ca2+ uptake were performed as described by Luo et
al. (14). The use of whole heart homogenates for the analysis of
Ca2+ uptake by SR vesicles has been discussed and validated
in previous studies (14-17). Duplicates of each sample
(n = 3 males and 3 females of each genotype) were
analyzed and the values presented are means ± S.E. The
Vmax and Ca2+ concentration required
for half-maximal activation (K0.5) were calculated by non-linear regression analysis using MicroCal Origin software. Statistical analyses were performed by unpaired two-tailed Student's t test.
Analysis of Left Ventricular Function and Blood
Pressure--
Anesthesia, surgical procedures, and collection of data
for analysis of cardiac performance in the intact closed-chest mouse were carried out essentially as described previously (18, 19). 12-14-week-old male wild-type (n = 11) and
heterozygous mutant (n = 10) mice were analyzed. Each
anesthetized mouse was placed on a thermally controlled surgical table,
and a tracheotomy was performed. The right femoral artery was
cannulated and connected to a COBE CDXIII pressure transducer (COBE
Cardiovascular, Arvada, CO) for measurement of arterial blood pressure.
The right femoral vein was cannulated and used for infusion of the
-adrenergic agonist, dobutamine (Abbott Laboratories, North Chicago,
IL) or the
-adrenergic antagonist, propranolol (Sigma). The right
carotid artery was cannulated with a Millar Mikro-Tip transducer with an outer diameter of 0.47 mm (model SPR-612, Millar Instruments, Houston, TX), which was then advanced into the left ventricle. After
the animal had stabilized for ~30 min, measurements of ventricular function in the absence of stimulation were performed. Cardiovascular performance in response to
-adrenergic stimulation was examined by
infusing increasing concentrations of dobutamine (1-32 ng/min/g of
body weight) over a 3-min period, with peak responses measured during
the final 30 s of the infusion period. At the end of each experiment, a bolus of propranolol (100 ng/g of body weight) was infused in order to reevaluate cardiac function at the end of each
experiment in the absence of
-adrenergic stimulation. Pressure signals from both the COBE and Millar transducers were recorded using a
MacLab 4/s data acquisition system (AD Instruments, Milford, MA) and a
Macintosh 7100/80 computer as described previously (18, 19). The
analysis software permits direct determination of arterial systolic and diastolic pressure, mean arterial pressure, heart rate,
left ventricular systolic pressure, developed pressure, and both
positive (dP/dtmax) and negative
(dP/dtmin)
dP/dt. Data were analyzed using a mixed,
two-factor analysis of variance with repeated measures on the second
factor. When necessary, post hoc comparisons were
performed by single degree-of-freedom contrasts.
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RESULTS |
Preparation of Mutant Mice and Gross Phenotype--
To
introduce a null mutation into the SERCA2 gene, we used a targeting
construct in which part of the promoter, the transcription initiation
site, the first two exons, and part of the third exon were deleted
(Fig. 1A). We anticipated that
homologous recombination of this construct with the SERCA2 gene would
eliminate transcription of the gene, thereby allowing unambiguous
determination of mRNA and protein levels produced by the single
remaining functional copy of the SERCA2 gene. After electroporation of
ES cells, 3 of 105 colonies that survived the selection procedure were
found to be correctly targeted when analyzed by Southern blot
hybridization. Fifteen male chimeric mice were obtained by
blastocyst-mediated transgenesis. When bred with Black Swiss females,
six of these chimeras yielded offspring carrying the mutant allele.

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Fig. 1.
SERCA2 gene targeting strategy and Southern
blot analysis. A, targeting strategy. Top,
restriction map and organization of the 5' end of the wild-type gene.
Exons 1-3 and location of the 5' and 3' probes used for Southern blot
analysis are indicated. Middle, targeting construct in which
1.5 kb of SERCA2 genomic sequence, containing exons 1 and 2 and part of
exon 3, was replaced by the neo gene. The herpes simplex virus
thymidine kinase gene was placed 5' to the SERCA2 genomic sequence.
Bottom, targeted SERCA2 allele, with BamHI
restriction fragments identified by 5' and 3' probes indicated
below. Restriction enzyme sites: B,
BamHI; Bg, BglII,
Sp, SpeI; S, SacI.
B, Southern blot analysis of tail DNA from offspring of
heterozygous matings. The samples were digested with BamHI
and hybridized with the 5' and 3' probes, which identify a 14.3-kb
fragment in the wild-type allele and 8.1- and 6.3-kb fragments,
respectively, in the mutant allele. Only wild-type (+/+) and
heterozygous (+/ ) mice were observed.
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SERCA2 heterozygous mice were mated and the genotypes of offspring were
determined by Southern blot analysis (Fig. 1B). Among 116 offspring, 41 were wild-type (35%) and 75 (65%) were heterozygous, with a similar percentage of males and females (53% and 47%,
respectively). Homozygous mutants were not observed, and the ratio of
wild-type:heterozygous offspring was very close to the 1:2 Mendelian
ratio that would be expected if loss of homozygous mutants was
occurring during embryonic development. Heterozygous mutants were
viable, appeared healthy, and exhibited no outward manifestations of a
disease phenotype. There was no evidence of cardiac hypertrophy in the mutant mice. The heart weight/body weight (mg/g) ratios of the 15-16-week-old mice used below for analysis of SERCA2a levels and
Ca2+ uptake activity in heart was 4.33 ± 0.18 for
wild-type mice and 4.33 ± 0.16 for heterozygous mutants.
SERCA2 mRNA Levels Are Reduced in Hearts of Heterozygous Mutant
Mice--
To determine whether the loss of one copy of the SERCA2 gene
causes a reduction in the levels of SERCA2 and phospholamban mRNAs,
RNase protection analysis was performed. The SERCA2 probe does not
distinguish between SERCA2a and SERCA2b mRNAs. However, because
previous nuclease protection studies have shown that ~95% of the
SERCA2 mRNA in heart encodes SERCA2a (5), only a small fraction of
the mRNA detected in these experiments would be due to SERCA2b. As
shown in Fig. 2A, there
appeared to be a marked reduction of SERCA2 mRNA, but not
phospholamban mRNA, in the hearts of heterozygous mutants.
Quantitation of the hybridization signal intensities by PhosphorImager
analysis and normalization with an internal control (Fig.
2B) showed that SERCA2 mRNA levels were reduced to
55 ± 2% of wild-type levels, whereas phospholamban mRNA
levels were the same in both genotypes.

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Fig. 2.
RNase protection analysis of SERCA2 and
phospholamban mRNAs in wild-type and heterozygous mutant
hearts. A, total heart RNA (5 µg/lane) was hybridized
with riboprobes for SERCA2, phospholamban (PLB), and the L32
ribosomal subunit. The samples were then treated with RNase A and RNase
T1, separated by polyacrylamide gel electrophoresis, and visualized by
autoradiography. Each pair of lanes, L-1-L-7, contains
samples from 12-14-week-old, age- and sex- matched wild-type
(W) and heterozygous mutant (H) mice;
L-3 and L-5 were female pairs, and the others
were male. B, the autoradiogram in panel A was
examined by PhosphorImager analysis and the hybridization signals for
SERCA2 and phospholamban mRNAs in hearts from wild-type (+/+) and
heterozygous (+/ ) mice were quantitated relative to that of the L32
ribosomal subunit. The data are expressed as means ± S.E. *,
significantly different (p < 0.001) from wild-type as
determined by Student's t test.
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SERCA2a Protein and Ca2+ Uptake Activity Are Reduced in
Hearts of Heterozygous Mutant Mice--
Western blot analysis of whole
heart homogenates was performed using an anti-SERCA2a antibody, and the
levels of
-actin were analyzed as a control. SERCA2a in heterozygous
mutant hearts was reduced to 63 ± 6% of wild-type levels
(p < 0.05), whereas
-actin levels were the same in
mutant and wild-type hearts (Fig. 3).

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Fig. 3.
Western blot analysis of SERCA2 protein
levels in heart. Increasing amounts (1-5 µg) of pooled
(n = 6) wild-type cardiac homogenates (which served as
a standard curve) and duplicate samples (5 µg) of individual
heterozygous mutant (HET) cardiac homogenates were separated by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose
membranes, and probed with anti-SERCA2 and anti- -sarcomeric actin
antibodies. The left panel shows a representative blot of
one of the six heterozygous mutant samples that were analyzed. The
bar graph shows the mean ± S.E. of six individual
heterozygous hearts.
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Analyses of both the maximum rate and Ca2+ dependence of
Ca2+ uptake activity were performed using the same cardiac
homogenates used for Western blot analysis. The maximum velocity of
Ca2+ uptake (Fig.
4A) was significantly reduced
(p < 0.01) in cardiac samples from heterozygous
mutants (to ~66% of wild-type levels), with a
Vmax of 46.5 ± 2.5 and 30.6 ± 1.7 nmol/mg/min in wild-type and heterozygous hearts, respectively. In
contrast, the Ca2+ dependence of Ca2+ uptake
activity (Fig. 4B) was essentially the same in both sets of samples,
with a K0.5 of 0.238 ± 0.009 µM in wild-type hearts and 0.242 ± 0.003 µM in heterozygous hearts.

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Fig. 4.
Ca2+ uptake by sarcoplasmic
reticulum in cardiac homogenates from wild-type and heterozygous mutant
mice. Ca2+ uptake assays were performed using whole
heart homogenates from 15-16-week-old mice (n = 6 for
each genotype). A, Ca2+ uptake rates (nmol of
Ca2+/mg of protein/min) at varying Ca2+
concentrations. B, Ca2+ dependence of
Ca2+ uptake; mean values at each Ca2+
concentration are from the experiment shown in panel A and
are expressed as a percentage of Ca2+ uptake rates at
pCa 5.5.
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Cardiovascular Function in Wild-type and Heterozygous Mutant
Mice--
In these experiments, measurements of cardiac function were
recorded using a micro-tip transducer that was placed in the left ventricle of anesthetized mice via the right carotid artery. Blood pressure was recorded using a transducer that was inserted in the right
femoral artery, and dobutamine, a
-adrenergic receptor agonist, was
infused through a catheter in the right femoral vein. Representative
tracings of arterial blood pressure, left ventricular systolic
pressure, and left ventricular dP/dt under
non-stimulated conditions are shown in Fig.
5.

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Fig. 5.
Representative tracings of blood pressure,
left ventricular pressure, and the first derivative of the left
ventricular pressure wave. Pressure measurements were obtained
from anesthetized wild-type and heterozygous mutant (knockout) mice via
a COBE CDXIII transducer in the right femoral artery and a Millar
Mikro-Tip transducer in the left ventricle. Typical recordings of
femoral artery blood pressure, the left ventricular pressure wave, and
the first derivative of the left ventricular pressure wave
(dP/dt) from unstimulated wild-type
(left) and heterozygous mutant (right) mice are
shown. The bar in the left panel indicates the
time scale.
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To determine whether there were any differences in cardiovascular
function between the two genotypes under control conditions and at
increasing levels of
-adrenergic stimulation, dobutamine dose-response relationships were analyzed. Under non-stimulated conditions and at the lower doses of dobutamine, the mean value for
heart rate rate was slightly reduced in heterozygous mutants (Fig.
6A); however, the differences
were not statistically significant. Mean arterial blood pressure (Fig.
6B) and left ventricular systolic pressure (Fig.
6C) were significantly lower in heterozygous mutants than in
wild-type mice under non-stimulated conditions and at the two lower
doses of dobutamine, but not at higher levels of
-adrenergic
stimulation. The absolute values of both positive and negative
dP/dt were significantly lower in heterozygous
mutants under non-stimulated conditions and at all levels of
-adrenergic stimulation (Fig. 7). A
significant dobutamine dose response (p < 0.001) for
each of these measurements of cardiac performance was observed in both
wild-type and heterozygous mutant mice.

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Fig. 6.
Dobutamine dose-response relationships for
heart rate, mean arterial pressure, and left ventricular systolic
pressure. Heart rate (A), mean arterial pressure
(B), and systolic left ventricular pressure (C)
of 12-14-week-old wild-type and heterozygous mutant (knockout) male
mice were examined under control conditions and in response to
increasing levels of -adrenergic stimulation (n = 11 wild-type and 10 mutant mice). Values were measured during the final
30 s of each 3 min period in which increasing concentrations of
the -adrenergic agonist, dobutamine, were infused. BPM,
beats per minute; MAP, mean arterial pressure;
LVPsys, left ventricular systolic pressure. *,
mutants significantly different (p < 0.05) from
wild-type at corresponding dose levels, as determined by analysis of
variance.
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Fig. 7.
Dobutamine dose-response relationships for
minimum and maximum dP/dt. The
first derivative of the left ventricular pressure wave of the same
wild-type and heterozygous mutant (knockout) mice analyzed in Fig. 6
was determined at all levels of -adrenergic stimulation.
A, positive dP/dt
(dP/dtmax). B, negative
dP/dt
(dP/dtmin). , significantly
different from wild-type at all dose levels (p < 0.01), as determined by analysis of variance.
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DISCUSSION |
Our primary objective was to analyze the relationship between
cardiac performance and the levels of SERCA2 expression and activity in
heart. Because we anticipated that homozygous mutants would not survive
and that it would be necessary to perform our studies using
heterozygotes, a major concern, and an important issue in its own
right, was whether SERCA2 levels in heart were critically dependent on
the presence of two functional copies of the SERCA2 gene. Homozygous
mutants were not observed, demonstrating that one or both variants of
SERCA2 serve functions that are essential for life, and that other
pumps cannot provide sufficient compensation to rescue the null mutant.
The 2:1 genotype ratio of heterozygous and wild-type offspring
indicated that embryonic development of heterozygous mutants was
normal. Both young and adult heterozygous mutant mice appeared healthy,
grew normally, and were indistinguishable from wild-type mice in both
appearance and behavior. To gain a better understanding of the in
vivo functions of SERCA2, we conducted biochemical and
physiological analyses of heterozygous mutants, with the expectation
that SERCA2 mRNA, protein, and activity levels would be reduced and
that this would produce an informative cardiac phenotype.
There are now many examples of mouse knockout models in which mRNA
and protein levels are reduced in heterozygous mutants, with a
corresponding alteration of physiological function. A particularly relevant example is the phospholamban-deficient mouse (14, 19, 20), in
which heterozygotes display an ~50% decrease in phospholamban mRNA and protein levels and an increase in cardiac contractility and relaxation. Nevertheless, for proteins that play a central role in
critical physiological processes, such as the cardiac SR
Ca2+ pump in excitation-contraction coupling, it is not
unreasonable to anticipate that homeostatic mechanisms within the cell
might sense perturbations and adjust its levels of expression. In fact, recent studies have shown that transgenic mice with a 2.6-fold increase
in SERCA2a mRNA in heart have only a 20% increase in SERCA2a
protein (21) and that transgenic overexpression of SERCA1 in mouse
heart leads to a ~50% reduction in the levels of endogenous SERCA2a
(12). The results of those studies indicate the existence of mechanisms
that can limit SERCA2a expression in cardiac myocytes when
Ca2+ pump activity is greater than normal. The development
of the SERCA2-deficient mouse has allowed an investigation of whether the amount of SERCA2a mRNA and protein produced from a single copy
of the gene can be up-regulated in response to a deficiency in
Ca2+ pump activity.
To allow unambiguous determination of the levels of mRNA and
protein produced from the remaining functional copy of the SERCA2 gene
in heterozygous mutants, we prepared the model using a targeting construct in which 1.5 kb from the 5' end of the gene was replaced with
the neomycin resistance gene. Homologous recombination between the
construct and the SERCA2 gene eliminated the TATA box, both the
transcription and translation initiation sites, and sequences encoding
the N-terminal cytoplasmic domain of the protein. RNase protection
analysis revealed that steady-state levels of SERCA2 mRNA in hearts
of heterozygous mice were ~55% of wild-type levels. These data
indicate that the expression of normal levels of SERCA2 mRNA in
heart is dependent on the presence of two functional copies of the
SERCA2 gene, and that transcriptional up-regulation of the SERCA2 gene
in response to a deficit in Ca2+ sequestering activity, if
it occurs at all, is very limited. SERCA2a protein, however, was
~65% of wild-type levels, indicating an ~30% increase in the
amount of protein produced from the single functional copy of the gene
in heterozygous mutants. The results of these experiments show that
both copies of the SERCA2 gene are required to maintain normal SERCA2
mRNA and protein levels, although some compensatory up-regulation
of SR Ca2+ pump expression from the remaining gene does
occur in heterozygotes. The mechanisms underlying the modest
compensatory up-regulation are unclear, but may occur at several
levels, including increased transcription and/or mRNA stability and
increased translation and/or a reduction in the rate of SERCA2a protein degradation.
When compared with the levels in wild-type mice, the
Vmax of Ca2+ uptake activity and
SERCA2a protein in heart samples from heterozygous mutants were reduced
to similar extents, confirming that there is a direct relationship
between SERCA2a levels and maximum rates of Ca2+
sequestration. The Ca2+ dependence of Ca2+
uptake activity was the same in mutant and wild-type mice, suggesting that under basal conditions there is no compensatory up-regulation of
pump activity in mutant hearts via an increased affinity for Ca2+. Such compensation could occur, for example, by
phosphorylation of phospholamban (which represses cardiac contractility
and relaxation when it is in its non-phosphorylated state), thereby
relieving its inhibition of pump activity (14, 19, 20). The lack of an
alteration in Ca2+ dependence of Ca2+ uptake
activity was somewhat surprising, as phospholamban mRNA levels were
the same in wild-type and heterozygous mutant hearts. If the levels of
phospholamban protein and its phosphorylation state are unchanged in
heterozygous hearts, which were not examined in this study, then the
increase in phospholamban/SERCA2 ratios in heterozygous mutants would
be expected to cause a reduction in the affinity of the pump for
Ca2+, as observed in hearts of transgenic mice in which
phospholamban was overexpressed (22). Additional studies will be needed
to determine whether phospholamban protein levels and its
phosphorylation state under basal conditions are altered in
heterozygous mutant hearts.
A clear cardiovascular phenotype was observed in heterozygous mutants,
demonstrating that the ~35% reduction in SERCA2a levels and
Ca2+ uptake activity resulting from the loss of one
functional copy of the SERCA2 gene has a significant physiological
effect. Under basal conditions, mean arterial blood pressure and left
ventricular systolic pressure were significantly lower in heterozygotes
than in wild-type mice. However, during maximum
-adrenergic
stimulation, a condition in which inhibition of the pump by
phospholamban is relieved (14, 19, 20), both genotypes exhibited
similar arterial and left ventricular pressures. The reduced levels of SERCA2a protein and activity would be expected to lead to both a
reduction in SR Ca2+ stores, which would decrease the
amount of Ca2+ available for excitation-contraction
coupling, and a reduction in the rate of Ca2+
sequestration, which would reduce the relaxation rate. Consistent with
this expectation, the absolute values of both maximum and minimum
dP/dt were significantly lower in mutant mice
under basal conditions and at all levels of
-adrenergic stimulation.
These results, along with those of previous studies showing that
overexpression of SERCA2a (21) or SERCA1 (12) in transgenic mouse
hearts increases cardiac contractility and relaxation, demonstrate that SR Ca2+ pump levels are a major determinant of cardiac
muscle contractility and relaxation under both basal and stimulated conditions.
Studies of human heart disease have revealed reductions in the levels
of SERCA2a mRNA, protein, and activity (6, 7, 23-28), and similar
observations have been made in animal models of heart disease (29-33).
In the failing human myocardium, Ca2+ transients are
markedly prolonged in both Ca2+ release and uptake phases
(34), and there is evidence that the reduction in SERCA2a levels is
involved in the altered force-frequency relationship (27). On the basis
of these and other studies, it has been suggested that SR dysfunction
resulting from decreased levels of the SR Ca2+ pump
contributes to the pathogenesis of heart disease (6, 23-25). The
experiments described here have shown a direct relationship between
SERCA2a levels and Ca2+ uptake activity, and have also
shown that a reduction in pump activity impairs cardiac contractility
and relaxation. However, the 12-16-week-old mutant mice that we
analyzed appeared to be healthy, and overt evidence of heart disease,
such as cardiac hypertrophy, was not observed. This indicates that an
~35% deficit in Ca2+ sequestering activity, such as that
resulting from the loss of one copy of the SERCA2 gene, is not a
sufficient perturbation to cause heart disease in the relatively young
adult mice analyzed here. However, it remains to be determined whether
heterozygous mutants may be more prone to heart disease as they age or
when subjected to rigorous exercise or other experimental protocols.
 |
ACKNOWLEDGEMENTS |
We thank Evangelia Kranias for critical
review of the manuscript and David Hellard for expert technical assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL41496, HL52318, DK50594, and TG HL07382.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.
To whom correspondence should be addressed: Dept. of Molecular
Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Bethesda Ave., ML 524, Cincinnati, OH 45267-0524. Tel.: 513-558-0056; Fax: 513-558-1885; E-mail:
shullge{at}ucmail.uc.edu.
The abbreviations used are:
SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SR, sarcoplasmic
reticulum; neo, neomycin resistance; kb, kilobase(s); bp, base pair(s); dP/dtmin, minimum
dP/dt; dP/dtmax, maximum
dP/dt; PCR, polymerase chain reaction.
 |
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