Vol. 279, Issue 3, H1307-H1318, September 2000
Dilated cardiomyopathy in transgenic mice expressing a mutant
A subunit of protein phosphatase 2A
Neil
Brewis1,*,
Kim
Ohst1,*,
Katherine
Fields1,
Antonio
Rapacciuolo3,
Danny
Chou1,
Colin
Bloor1,
Wolfgang
Dillmann2,
Howard
Rockman3, and
Gernot
Walter1
1 Department of Pathology and 2 Department of
Medicine, University of California San Diego, La Jolla, California
92093; and 3 Department of Medicine, Duke University Medical
Center, Durham, North Carolina 27710
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ABSTRACT |
The protein phosphatase 2A (PP2A)
holoenzyme consists of a catalytic subunit, C, and two regulatory
subunits, A and B. The PP2A core enzyme is composed of subunits A and
C. Both the holoenzyme and the core enzyme are similarly abundant in
heart tissue. Transgenic mice were generated expressing high levels of
a dominant negative mutant of the A subunit (A
5) in the heart,
skeletal muscle, and smooth muscle that competes with the endogenous A
subunit for binding the C subunit but does not bind B subunits. We
found that the ratio of core enzyme to holoenzyme was increased in
A
5-expressing hearts. Importantly, already at day 1 after
birth, A
5-transgenic mice had an increased heart weight-to-body
weight ratio that persisted throughout life. Echocardiographic analysis
of A
5-transgenic hearts revealed increased end-diastolic and
end-systolic dimensions and decreased fractional shortening. In
addition, the thickness of the septum and of the left ventricular
posterior wall was significantly reduced. On the basis of these
findings, we consider the heart phenotype of A
5-transgenic mice to
be a form of dilated cardiomyopathy that frequently leads to premature death.
protein phosphatase 2A holoenzyme; protein phosphatase 2A core
enzyme; heart weight-to-body weight ratio; muscle-specific gene
expression
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INTRODUCTION |
THE FUNCTIONS
OF SERINE/THREONINE KINASES in cardiac gene expression and
hypertrophy have been studied extensively, in particular those of
protein kinase C, protein kinase A, and members of the mitogen-activated protein (MAP) kinase pathways (37).
However, the functions of serine/threonine phosphatases in these
processes are less well understood. Recently, calcineurin (protein
phosphatase 2B) has been shown to play a role in the induction of
cardiac gene expression and hypertrophy through a mechanism that
involves dephosphorylation of the transcription factor NF-AT3 (nuclear factor of activated T cells-3) (24, 40). Other
serine/threonine phosphatases are also involved in cardiac function.
This became apparent when it was discovered that okadaic acid, an
inhibitor of protein phosphatase 1, protein phosphatase 2A (PP2A), and
related serine/threonine phosphatases, induces a shortening in
contraction time of isolated cardiac muscle (13). The
effects of okadaic acid are similar to those induced by
-adrenergic
receptor agonists. It has been suggested that they are mediated by
increased phosphorylation of the cardiac-specific proteins
phospholamban and troponin I (27, 39). Because okadaic
acid inhibits several serine/threonine phosphatases (6,
7), it is difficult to correlate the biological effects of
okadaic acid with the inhibition of a specific phosphatase. However,
there is biochemical evidence that protein phosphatase 1 is mainly
responsible for the dephosphorylation of phospholamban (18), whereas dephosphorylation of troponin I might be
carried out by PP2A (25). PP2A is also involved in
regulating ATP-sensitive potassium channels, as demonstrated by single
channel recordings on inside-out membrane patches from ventricular
myocytes (16). Furthermore, PP2A may modulate MAP kinase
pathways, which are activated in response to treatment with phorbol
ester, endothelin-1, or phenylephrine (3, 30, 43). As
shown for cardiomyocytes (4) and other systems (38,
45), PP2A can either enhance or reduce signaling through the MAP
kinase pathway.
PP2A exists in cells as two forms: the heterodimeric core enzyme,
composed of the catalytic subunit C and the regulatory subunit A, and
the heterotrimeric holoenzyme, composed of the core enzyme and a second
regulatory subunit, subunit B. The A subunit polypeptide consists of 15 nonidentical repeats. The B subunit binds to repeats 1-10, and the
C subunit binds to repeats 11-15 of the A subunit (Fig.
1, see Ref. 26 for review). The
core enzymes and holoenzymes are expressed in approximately equal
quantities (15), and they differ in substrate specificity
(26). B subunits fall into three families, designated B
(10, 20, 29, 46), B' (or B56) (8, 22, 23, 41,
42), and B'' (5), which are unrelated by primary
sequence. The B' family consists of numerous isoforms and splice
variants that determine the specificity and intracellular localization
of the holoenzyme (22, 42). In addition, there is
tissue-specific expression of B' subunits (8, 22, 23, 42).
Importantly, B56
- and B56
-subunits are preferentially expressed
in heart and skeletal muscle, suggesting that they direct the
holoenzyme to a specific subcellular location in these tissues and/or
toward a specific substrate.

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Fig. 1.
Model of protein phosphatase 2A (PP2A) and location of
epitopes on the A subunit. The B subunits and T antigens bind to the
NH2-terminus, and the C subunit binds to the COOH-terminus
of the A subunit (34, 35). The regions where the epitopes
of the 6F9 and 5H4 antibodies are located are indicated by horizontal
bars. Figure modified from Ref. 15.
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We have previously demonstrated that expression of the A
5 subunit, a
NH2-terminal mutant of the A subunit of PP2A with a deletion of repeat 5 [which binds the catalytic C subunit but none of
the regulatory B subunits (Fig. 1)], causes an increase in core
enzymes and a decrease in holoenzymes in tissue culture cells
(32). As a result of this change in the core
enzyme-to-holoenzyme ratio, the Tat protein-stimulated transcription
from the human immunodeficiency virus-1 (HIV-1) long-terminal repeat
and HIV-1 virus production are strongly inhibited. On the basis of this result, we asked whether expression of the mutant A
5 subunit in
animals would alter PP2A activity and provide information about the
function of PP2A. Here we show that mice expressing the mutant A
5
subunit under the combined control of the chicken
-actin promoter
and the cytomegalovirus (CMV) enhancer, yielding high gene expression
in muscle tissue, have an increased heart weight-to-body weight ratio
early in life and develop a form of dilated cardiomyopathy.
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MATERIALS AND METHODS |
DNA construction.
PCR was used to engineer a sequence encoding the nine amino acid
epitope tag EEEEYMPME (EE) at the 3' end of the human A subunit-cDNA open reading frame (33). The construct was subcloned into
the ecdysone-responsive expression plasmid pIND (Invitrogen) using EcoR I and Xho I. This vector was termed
pINDA
5EE. To create an EcoR I site at the end
of the A
5EE sequence, the vector pINDA
5EE
was digested with Xho I, treated with Klenow, and blunt-end
ligated to an EcoR I linker. The EcoR
I-EcoR I insert of A
5EE was ligated into the
EcoR I site of the pCAGGS vector (28) resulting
in pCAGGSA
5EE (Fig. 2).
The pCAGGS vector containing the CMV enhancer, the chicken
-actin
promoter, and the rabbit
-globin polyadenylation signal was used
because this vector yields high gene expression in muscle tissue
(19).

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Fig. 2.
Construct containing mutant A 5EE subunit (the
A subunit with repeat 5 deleted) used to generate transgenic mice. This
construct was generated by inserting the glutamic acid-glutamic acid
epitope (EE)-tagged A 5 (A 5EE) mutant into the vector
pCAGGS, which contains the cytomegalovirus (CMV) enhancer, the chicken
-actin promoter, and the rabbit -globin polyadenylation signal.
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Generating transgenic mice.
The vector pCAGGSA
5EE was cut with Ssp I and
BamH I. The 3.7-kb fragment (shown in Fig. 2) was purified
from the gel using a Qiagen kit and then concentrated with an Elutip
(Schleicher & Schuell). The DNA was ethanol precipitated and dissolved
in 5 mM Tris (pH 7.5) and 0.1 mM EDTA at a concentration of 2.0 µg/ml.
To develop transgenic mice, pronuclei of fertilized eggs from CB6
F1 mice were microinjected with the 3.7-kb fragment at a concentration of 2.0 µg/ml. Surviving embryos were transferred into
the oviducts of pseudopregnant ICR mice. Litters were delivered after
19-20 days of gestation. After the mice were 3 wk old, tail clippings were taken, and the DNA was extracted and digested with Apa I. The digested DNA (15 µg) was loaded on a 0.7%
agarose gel in Tris·borate·EDTA and transferred to Hybond-N nylon
membrane (Amersham). The blot was then hybridized with a
32P-labeled probe. The probe used was the entire injected
fragment from Ssp I to BamH I. Of the 40 offspring born, 8 had integrated the transgene and were considered the
founders. The founders were bred with mice of the same strain, and the
resultant litters were also analyzed by Southern blotting as described
above. Transgenic lines were established with the two founders that
expressed the protein in their tissue (lines I1 and I4) in addition to
being positive by Southern blotting. The approximate transgene copy numbers in the I1 and I4 lines, as determined by Southern blots, were 7 and 25, respectively (data not shown). Heterozygous and homozygous I1-
and I4-transgenic mice were distinguished by Southern blotting.
Analyzing mouse organs for expression of A
5EE
protein.
The mice were euthanized by CO2 inhalation for
30 s. The different tissues were removed, frozen on dry ice, and
weighed. Protein was extracted by grinding the defrosting tissue with a
pestle and then grinding with 10 µl of SDS sample buffer [2% SDS,
5%
-mercaptoethanol, 5% glycerol, 10 mM Tris (pH 6.8), 10 mM
dithiothreitol, and 0.025% bromophenol blue] per milligram of
tissue. An 18-gauge needle was used to further dissociate the tissue,
and the lysate was then boiled for 5 min, diluted, and boiled again.
For protein determination, aliquots of solubilized tissues were
precipitated with trichloroacetic acid. Protein (10-20 µg) was
analyzed on a 10% acrylamide gel and transferred to a polyvinylidene
difluoride membrane (Immobilon-P, Millipore). Western blotting was done
using the 6G3 antibody (15).
Heart weight-to-body weight ratios and histology.
Mice were euthanized using CO2 inhalation as described in
Analyzing mouse organs for expression of A
5EE
protein, and the mice were weighed. Their hearts were removed, blotted on filter paper, and weighed. For histology, hearts were fixed
in 10% buffered Formalin for at least 48 h, cut longitudinally or
transversely, dehydrated, and infiltrated with paraffin. Sections (5 µm) were cut after embedding the hearts in paraffin. Sections were
floated onto slides and dried overnight. The sections were stained with
hematoxylin and eosin or with Masson trichrome stain.
Immunoprecipitation of PP2A.
Mice were euthanized by 30 s of CO2 inhalation, and
their hearts were removed and weighed. Sections of 100-150 mg of
heart were frozen in liquid nitrogen and ground with a mortar and
pestle and lysis buffer [50 mM Tris · HCl (pH 8.0), 150 mM
NaCl, 3 mM MgCl2, 1 mM Pefabloc SC (Boehringer), 50 µM
leupeptin (Sigma), 0.2 mg/ml soybean trypsin inhibitor (Calbiochem), 1 µg/ml aprotinin, and 1 mM dithiothreitol]. The lysate was spun at
16,000× g at 4oC, and the supernatant protein
concentration was determined with Pierce's Coomassie Plus Assay. Over
95% of PP2A was solubilized under these conditions, as determined by
Western blotting. Addition of 0.5% Triton X-100 to the lysis buffer
did not increase the amount of PP2A in the supernatant. For
immunoprecipitation, 300 µg of lysate was incubated for 2.0 h at
4oC with 360 µg of anti-EE, 300 µg of 6F9, or 200 µg
of 5H4 monoclonal antibodies coupled to Gamma Bind Plus sepharose beads
(Pharmacia) (15). After centrifugation, we
incubated the supernatant with the next antibody. The
immunoprecipitates were washed three times with 1 ml of wash buffer
(above lysis buffer with 0.5% Triton X-100 without Pefabloc,
aprotinin, and soybean trypsin inhibitor) and boiled in SDS sample
buffer. One-half of the immunoprecipitate was loaded on a 10% high-bis
gel (19 × 16 cm). The protein was transferred to the
polyvinylidene difluoride membrane, and antibody detection was carried
out with rat monoclonal anti-A subunit (6G3), rabbit anti-B
-subunit,
and mouse monoclonal anti-C subunit antibodies, as described
(32).
Culturing and immunostaining of cardiomyocytes and heart
fibroblasts.
For the preparation of fibroblasts, hearts from 4-mo-old animals were
rinsed in cold PBS, the atria and aorta were removed, and the
ventricles were cut into small pieces and placed into 37oC
buffer containing 18.3 mM HEPES, 0.8 mM MgSO4, 1.0 mM
NaH2PO4, 116.4 mM NaCl, 5.5 mM
D-glucose, and 5.4 mM KCl. The hearts were digested six
times for 20 min at 37oC with 100 U/ml collagenase type 2 (Worthington Biochemicals) and 0.6 mg/ml pancreatin (GIBCO) in the same
buffer (5.6 ml/heart). The cells dissociated from the last five
digestions were pooled and plated in DMEM, 10% FCS, and 1%
penicillin-streptomycin solution. Cells were transferred once
and plated on tissue culture plates or coverslips. Cells were fixed for
7 min with 4% p-formaldehyde and permeabilized with 1%
NP-40/PBS for 1 min. The cells were blocked for 30 min in 1% BSA/PBS,
incubated for 1 h with anti-glutamic acid-glutamic acid (EE) tag
antibody (50 µg/ml of 1% BSA/PBS), washed with PBS, incubated for
1 h with anti-mouse horseradish peroxidase (1:250 Jackson Immuno
Research), washed with PBS, and incubated with
diaminobenzidine substrate (Vector). Cardiomyocytes were
prepared from five 1-day-old mouse hearts, as described above except
that 0.3 ml of enzyme solution (0.8 mg/ml pancreatin and 0.55 mg/ml
collagenase type 2) was used per heart. The myoblasts were plated into
media containing 13.9 g/l DMEM, 3.02 g/l medium 199, 3.4 g/l sodium
bicarbonate, 10% horse serum, and 5% fetal calf serum (11,
14). The reason why cardiomyocytes were prepared from
1- to 5-day-old mice and not from adults was because adult cardiomyocytes are difficult to isolate and to maintain in culture for immunostaining.
Phosphatase assays.
Phosphorylase A and Rb peptide were used as substrates and labeled as
described (32). Heart extracts were made as described for
Immunoprecipitation of PP2A, and the protein contents of the heart extracts were immediately determined and adjusted to be equal.
Extracts containing equal amounts of protein were diluted 1:40 in 0.1%
BSA, 50 mM Tris · HCl (pH 7.5), 0.1 mM EGTA, and 0.1%
2-mercaptoethanol. Extracts (5 µl) containing 0.6 µg of total protein were assayed in the presence of protein phospatase 1 inhibitor 2 as described (32). The reaction time was 10 min. The
assay was linear for at least 10 min and directly dependent on the
amount of extract used. The amounts of catalytic C subunit in the
wild-type and I1 heart extracts used for the assay were identical as
determined by Western blotting.
Transthoracic echocardiography.
Echocardiography was performed in intact anesthetized mice (2.5%
Avertin, 14 µl/g ip) using an Apogee CX echocardiograph
(Interspec-ATL, Bothell, WA) as previously described (31).
Wild-type and homozygous I1-transgenic mice were studied at age
4-7 mo. The average heart weight-to-body weight ratios of the
I1-transgenic and wild-type mice were 6.7 and 5.0, respectively (data
not shown). The operator who performed and measured the echocardiograms
was blinded to the genotype of the animals. The following parameters
were measured: left ventricular end-diastolic dimension (LVEDD),
left-ventricular systolic dimension (LVESD), percent fractional
shortening [calculated as (LVEDD
LVESD)/LVEDD × 100],
septal wall thickness, posterior wall thickness, and heart rate.
Arterial blood pressure.
Mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg
ip). A polyethylene-50 flame-stretched, fluid-filled catheter was
introduced through a cervical incision into the carotid artery and
attached to a modified P50 Statham transducer. Systolic and diastolic
blood pressure were recorded.
Northern blot analysis.
Two- to three-month-old wild-type and homozygous I1-transgenic mice
were euthanized for 30 s with CO2 inhalation. The
hearts were removed, frozen quickly in liquid nitrogen, and kept on dry ice until homogenized. To avoid RNA degradation, the heart weight was
not determined. Total RNA was isolated using the RNeasy Midi Kit
(Qiagen) following the manufacturor's instructions. The RNA concentration was determined by measuring optical density at 260 nm. RNA (10 µg) was loaded onto a 1% agarose gel under
denaturing conditions, as described by Qiagen. The RNA was transferred
onto a Magna Graph nylon membrane (MSI) overnight with 20×
saline-sodium citrate buffer and hybridized with probes.
Myosin heavy chain-
(
-MHC) and myosin heavy chain-
(
-MHC)
oligonucleotides were end labeled with deoxynucleotide transferase.
Hybridization was carried out overnight at 65oC. The mouse
-MHC oligonucleotide had the sequence
AACGTTTATGTTTATTGTGGATTGGCCACAGCGAGGGTCT derived from the 3'
untranslated region that does not show homology to the
-form. The
oligonucleotide from rat
-MHC was purchased from Calbiochem (cat.
no. ON366, sequence unknown). Atrial natriuretic factor, calsequestrin,
sarco(endo)plasmic reticulum Ca2+-ATPase, phospholamban,
and the ryanodine receptor RNAs were labeled with Amersham's
multiprime kit. Hybridization was carried out overnight at
42oC, as described by He et al. (9).
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RESULTS |
Expression of A
5EE subunit in muscle tissue of
transgenic mice.
As previously described (11), transgene expression
controlled by the CMV enhancer and the chicken
-actin promoter
combined, as in the vector pCAGGS (28), is very high in
muscle tissue and low or undetectable in other tissues. We chose this
promoter because the endogenous level of the A subunit of PP2A is very high (~0.1% of total protein) in all cells and tissues that have been studied (15, 17, 36). Therefore, a dominant negative mutant of the A subunit, such as the A
5 subunit (35),
can only compete with the endogenous A subunit if the mutant subunit is expressed as high or higher than the endogenous protein. We found two
transgenic founder mice that expressed a high amount of the A
5EE protein in the heart, skeletal muscle, and smooth
muscle (stomach). These mice were bred to generate the lines I1 and I4.
As shown in Fig. 3, the approximate
ratios of the A
5EE subunit to the endogenous A subunit
for the I1 line are 4:1 for heart, 8:1 for skeletal muscle, and 1:1 for
stomach. The approximate ratios for the I4 line are 1:1 for heart, 1:2
for skeletal muscle, and 1:8 for stomach. Note that the amounts of the
A subunit and the A
5EE subunit cannot be compared
between lanes because unequal amounts of protein were loaded. In
addition, to optimize visualization and quantitation of the ratio of
the wild-type A subunit to the A
5EE subunit, different
exposure times were chosen for different lanes. Importantly, expression
of the A
5EE subunit had no significant effect on the
level of the endogenous A subunit in the heart (data not shown). Only
trace amounts of the A
5EE subunit were found in the
kidney, liver, and brain (data not shown). During the first year after
these two mouse lines were established, the expression of the
A
5EE subunit dropped. In hearts from I1-transgenic mice,
which were used for the majority of our experiments, the ratio of the
A
5EE subunit to the endogenous A subunit reached a level
of 2:1. The A
5EE and endogenous A subunits were
evenly distributed between the ventricles and atria of I1- and
I4-transgenic mouse hearts (data not shown). Importantly, the
A
5EE subunit was only expressed in cardiomyocytes but
not in heart fibroblasts, as shown in Fig.
4.

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Fig. 3.
Expression of mutant A 5EE protein in
transgenic mice. Western blot of wild-type A subunit and mutant
A 5EE subunit in the heart (He), skeletal muscle (Sk),
and stomach (St) from two independent lines of trangenic mice, I1 and
I4. Monoclonal antibody 6G3 was used as described in MATERIALS
AND METHODS.
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Fig. 4.
Expression of A 5EE subunit in
cardiomyocytes but not in heart fibroblasts. Cardiomyocytes were
prepared from 1-day-old mice, and heart fibroblasts were prepared from
4-mo-old mice. The cells were fixed with paraformaldehyde and stained
with antibodies against the EE tag, as described in MATERIALS AND
METHODS. Only cardiomyocytes from I1-transgenic mice (top
right) were stained with the antibodies. I1, I1-trangenic mice;
WT, wild-type mice.
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Transgenic expression of A
5EE subunit causes
increase in heart weight-to-body weight ratio.
We discovered that the hearts of transgenic animals were significantly
larger than normal hearts and that there was no sign of interstitial
fibrosis (Fig. 5). Furthermore, the heart
weights and heart weight-to-body weight ratios of the I1-transgenic
mice were already increased on day 1 after birth and
continued to be high throughout life. These data are presented in Figs.
6 and 7,
showing the values of individual animals in the different age groups
(plus signs, closed triangles, and open squares for I1-transgenic, I4-transgenic, and wild-type mice, respectively), and in Table 1, presenting the average values. Similar
results to the I1-transgenic mice were obtained for the I4-transgenic
mice, although the increase in heart weight and heart weight-to-body
weight ratio was generally smaller than for I1-transgenic mice. This
latter finding is consistent with the observation that I4-transgenic
mice expressed less A
5EE subunit than I1-transgenic
mice. Heterozygous I1- and I4-transgenic mice had lower heart
weight-to-body weight ratios than the corresponding homozygous mice (as
shown in Fig. 8), further indicating that the heart weight and heart weight-to-body weight ratio correlate with
the level of A
5EE subunit expression. These results
strongly suggest that the A
5EE subunit is the
cause for the increased heart weight of the
A
5EE-transgenic mice. There was no apparent sex
difference in heart weight and heart weight-to-body weight ratio.
Interestingly, starting at 3-4 mo of age, I1-transgenic mice with
extremely large hearts and heart weight-to-body weight ratios (between
two- and fivefold above the norm) were observed. These mice
represented ~25% of all animals in the 7- to 12-mo age group. They
were characterized by general weakness, slow movements, and increased
respiratory rate, and they died within a few weeks after showing these
symptoms. A summary of all data is presented in Fig. 7 and Table 1.
Note that the P values for the heart weights and the heart
weight-to-body weight ratios of I1-transgenic mice in the 3- to 4-, 5- to 6-, and 7- to 12-mo-old age groups are skewed by the mice with
extremely high ratios. For example, the increased ratio of 3- to
4-mo-old mice appears statistically nonsignificant due to the
presence of two animals with extreme ratios, although the
increase is highly significant, as illustrated in Fig. 6.

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Fig. 5.
Increase in heart size of A 5EE-transgenic mice and
lack of interstitial fibrosis. Hearts from a normal (A) and
a A 5EE-transgenic mouse (B) were sectioned
transversely and stained with Masson trichrome stain. The scale is 2 mm.
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Fig. 6.
Increase in heart weight of
A 5EE-transgenic mice on postnatal day 1 (A) and throughout life. Body weights and heart weights of
WT ( ), A 5EE I1-trangenic (+), and
A 5EE I4-transgenic ( ) mice were
determined at different ages [10-day-old mice (B), 1- to
2-mo-old mice (C), 3- to 4-mo-old mice (D), 5- to
6-mo old mice (E), and 7- to 12-mo-old mice
(F)]. G and H: comparison of body
weight to heart weight in male (G) and female (H)
WT, I1-, and I4-transgenic mice. I: comparison of body
weight to heart weight in all of the mice. This figure illustrates the
general increase in heart weight of transgenic compared with WT animals
and the appearance of animals with extremely large hearts beginning at
3-4 mo of age. It also illustrates the general tendency of
transgenic hearts toward a slightly decreased body weight compared with
WT hearts.
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Fig. 7.
Increase in heart weight-to-body weight ratio of
A 5EE-transgenic mice. The heart weight-to-body weight
ratio was plotted for the different age groups. This figure illustrates
the general increase in the ratio for transgenic animals and the
extremely high ratio for a fraction of older animals. Also note that
the ratio is constant for WT mice at all ages. , WT; +,
I1-transgenic mice; , I4-transgenic mice.
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Fig. 8.
Homozygous A 5EE-transgenic mice have a
larger heart weight-to-body weight ratio than heterozygous mice. The
P values (means ± SD) for homozygous (Homo) I1- and
I4- and heterozygous (Hetero) I1- and I4-transgenic mice compared with
WT mice are 0.0005, 0.0054, 0.0011, and 0.24. The P value
for I1 Homo- compared with I4 Homo-transgenic mice is 0.0178; for I1
Hetero compared with I4 Hetero, 0.0077; for I1 Homo compared with I1
Hetero, 0.0062; and for I4 Homo compared with I4 Hetero, 0.0235.
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I1-transgenic mice have a general tendency toward a lower body weight
compared with wild-type mice of the same age group. This was most
pronounced at 7-12 mo of age, when some control mice became very
large (while maintaining a constant heart weight-to-body weight ratio),
whereas I1-transgenic mice never surpassed a weight of 30 g (Fig.
6). However, despite the decrease in body weight, it is clear that the
increase in heart weight is by far the predominant factor for the
increased heart weight-to-body weight ratio of A
5EE-transgenic mice. An exception was 1- to 2-mo-old
A
5EE-transgenic mice whose hearts were not
significantly larger than control hearts, whereas their body weights
were reduced by 19%, and this gave rise to an increase in the heart
weight-to-body weight ratio. These results suggest that between 10 days
and 1-2 mo, control mice grew slightly more than transgenic mice.
During the same period, transgenic hearts, which are significantly
larger than the controls at 1 and 10 days, grew slightly less than the controls. After 1-2 mo, the development of progressive
cardiomyopathy began and eventually led to death.
Heart function of A
5EE-transgenic mice is
impaired, and blood pressure is unchanged.
Echocardiography was used to evaluate cardiac chamber size and function
in vivo in wild-type and homozygous A
5EE-transgenic
mice. As shown in Table 2, LVEDDs for
I1-transgenic mice were significantly greater compared with wild-type
mice (4.5 vs. 3.5 mm, respectively), indicating chamber enlargement of
the I1-transgenic mouse hearts. Furthermore, a significant increase in
LVESD was found in the I1-transgenic mouse hearts compared with
wild-type mouse hearts (3.5 vs. 2.2 mm, respectively). Calculated fractional shortening (end-diastolic dimension
end-systolic dimension × 100%/end-diastolic dimension), a measure of systolic function, indicates depressed cardiac performance in vivo in the I1-transgenic mice (21%) compared with wild-type mice (36.5%). Another important result from the echocardiographic analysis was that
the thickness of the septum and of the left ventricular posterior wall
was significantly reduced in transgenic mice. Taken together, these
data of chamber enlargement, reduced systolic function, and wall
thinning are consistent with a phenotype of dilated cardiomyopathy.
The results from echocardiography appear to contradict the histological
data (Fig. 5) showing a mild increase in wall thickness and normal
chamber size in A
5EE-transgenic mice. This difference
between echocardiography and histology is due to a fixation artifact.
Because the purpose of the histology was only to look for fibrosis, the
hearts were not arrested in diastole by perfusing a hyperkalemic
solution to prevent systolic contracture. Indeed, to measure cardiac
chamber size and function, we performed the more accurate method of
noninvasive echocardiography.
The blood pressure was determined by recording the left ventricular
systolic pressure (which equals aortic pressure) of anesthetized mice.
The left ventricular systolic pressure of wild-type versus I1-transgenic mice was 113.1 ± 11.2 versus 101.6 ± 9.1 (means ± SD) mmHg; P = not significant. This
result demonstrates that expression of A
5EE has no
effect on systolic blood pressure.
A
5EE subunit binds C subunit but not B subunit:
similar abundance of core enzyme and holoenzyme in hearts.
The underlying assumptions on which the present work is based are that
1) the A
5EE mutant subunit competes with the
endogenous A subunit for binding of C subunit, 2) the
A
5EE subunit does not bind B subunits, 3) the
ratio of core enzyme to holoenzyme increases in
A
5EE-expressing tissue, and 4) the
phosphatase activity changes due to the increase in the core
enzyme-to-holoenzyme ratio. To test these assumptions, we carried out
immunoprecipitations with normal and transgenic heart extracts, using
monoclonal antibodies against the EE tag (anti-EE) and monoclonal
antibodies 5H4 and 6F9 directed against the wild-type A subunit. These
latter antibodies distinguish between the core enzyme and holoenzyme.
Whereas 5H4 recognizes the core enzyme but not the holoenzyme, 6F9
precipitates both forms (Fig. 1) (15). To achieve
quantitative precipitations, two consecutive precipitations were
carried out with each antibody. The amounts of the A, B, and C subunits
in immunoprecipitates were determined by Western blotting with
antibodies against the individual subunits as described in
MATERIALS AND METHODS.
As shown in Fig. 9 (lanes 1 and 3), more C subunit was bound to the A
5EE
subunit (precipitated with anti-EE antibody) than to the endogenous A
subunit (precipitated with 5H4), demonstrating that the
A
5EE subunit effectively competes with the wild-type A
subunit for binding of the C subunit. Importantly, there was no
B subunit bound to the A
5EE-C (lane 1) and
A-C core enzymes (lane 3). To determine whether the ratio of
core enzyme (A
5EE-C plus A-C subunits) to holoenzyme was
changed in transgenic heart tissue, heart extracts were first
precipitated with anti-EE (lanes 1 and 2) and 5H4
(lanes 3 and 4) antibodies to remove all core
enzyme, followed by precipitation of holoenzyme with the 6F9 antibody
(lanes 5 and 6). As a control, extracts from
wild-type hearts were precipitated with the 5H4 antibody (lanes
7 and 8) followed by the 6F9 antibody (lanes
9 and 10). In A
5EE-transgenic heart
extracts, there was more C subunit in the core enzyme
[A
5EE-C (lanes 1 and 2) and A-C
(lanes 3 and 4) core enzymes combined] than in
the holoenzyme (lanes 5 and 6). In contrast, in
extracts from wild-type hearts, there was more holoenzyme (lanes
9 and 10) than core enzyme (lanes 7 and
8), on the basis of the amount of C subunit. A similar
result was obtained when the I1-transgenic heart extract, first
precipitated with the 5H4 antibody (lanes 11 and
12) followed by the 6F9 antibody (lanes 13 and
14), was compared with wild-type heart extract, which was
precipitated with the 5H4 (lanes 7 and 8) and 6F9
antibodies (lanes 9 and 10). These
experiments demonstrate that the core enzyme and holoenzyme are
similarly abundant in the heart and that A
5EE subunit
expression increases the level of core enzyme and decreases the level
of holoenzyme, as expected. Because precise quantitation of Western
blots is difficult (in particular when using the enhanced chemiluminescence system), we also carried out phosphatase
assays (see below).

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Fig. 9.
Abundance of holozyme and core enzyme in heart tissue of
WT and A 5EE-transgenic mice. Lanes 1-6,
extracts from I1-transgenic mouse hearts were incubated sequentially
with antibodies against the EE tag precipitating A 5EE-C
core enzyme (lanes 1 and 2), 5H4 precipitating WT
core enzyme (AC) (lanes 3 and 4), and 6F9
precipitating holoenzyme (ABC) (lanes 5 and 6).
Extract from WT hearts (lanes 7-10) and extract from
I1-transgenic mouse hearts (lanes 11-14) were incubated
sequentially with 5H4 (lanes 7, 8, 11,
and 12) and 6F9 (lanes 9, 10,
13, and 14). To deplete the extract of the
different forms of PP2A, each antibody was used twice. The precipitates
were analyzed by Western blotting as described in MATERIALS AND
METHODS. The positions of the A, B, and C subunits are indicated
on the right.
|
|
If the A
5EE subunit can only bind the C subunit, one
would expect that the 5H4 antibody removes all the
A
5EE-C subunits from cell extracts. However, this was
not the case. As shown in Fig. 9 (lanes 13 and
14), the 6F9 antibody precipitated ~30% of the
A
5EE subunit despite exhaustive prior precipitation with
the 5H4 antibody (lanes 11 and 12). This
indicates that, in a fraction of the A
5EE subunits, the
5H4 epitope was covered up by another protein. This protein could not
have been the B
-subunit because there was no B
-subunit in
precipitates of the A
5EE subunit with the anti-EE
antibody. It also could not have been one of the other known forms of B
subunit (B' or B'') because none of these binds to the
A
5EE subunit, as shown previously (33,
34). Alternatively, there is a remote possibility that
the 5H4 epitope was denatured and therefore not recognized by the 5H4
antibodies in a fraction of the A
5EE subunits. However,
because the 6F9 epitope was intact in A
5EE subunits, the
latter possibility seems less likely.
Reduction of holoenzyme activity in transgenic hearts.
To determine whether a decrease in holoenzyme activity and an increase
in core enzyme activity in fact occurs as a result of
A
5EE subunit expression, we used phosphorylase
a, phosphorylated by phosphorylase kinase, and Rb peptide,
phosphorylated by cdk1 kinase/cyclin B, as substrates. The holoenzyme
is 100-fold more active than the core enzyme in dephosphorylating Rb
peptide (1), whereas the core enzyme activity toward
phosphorylase a is ~2.5-fold higher than that of the holoenzyme
(44). As shown in Table 3,
the phosphatase activity toward the Rb peptide was reduced by 29% in
extracts from I1-transgenic mouse hearts compared with extracts from
control hearts, whereas the activity toward phosphorylase a increased
by 24%. This change in activity is reflected in a drop in the ratio of
the Rb peptide phosphatase activity to the phosphorylase a
phosphatase activity from 3.7 to 2.1. Because the activity toward the
Rb peptide comes almost exclusively from the holoenzyme, these results
indicate a 29% drop in the level of the holoenzyme; and, because
holoenzyme represents approximately two-thirds (66%) of the total PP2A
in the wild-type heart extract (Fig. 9), a 29% drop would result in a
final amount of 42% holoenzyme in I1-transgenic mouse heart extract.
Furthermore, because the decrease in holoenzyme leads to a
corresponding increase in the core enzyme, the level of core enzyme is
expected to rise from ~33% in wild-type heart extract (Fig. 9) to
58% in I1-transgenic mouse heart extract. Because the core enzyme is
2.5 times more active than the holoenzyme, one would expect a 25%
increase toward phosphorylase a, which is very close to the
experimental value of 24%. Thus the results obtained from phosphatase
assays confirm those obtained by immunoprecipitation.
Increased
-MHC gene expression in A
5-transgenic hearts.
A characteristic of cardiac hypertrophy is the reactivation of genes
whose expression is normally restricted to fetal development, e.g., the
-MHC and the atrial natriuretic factor gene. On the other hand, the
transcription of genes encoding sarco(endo)plasmic reticulum
Ca2+-ATPase, phospholamban, and
-MHC has been reported
to be suppressed in hypertrophic hearts (see Refs. 2 and 14 for
review). As demonstrated by Northern blotting, the level of
-MHC
transcripts in homozygous A
5-transgenic hearts was significantly
increased, whereas no change in atrial natriuretic factor,
sarco(endo)plasmic reticulum Ca2+-ATPase, phospholamban,
and
-MHC expression was observed (Fig. 10A). The increase in
-MHC transcripts in A
5-transgenic hearts was fourfold compared
with control animals (Fig. 10B). Elevated levels of
-MHC
were also observed in transgenic mice that developed cardiac
hypertrophy and ventricular dilation due to overexpression of the
calcium-dependent phosphatase calcineurin in the heart (24). We also found no change in calsequestrin and
ryanodine receptor gene expression.

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Fig. 10.
Cardiac gene expression in I1-transgenic mice.
A: Northern blot showing cardiac expression in both
I1-transgenic and WT mouse hearts. RNA was isolated and hybridized with
specific probes as described under MATERIALS AND METHODS.
B: quantitative comparison of transcripts from I1-transgenic
and WT mice. WT gene expression was set to 100%. Numbers (means ± SD) in arbitrary units were obtained from 3 animals. MHC, myosin
heavy chain; CSQ, calsequestrin; SERCA, sarco(endo)plasmic reticulum
CA2+-ATPase; PLB, phospholamban; RYR2, ryanodine receptor
2; ANF, atrial natriuretic factor. The measured values (in arbitrary
units) for WT mice are the following: -MHC, 4.6 ± 1.3; ANF,
260 ± 79; CSQ, 19.4 ± 1.6; -MHC, 310 ± 49.6;
SERCA, 141.9 ± 8.5; PLB, 793.8 ± 50.9; RYR2, 1.6 ± 0.4. The measured values for I1-transgenic mice are the following:
-MHC, 18.2 ± 6.3; ANF, 350 ± 170; CSQ, 21.2 ± 3.8;
-MHC, 324.5 ± 95.5; SERCA, 120.4 ± 20.6; PLB, 705.6 ± 101.8; and RYR2 1.3 ± 0.7. Note that the level of expression
varies greatly between the different genes. The exposure times shown in
A were chosen to best visualize the WT and I1 signals in
each case.
|
|
 |
DISCUSSION |
In this report, we have shown that transgenic expression of a
dominant negative mutant (A
5) of the regulatory A subunit of PP2A,
which is defective in binding regulatory B subunits but normal in
binding the catalytic C subunit, leads to a significant increase in
heart weight and heart weight-to-body weight ratio. Previous
experiments with tissue culture cells (32) have
demonstrated that expression of the A
5 subunit causes an increase in
the amount of the core enzyme and a decrease in the amount of the
holoenzyme, resulting in a change in PP2A activity. In the heart,
expression of the A
5 subunit also caused an increase in the ratio of
the core enzyme to the holoenzyme and a change in activity, and this change is responsible for the observed increase in heart weight and the
depressed cardiac function. At present, we do not know what causes the
dramatic increase in heart weight at older age. It is conceivable that
this is due to genetic or environmental factors in addition to
expression of the A
5 subunit. The increase in heart weight was
proportional to the expression of the A
5 subunit, because
I4-transgenic mice, expressing less of the A
5 subunit than
I1-transgenic mice, had smaller hearts than the I1-transgenic mice.
Furthermore, heterozygous I1-trangenic mice had smaller hearts than
homozygous mice. These observations further indicate that the A
5
subunit is the actual cause for the observed phenotype. Echocardiographic analyses revealed that transgenic hearts were significantly dilated, the walls of the hearts were thinner, and the
capacity of the hearts to contract was considerably reduced. These
findings are consistent with a phenotype of dilated cardiomyopathy. The
A
5-transgenic hearts show only a modest increase in
-MHC expression and normal atrial natiuretic factor expression. These findings are compatible with the echocardiography results showing no
increase in wall thickness. In addition, no significant interstitial fibrosis occurred.
We have shown that the heart weight of the A
5-transgenic animals was
significantly increased at birth. Because up to this time in
development the heart grows by an increase in the number of
cardiomyocytes, this finding might suggest that expression of the A
5
subunit stimulates cardiomyocyte proliferation (hyperplasia) during
fetal development. Alternatively, the cardiomyocytes in A
5-transgenic mice may be abnormally large at birth, whereas the
number of cardiomyocytes is unchanged. Transgenic overexpression of the
c-myc proto-oncogene also causes cardiac enlargement, which in this case is due to myocyte hyperplasia during fetal development (12).
We have demonstrated that the core enzyme and holoenzyme are both
similarly abundant in the heart, as shown previously for bovine hearts
(25) and for several lines of cultured fibroblasts (15). This was achieved by quantitative
immunoprecipitation of the core enzyme and holoenzyme with specific
monoclonal antibodies. The conditions of extract preparation and
immunoprecipitation were chosen such that proteolysis and dissociation
of the holoenzyme into the core enzyme and B subunit were minimized.
Our data contradict the commonly held view that the holoenzyme is the
only form of PP2A in intact cells and that the core enzyme is an
artifact of enzyme purification (for discussion, see Ref. 15). A simple calculation shows that the change in the amounts of the core enzyme and
holoenzyme, which would result from a twofold A
5 subunit overexpression, are relatively small (in the order of 30%) and fall
within close range of the observed values of 20-30%. If core enzymes were not initially present in cardiomyocytes, the expected increase in core enzyme activity due to the formation of the A
5-C subunit would be dramatic (50-fold). That this formation is, in fact,
only 24% further supports the notion that the core enzyme is as
similarly abundant as the holoenzyme. Our findings are
important because the core enzyme differs significantly in substrate
specificity from the holoenzyme. In addition, the core enzyme may serve
as a reservoir for the binding of various regulatory B subunits. Besides the core enzyme and B
-subunit-containing holoenzyme, at
least three additional heart-specific forms of the holoenzyme can be
inferred from the fact that three additional B subunits are expressed
in the heart. The B56
- and B56
-subunits (members of the B'
family) are preferentially expressed in the heart and may have
cytosolic and nuclear functions, respectively (22, 42).
The 74-kDa B'' subunit is highly expressed in the heart and brain
(5). It is likely that these B subunits do not exist as
free monomers but are bound to excess core enzymes forming distinct
holoenzymes. The phenotype of A
5-transgenic mice may be attributed
either to an increase in core enzymes or a decrease in one or several
forms of holoenzyme. Because the A
5 mutant protein does not bind any
form of the B subunit, we assume that transgenic expression of the
A
5 subunit caused a decrease in all forms of holoenzyme. Hence, it
is not possible to link the phenotype of the A
5-transgenic mice to
the decrease of a particular form of the holoenzyme. To identify the
heart-specific substrates of the core enzyme and the various
holoenzymes is an important goal. One candidate is MAP kinase, a known
substrate of PP2A (38), which is involved in the
hypertrophic response of cardiomyocytes to a variety of factors and
stimuli. Interestingly, there is an inverse relationship between
increasing MAP kinase activity and decreasing PP2A phosphatase activity
in cultured cardiomyocytes upon treatment with phorbol ester, an
inducer of protein kinase C and of hypertrophic response
(4).
It is intriguing that the genes encoding the B56
- and
B56
-subunits have been mapped to regions on the human chromosome
linked to heart disease (21). The B56
-subunit gene is
located on chromosome 1q41, a region linked to rippling muscle disease
and ventricular cardiomyopathy, and the gene for the B56
-subunit is
located on 3p21, a region linked to a form of familial cardiomyopathy.
However, mutations in the B56
- or B56
-subunit genes have not been
reported in patients with heart disease. It is conceivable that
mutations of the A subunit, which impair binding of the B
-, B56
-,
B56
-subunits, or the 74-kDa B'' subunit to the core enzyme, are
genetically linked to heart disease, in particular because a large
number of point mutations in the A subunit abolish binding of specific B subunits (33). We recently constructed mutants
that do not bind B' (B56) subunits but bind B
- and B'' subunits
normally (33). The expression of these mutants in the
heart is expected to shed light on the function of B' subunits,
including the B56
- and B56
-subunits.
 |
ACKNOWLEDGEMENTS |
We acknowledge the Transgenic Mouse Core at UCSD for carrying out
microinjections of pronuclei and the development of founder mice. We
thank Larry Brunton, Gary Meszaros, and Åsa Gustafsson for help in
setting up cardiomyocyte and heart fibroblast cultures, Eric Swanson
for providing probes for Northern blotting, and Lana Nimmo for teaching
us histology.
 |
FOOTNOTES |
*
Authors have contributed equally to this paper.
This work was supported by Public Health Service Grant CA 36111 (to G. Walter) and National Heart, Lung, and Blood Institute Grant HL-61558
(to H. Rockman).
Present address of N. Brewis: Marie Curie Research Institute, The
Chart, Oxted, Surrey, RH8 OTL, UK
Address for reprint requests and other correspondence: G. Walter, Dept.
of Pathology 0612, Univ. of California San Diego, 9500 Gilman Dr., La
Jolla, CA 92093-0612 (E-mail: gwalter{at}ucsd.edu).
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.
Received 29 November 1999; accepted in final form 4 April
2000.
 |
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