Cell-specific promoter in adenovirus vector for transgenic
expression of SERCA1 ATPase in cardiac myocytes
G.
Inesi1,2,
D.
Lewis1,2,
C.
Sumbilla2,
A.
Nandi2,
C.
Strock2,
K. W.
Huff2,
T. B.
Rogers2,
D. C.
Johns3,
P. D.
Kessler3, and
C. P.
Ordahl1
1 Department of Anatomy and
Cardiovascular Research Institute, University of California School
of Medicine, San Francisco, California 94143;
2 Department of Biochemistry and
Molecular Biology, University of Maryland School of Medicine,
Baltimore, 21201; and 3 The Peter
Belfer Cardiac Laboratory and Department of Medicine, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
Adenovirus-mediated transfer of cDNA encoding the chicken
skeletal muscle sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA1) yielded
selective expression in cultured chick embryo cardiac myocytes under
control of a segment (
268 base pair) of the cell-specific
cardiac troponin T (cTnT) promoter or nonselective expression in
myocytes and fibroblasts under control of a constitutive viral
[cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to
express transgenic SERCA1 ATPase. Expression was targeted to
intracellular membranes and was recovered in subcellular fractions with
a pattern identical to that of the endogenous SERCA2a ATPase. Relative
to control myocytes, transgenic SERCA1 expression increased up to four
times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell
homogenates. Although the CMV promoter was more active than the cTnT
promoter, an upper limit for transgenic expression of functional enzyme
was reached under control of either promoter by adjustment of the
adenovirus plaque-forming unit titer of infection media. Cytosolic
Ca2+ concentration transients and
tension development of whole myocytes were also influenced to a similar
limit by transgenic expression of SERCA1 under control of either
promoter. Our experiments demonstrate that a cell-specific protein
promoter in recombinant adenovirus vectors yields highly efficient and
selective transgene expression of a membrane-bound and functional
enzyme in cardiac myocytes.
sarco(endo)plasmic reticulum calcium-adenosinetriphosphatase; transfected adenosinetriphosphatase gene; calcium
transport
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INTRODUCTION |
THE CONTRACTION AND relaxation cycle of muscle fibers
is controlled by a sequential rise and fall of the cytosolic
Ca2+ concentration
([Ca2+]i).
In this regard, the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) isoforms of
skeletal (9, 14) and cardiac (5, 10, 16) muscle play an
important role by sequestering cytosolic
Ca2+ in intracellular stores from
which it can be subsequently released. The prominent role of SERCA in
cardiac muscle is emphasized by its involvement in the inotropic
response to sympathetic stimulation through the phospholamban
regulatory mechanism (18, 28, 35). Furthermore, selective inhibition of
SERCA by thapsigargin is followed by reduction of intracellular
Ca2+ transients, tension development, and relaxation
kinetics in cardiac myocytes, without alterations of plasma membrane
electrical parameters (24).
The availability of SERCA1 and SERCA2a cDNA clones, encoding the two
ATPase isoforms that are specific for skeletal and cardiac muscle,
respectively (4, 22, 27, 30, 32, 37), has rendered possible their
expression in COS-1 cells for mutational analysis (17, 29). Most
importantly, initial reports indicate that contractile parameters of
rat cardiac myocytes may be influenced by overexpression of SERCA2a
ATPase by gene transfer in cultured myocytes (13) or by whole mouse
transgenic procedures (11, 15). It is noteworthy, in this regard, that
various transfection methods differ widely in their ability to affect a
significant number of cells in culture. Furthermore, transfection
constructs containing viral promoters override specific transcriptional
controls and are constitutively effective not only in myocytes, but
also in other cell types.
We considered that, in attempts to influence
Ca2+ homeostasis or other
functions by gene transfer into heterogeneous cell populations or whole
muscle, it is desirable to achieve effective transfection of the
majority of muscle cells and only of muscle cells. Therefore, with the
experiments reported here, we have evaluated various methods of gene
transfer in cell cultures, using viral and muscle-specific promoters.
We investigated whether these promoters retain exclusive transcriptional control of the ATPase gene, independent of intrinsic adenovirus promoters, and compared their efficiency in control of
reporter gene and ATPase gene expression. For this purpose, we used
LacZ, enhanced green fluorescence protein (EGFP), or avian SERCA1 cDNA,
under control of the constitutive cytomegalovirus (CMV) promoter or the
cardiac muscle-specific cardiac troponin T (cTnT) promoter (31) for
transfection of chick embryo myocytes and fibroblasts in culture. We
evaluated the percentage of cells effectively transfected, the extent
of preferential expression in myocytes over fibroblasts, the
intracellular membrane targeting of the transgenic ATPase, the yield of
Ca2+ transport activity in cell
homogenates, and the effect on
[Ca2+]i
transients and contractile dynamics in intact myocytes.
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METHODS |
DNA constructs and vectors.
Chicken fast-twitch muscle SERCA1 (22) cDNA was initially placed in the
pUC19 plasmid for amplification and then subcloned into the shuttle
plasmid p
E1sp1A (Microbix Systems). In the final constructs, the
cDNA was preceded by the constitutive CMV promoter or by the cTnT (31)
muscle-specific promoter and was followed by a simian virus 40 polyadenylation signal. LacZ (
-galactosidase) and EGFP reporter
genes, obtained from Clontech (Palo Alto, CA), were also subcloned into
the p
E1sp1A shuttle plasmid. The shuttle plasmids were either used
directly for transfections of myocytes and fibroblasts or alternatively
for cotransfection of HEK-293 cells in conjunction with the
replication-defective viral plasmid pJM17 (Microbix Systems) to obtain
recombinant adenovirus vectors (12). The shuttle vector was constructed
such that homologous recombination resulted in antisense direction of
the gene of interest with respect to the adenovirus E1 gene promoter.
The recombinant products were plaque and band purified, yielding
concentrations in the order of
1010 plaque-forming units
(PFU)/ml.
Cell cultures.
Primary cultures of cardiac myocytes were obtained from pooled hearts
of day 8 chicken embryos, which were first placed in cold
heart medium [500 ml medium 199 (M199) plus Earle's
balanced salts, 25 ml fetal bovine serum (FBS), 5 ml
penicillin-streptomycin, and 5 ml Fungizone]. After
we had removed atria and pericardial membranes with the aid of a
dissecting microscope and gently teased the muscle tissue apart, the
fragments obtained from 20 to 40 hearts were washed in Hanks' buffered
salt solution and then subjected to digestion in 5.0 ml of trypsin
solution (0.05 g trypsin, 0.2 g EDTA, 1 g glucose, 0.58 g
NaHCO3, and 4.5 mg/l phenol red)
stirred with a magnetic bar at room temperature. After 10 min of
digestion, the medium was discarded and the muscle fragments were then
subjected to six consecutive trypsinizations of 10 min each. At the end of each trypsinization, the supernatant was collected and added to an
equal volume of cold heart medium to prevent further trypsinization of
the collected cells. The pooled supernatants were then centrifuged for
5 min at 2,500 g in a refrigerated
centrifuge. The sedimented cells were resuspended in 10 ml of heart
medium and preplated for 1 h in a 100-mm culture dish at 37°C in
5% CO2. One hour after preplating
on uncoated dishes (for selected attachment of fibroblasts), the
detached myocytes were collected in 10 ml heart medium. This suspension
was diluted again to plate ~2 × 106 cells/35-mm dish on
collagen-coated dishes. Twenty-four hours after the initial plating,
detached myocytes were removed by changing the medium. The remaining
30-40% confluent cultures (mostly myocytes and a few remaining
fibroblasts) were used for transfections. Sterile conditions were
maintained as much as possible throughout these procedures.
Similar methods were used for primary cultures of chicken embryo skin
fibroblasts. The culture medium for fibroblasts was made by adding 50 ml tryptose phosphate, 10 ml chick serum, 50 ml FBS, 5 ml
penicillin-streptomycin, and 5 ml Fungizone to 500 ml M199 plus
Earle's balanced salts with glutamine.
Transfections.
Transfections were carried out on cell cultures (30-40% confluent
fibroblasts or nearly confluent myocytes) by calcium phosphate (6) or
liposome (PerFect transfection kit, Invitrogen) methods. Alternatively,
the adenovirus-polylysine component method (7, 36), based on physical
aggregation of adenovirus, polylysine, and transfection plasmid, was
used as described by Kohout et al. (26). For this purpose we used
replication-defective adenovirus type 5 mutant Ad5dl312, kindly
supplied by Dr. Thomas Shenk (19, 20), propagated in HEK-293 cells, and
purified by CsCl density gradient centrifugation before mixing with
polylysine and transfection plasmid.
Recombinant adenovirus vectors were used as follows: lawns of cultured
cells were first rinsed with phosphate-buffered saline (PBS) and then
layered with serum-free medium containing adenovirus titers of
0.8-50.0 PFU/seeded cell. Ninety minutes thereafter, the infection
medium was diluted by adding medium containing serum and no virus. Two
days after the infection, the cells were harvested for immunostaining
or functional assays.
Immunostaining.
The lawns of cultured cells were first washed with PBS and then fixed
with 4% formaldehyde for 10 min. After repeated washings with PBS,
blocking was produced by 10 min of incubation with 1% serum albumin
and 0.5% lysine in PBS, followed by a 45-min incubation with the
primary antibody at a concentration of 5-10 µg/ml of PBS
containing 1% albumin, 0.5% lysine, and 0.25% saponin
(permeabilization medium). After being washed with PBS, the cells were
incubated for 45 min with biotinylated anti-mouse secondary antibody
(Vector Laboratory, Burlingame, CA) at a concentration of 5 µg/ml
permeabilization medium. The cells were then washed with PBS and
incubated for 20 min with fluorescein streptavidin (Amersham) at a
concentration of 5 µg/ml permeabilization medium. The sample was then
washed again with PBS, 70% ethanol, and 90% ethanol, allowed to dry, and processed for fluorescence microscopy using a Zeiss Axiophot microscope equipped with a mercury vapor lamp, excitation filters, and
digital video acquisition.
Cell homogenates, protein determinations, and Western blots.
The myocytes on a 100-mm culture dish were rinsed with 10 ml PBS and
collected by scraping with a spatula in 10 ml of a cold medium
containing 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS; pH 7.0), 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 0.4 mM Pefabloc, 10 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 µg/ml pepstatin A. The
cells were then sedimented by centrifugation at 2,500 g for 2 min, resuspended in 1 ml of
the same medium, frozen in liquid nitrogen, and stored at
70°C. Within 2 wk of storage, the frozen cells were thawed
and homogenized with 80 strokes of a hand-held homogenizer immediately
before their use for Ca2+ uptake
measurements. The total protein concentration was determined by
measurements of ultraviolet absorption (280 nm) in 0.1% sodium dodecyl
sulfate (SDS), using bovine serum albumin as a standard. Samples were
also subjected to SDS gel electrophoresis for determination of ATPase
by Western blots. For these experiments, myocytes were collected using
PBS containing 1 mM EDTA and the protease inhibitors indicated above.
The cells were then pelleted, resuspended in the same solution, and
homogenized by sonication. The protein concentration of the homogenates
was determined by the bicinchoninic acid assay method (Pierce kit), and
SDS was added (1%). The ATPase was then resolved in SDS gels,
transferred to nitrocellulose membranes, and probed with monoclonal
antibodies specifically reactive to the chicken SERCA1 (CaF3-5C3; Ref.
22) or to the chicken SERCA2a ATPase (CaS-3H2; Ref. 21). The secondary
antibody was goat anti-mouse horseradish peroxidase-conjugated
(Bio-Rad), and the reactive bands were detected using the enhanced
chemiluminescence Western blotting kit (Amersham).
Ca2+
transport in cell homogenates.
The Ca2+ uptake medium contained
40.0 mM MOPS (pH 7.0), 100.0 mM KCl, 5.0 mM
MgCl2, 5.0 mM
NaN3, 0.2 mM ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 1 µM ruthenium red, 0.2 mM [45Ca]CaCl2,
and 100-150 µg/ml cell homogenate protein. The reaction was
started by the addition of 5.0 mM potassium oxalate and, after 2 min,
5.0 mM ATP at 37°C. Samples were collected before and, at serial
times, after the addition of ATP. The samples (1 ml each) were passed
through 0.45-µm Millipore filters, which were washed with 10.0 ml of
2.0 mM LaCl3 in 10 mM MOPS (pH
7.0), blotted, and placed in scintillation vials for determination of
radioactivity.
[Ca2+]i
transients and contractility of intact myocytes.
Myocytes cultured on glass coverslips were loaded with fluo 3 (Molecular Probes) by incubation for 15 min with 5 µM fluo
3-acetoxymethyl ester from a 442 mM stock in dimethyl sulfoxide and
20% (wt/wt) Pluronic F-127. The cells were then placed in a
superfusion bath on the stage of a fluorescence microscope and were
superfused (1 ml/min) with buffer containing (in mM) 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 125 NaCl, 5 KCl, 20 glucose, 0.8 MgSO4, 1 Na2PO4,
and 1.8 CaCl2 (pH 7.4) at
30°C. Cells were field stimulated at 2 Hz using 5-ms pulses with a
magnitude of 1.5 times threshold. Fluorescence was measured on a Nikon
diaphot microscope using a commercially available fluorescence
detection system [Photon Technology International (PTI), South
Brunswick, NJ]. A 75-W xenon lamp was used as the excitation
source, and the excitation wavelength (488 nm) was selected with a
monochromator and a 510-nm dichroic long band-pass (DCLP) mirror.
Emission (510-610 nm) was collected with a 610-nm DCLP mirror
mounted at a 45° angle to the photomultiplier tube. Fluo 3 emission
was digitized and collected at 200 Hz using OSCAR software (PTI). The
resulting
[Ca2+]i
transients were reported as fluorescence emission following stimulation, relative to fluorescence emission at rest. In parallel experiments, the myocyte shortening dynamics were recorded by video
microscopy.
 |
RESULTS |
Vectors and transfection efficiency.
In preliminary experiments, we evaluated various procedures for gene
transfer, including physical aggregation with calcium phosphate
precipitates, liposomes, or adenovirus-polylysine aggregates, and
compared these methods with recombinant adenovirus vectors. The number
of cells expressing the transfected gene was evaluated by direct
microscopic visualization of intrinsic fluorescence or following
incubation with chromogenic substrates or immunofluorescent staining.
Consistent with previous reports (23, 25), we found that the
recombinant adenovirus is a highly efficient vector. An example of our
quantitative evaluation of transfection efficiency is shown in Table
1, where fluorescent cell counts as well as total fluorescence levels following infection with recombinant CMV-EGFP-adenovirus are reported. It is apparent that the percentage of
effectively transfected cells increases as the adenovirus PFU level is
raised. In fact, the percentage increases steeply as the PFU level is
raised from 0.08 to 0.8 PFU/cell, and then reaches an asymptotic level
near 100% as the PFU level is raised further. On the other hand, the
total fluorescence continues to increase in proportion to the PFU
level, likely due to a higher number of gene copies introduced in each
cell.
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Table 1.
Transfection efficiency in chick embryo cardiac myocyte culture
infected with recombinant CMV-EGFP adenovirus
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In parallel experiments, we found that recombinant adenovirus vectors
displayed slightly higher transfection efficiencies in cardiac myocytes
compared with (separately cultured) skin fibroblasts, yielding a
1.34-to-1.0 ratio for the number of transfected myocytes to the number
of transfected fibroblasts under the same conditions.
Among the transfection methods based on physical aggregation of plasmid
with various systems, we found liposomes to be most convenient. We then
settled on lipid no. 8 of the Invitrogen transfection kit and (under
the conditions given by the kit instructions) obtained transgene
expression in 6.7 ± 1.6% (n = 9)
of the cells in our cardiac muscle cultures.
Expression of transgenic SERCA1 ATPase.
For the experiments on transgenic ATPase expression, we made two
recombinant adenovirus constructs containing SERCA1 cDNA inserts that
were placed under the control of either the muscle-specific cTnT
promoter or the constitutive CMV promoter and were followed by an
identical polyadenylation signal. An advantage of transfections with
SERCA1 cDNA is that expression of the skeletal ATPase isoform in
cardiac myocytes can be monitored with the monoclonal antibody CaF3-5C3
(22), which does not react with the endogenous SERCA2a enzyme. We then
found that the recombinant SERCA1-adenovirus vector is highly efficient
and yields ATPase expression, under control of either promoter, in the
great majority of cardiac myocytes in culture (Fig.
1). Expression of SERCA1
ATPase was also demonstrated by Western blots obtained with the
SERCA1-specific CaF3-5C3 monoclonal antibody (Fig.
2).

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Fig. 1.
ATPase expression in cardiac myocytes transfected with
sarco(endo) plasmic reticulum
Ca2+-ATPase (SERCA) 1 cDNA.
Chicken embryo cardiac myocytes were transfected with SERCA1 cDNA under
control of cardiac troponin T (cTnT) promoter by means of recombinant
adenovirus (rec-Adv) vector. Myocytes were processed for
immunofluorescent staining 48 h after transfection, using
SERCA1-specific antibody CaF3-5C3 (22).
Left: control cells that were stained
following a transfection procedure without SERCA1 cDNA.
Right: note high percentage of
transfected myocytes showing SERCA1 expression. Magnification:
×100.
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Fig. 2.
Western blots showing cTnT-SERCA1 and cytomegalovirus promoter
(CMV)-SERCA1 expression in chick embryo fibroblasts
(A) and cardiac myocytes
(B). Cultures of chicken embryo skin
fibroblasts or cardiac myocytes were infected with either cTnT-SERCA1
[lanes 3 and
4; 50 plaque-forming units
(PFU)/cell] or with CMV-SERCA1
(lanes 5 and
6; 10 PFU/cell) recombinant adenovirus
or were subjected to a sham transfection procedure
(lanes 1 and
2; no cDNA). Cells were harvested, and
samples from whole homogenates of fibroblasts or myocytes were
processed for Western blotting using a SERCA1-specific monoclonal
antibody. Note occurrence of CMV-SERCA1 expression in both fibroblasts
and cardiac myocytes. Note also occurrence of cTnT-SERCA1 expression
only in cardiac myocytes.
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Cell specificity and efficiency of the cTnT promoter.
The advantage of the cTnT promoter is its cell specificity (2, 31). In
comparative experiments with chick embryo cardiac myocytes and chick
embryo skin fibroblasts, we detected, by Western blots or microscopy,
no transgenic expression in fibroblasts under control of the
muscle-specific cTnT promoter, while obtaining high expression in
cardiac myocytes with the same promoter (Figs. 2 and
3). In addition to demonstrating the cell
specificity of the cTnT promoter, the lack of expression in fibroblasts
indicates that the transfected gene is not influenced by intrinsic
promoters of the recombinant adenovirus. It should be pointed out that
ATPase expression is obtained in both myocytes and fibroblasts when the transfected gene is placed under control of the constitutive CMV promoter (Figs. 2 and 3). We found that SERCA1 expression under control
of the CMV promoter in fibroblasts was only 65 ± 5% of that in
myocytes, when the same adenovirus vector was used at equal PFU titer.

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Fig. 3.
Lack of SERCA1 expression in fibroblasts following gene transfer under
control of cTnT promoter. Cultures of chicken embryo fibroblasts were
infected either with CMV-LacZ (left)
or cTnT-SERCA1 (right) recombinant
adenovirus. Cells were stained 48 h after gene transfer, either for
-galactosidase activity (left) or
for reactivity to SERCA1 monoclonal antibodies
(middle and
right).
Left: number of fibroblasts in culture
and their sensitivity to recombinant adenovirus infection are shown.
Right: no SERCA1 expression occurs in
fibroblasts under control of cTnT promoter. In these experiments,
specificity of promoters was identical whether LacZ or SERCA1 gene was
used. Magnification: ×100.
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Comparison of SERCA1 expression in myocytes under control of the CMV or
cTnT promoter requires assessment of the percentage of myocytes and
fibroblasts in culture, since the CMV promoter sustains expression in
fibroblasts, whereas the cTnT promoter does not. We therefore stained
specifically the cardiac myocytes with a monoclonal antibody (21) that
is reactive to the SERCA2a ATPase (which is not present in fibroblasts)
and counted the percentage of fluorescent cells under microscopic
observation. We found that myocytes account for 86.1 ± 9%
(n = 7) of cells in our chick embryo heart cultures 24 h after seeding, whereas, due to proliferation of
fibroblasts, the myocytes account for 62 ± 11%
(n = 41) at harvesting time (i.e., 72 h after seeding or 48 h after transfection).
Notwithstanding the lower strength of the cTnT promoter (31), we
obtained cell-specific SERCA1 expression at levels similar to those
obtained with the CMV promoter by raising the recombinant adenovirus
PFU levels. Densitometric evaluation of Western blots indicates that
SERCA1 expression under control of the CMV promoter was twofold higher
than that under control of the cTnT promoter when the PFU levels were
10 and 50 PFU/seeded cell, respectively. It should be pointed out,
however, that 38% of all cells in the cardiac culture are fibroblasts
(which do not sustain ATPase expression under control of the cTnT
promoter, but yield 65% as much expression as myocytes under control
of the CMV promoter). Therefore, in comparative experiments on SERCA1
expression under control of the cTnT promoter and the CMV promoter, the
observed levels of total expression under control of the CMV promoter
are likely to exceed by ~25% the actual levels expressed in
myocytes. At any rate, even though SERCA1 protein is produced in either
case, it is clear that the advantage of the cTnT promoter is its cell specificity.
Intracellular targeting of transgenic ATPase expression.
Immunofluorescent micrographs of myocytes expressing SERCA1 following
transfection by the liposome or recombinant adenovirus methods (Fig.
4) are consistent with transgenic SERCA1
targeting to intracellular membranes, independent of the transfection
procedure. We extended our experimentation to clarify whether the
membrane targeting of transgenic SERCA1 isoform expression is the same as that of the endogenous SERCA2a ATPase. To this aim, we subjected transfected cells to homogenization and differential centrifugation and
then obtained Western blots of the subcellular fractions, staining the
same samples in parallel with the monoclonal antibody CaF3-5C3, which
is specific for chicken SERCA1 ATPase (22), and with the monoclonal
antibody CaS-3H2, which is specific for the chicken endogenous SERCA2a
ATPase (4). It is shown in Fig. 5 that the
distribution of immunofluorescent label among various subcellular
fractions is identical for the transgenic and endogenous ATPases
following infection with cTnT-SERCA1 adenovirus. Both endogenous and
transgenic ATPases are prevalently associated with the microsomal
fraction (i.e., sarcoendoplasmic reticulum).

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Fig. 4.
Intracellular membrane targeting of ATPase expression in cardiac
myocytes transfected with SERCA1 cDNA. Chicken embryo cardiac myocytes
were transfected with SERCA1 cDNA under control of cTnT promoter,
either by means of liposome aggregates
(left) or recombinant adenovirus
vector (right). Cells were processed
for immunofluorescent staining 48 h after transfection, using CaF3-5C3
SERCA1-specific antibody (22). Magnification: ×400.
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Fig. 5.
Western blots showing localization of transgenic cTnT-SERCA1 and
endogenous SERCA2a (endoSERCA2a) in subcellular fractions of
transfected cardiac myocytes. Cultures of chicken embryo cardiac
myocytes were infected with cTnT-SERCA1 recombinant adenovirus. Two
days after infection, cells were harvested, homogenized, and subjected
to differential centrifugations, and subcellular fractions were
collected. Preparations were processed for Western blotting, and same
samples were probed in parallel with monoclonal antibodies specific for
SERCA1 (CaF3-5C3) and SERCA2a (CaS-3H2) isoform.
Lanes 1 and
5: whole homogenate;
lanes 2 and
6: first fraction (cell membranes and
nuclei); lanes 3 and
7: second fraction (mitochondria); and
lanes 4 and
8: third fraction (microsomes).
Lanes 1, 2,
3, and
4 were probed with CaF3-5C3, whereas
lanes 5, 6,
7, and
8 were tested with CaS-3H2. Note that
transgenic ATPase is prevalently associated with microsomal fraction
(i.e., sarcoendoplasmic reticulum), colocalizing with endogenous
SERCA2a.
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ATP-dependent
Ca2+ uptake.
Active transport of Ca2+ by SERCA
can be assessed by the use of cardiac muscle homogenates in a reaction
mixture containing radioactive calcium isotope and ATP. We found that
homogenates of chick embryo cardiac culture sustain ATP-dependent
Ca2+ uptake with an average
initial velocity of 3.7 nmol
Ca2+ · mg
protein
1 · min
1.
The activity is totally inhibited by 1 micromolar thapsigargin (Fig.
6), which is a highly specific inhibitor of
SERCA (33).

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Fig. 6.
Ca2+ uptake by homogenates of
control and SERCA1 transgenic myocytes. Cultures of cardiac myocytes
were infected with cTnT-SERCA1 (+) or CMV-SERCA1 ( , ) recombinant
adenovirus, with PFU levels of 100 (+), 3 ( ), or 10 ( ) PFU/seeded
cell. Myocytes were collected for measurements of ATP-dependent
Ca2+ uptake 48 h after infection.
Control cultures ( ) were subjected to gene transfer procedure
without cDNA. Note that Ca2+
uptake was totally inhibited by 1 µM thapsigargin ( ), which is a
specific inhibitor of SERCA enzymes. Uptake shown was not corrected to
exclude contribution of fibroblasts. Western blots
(inset) show levels of transgenic
SERCA1 and endogenous SERCA2a expression in control myocytes
(lane 1), in cells infected with
cTnT-SERCA1 adenovirus (lanes 2 and
3), and in cells infected with
CMV-SERCA1 adenovirus at lower (lane 4) and higher
(lane 5) PFU titer.
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We then studied the effect of SERCA1 gene transfer by means of
recombinant adenovirus vectors in chick embryo cardiac cultures. It is
of interest that, under conditions of optimal PFU titers, we found
similar rates (14.6 nmol · mg
protein
1 · min
1)
of Ca2+ uptake (Fig. 6) following
transgenic SERCA1 expression under control of either the cTnT promoter
(100 PFU/seeded cell) or the CMV promoter (either 3 or 10 PFU/seeded
cell). This is a fourfold increase relative to the rates sustained by
control samples and is likely to correspond to an upper limit for the
ability of myocytes to express functional protein. In fact, parallel
Western blotting analysis shows that the amount of total protein
expressed is higher when high CMV-SERCA1 adenovirus titer is used, even
though the Ca2+ uptake activity is
not increased proportionally. It is also of interest that the
expression of endogenous SERCA2a appears to be reduced by 30-60%
under the conditions used for transgenic SERCA1 expression, as shown by
densitometry of the Western blots in Fig. 6.
Effects of SERCA1 transfection on contractile behavior and
[Ca2+]i
transients of intact myocytes.
A series of experiments was performed to determine whether the increase
in Ca2+ transport in vesicles
isolated from SERCA1-transfected myocytes is reflected in changes of
contractile dynamics and/or
Ca2+-handling properties of intact
cells. As shown in Fig.
7A,
transfected cells display dramatically shortened twitches, due to a
reduction of both tension development and relaxation times. In fact,
waveform analysis demonstrated a reduction of the width at half height from 223 ± 10 ms (n = 24) for
control cells to 160 ± 13 ms (n = 15) for cTnT-SERCA1 transfected cells. Similar effects were noted on
the
[Ca2+]i
transients (Fig. 7B), as the first
order time constant of the decay phase was decreased by 40%, from 190 ± 18 ms (n = 27) in control to 113 ± 13 ms (n = 15) in
cTnT-SERCA1 transfected cells. Similar results were obtained with
transgenic expression of SERCA1 under control of the CMV promoter.
These effects, originally observed by Hajjar et al. (13) following
transfection of neonatal rat myocytes with heterologous SERCA2a under
control of a viral promoter, cannot be related quantitatively to
transgene expression as accurately as the transport measurements
described above. Nevertheless, the results shown in Fig. 7 demonstrate
that expression of transgenic ATPase under control of the cell-specific
cTnT promoter has a strong influence on the
[Ca2+]i
transients in situ, just as transgenic expression does when under
control of a viral promoter.

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Fig. 7.
Contractility (A) and cytosolic
Ca2+
([Ca2+]i)
transients (B) in control and
SERCA1-transfected cultured myocytes.
A: cultured myocytes were field
stimulated at 2 Hz, and contractile behavior was recorded by video
microscopy. B: fluo 3-loaded cultured
myocytes were field stimulated, and resulting
[Ca2+]i
transients were recorded and analyzed as described in
METHODS. Normalized signal averages
from 3 cells are shown for each curve. See text for statistical
analysis.
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 |
DISCUSSION |
Consistent with previous reports (1, 23, 25, 34), our experiments
demonstrate unambiguously that recombinant adenovirus is a very
efficient vector for gene transfer into myocytes and fibroblasts,
yielding transgene expression in nearly all cells exposed when the
multiplicity of infection is optimized. The efficiency of recombinant
adenovirus is much higher than that obtained with methods based on
aggregation of transfection plasmids with calcium phosphate, liposome,
or adenovirus-polylysine complex.
Independent of the transfection vector, our findings contribute to
characterization of the cTnT promoter. We used the
268-base pair
(bp) segment (Fig. 8) of the chicken cTnT
promoter (31), which includes tandem M-CAT, "CarG,"
"MEF-1," and SP1 motifs in the proximal region
(
129 to
49 bp), and a cardiac element in the distal
region (
269 to
201 bp). Similar motifs are also present in a proximal (
284 to
72 bp) and a distal (
1810 to
1110 bp) segment of the SERCA2 promoter (3). We
used the cTnT promoter for its compact size and very strong
specificity. In fact, in our experiments, the
268-bp segment of
the cTnT promoter proved to be highly specific for myocytes, with no
appreciable leak in fibroblasts. Although the
268-bp segment of
the cTnT promoter is significantly weaker than the CMV promoter, we
were able to obtain similar levels of functional ATPase expression by
adjusting the PFU levels of recombinant adenovirus vectors containing
the two different promoters.

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|
Fig. 8.
Functional elements of 268-bp segment of cardiac troponin T
(cTnT) promoter. Elements involved in transcription regulation are
underlined (31).
|
|
It is noteworthy that previous studies of this cell-specific promoter
were performed with reporter genes by transfection methods involving a
small percentage of cells in heterogeneous cultures. In our
experiments, we have extended the characterization of a short segment
(
268 bp) of the cTnT promoter by the use of an isomorphic
endogenous gene that requires specific intracellular targeting for its
function. We have also used a recombinant adenovirus vector that
mediates gene transfer into the majority of cells in culture and
demonstrated that in the recombinant virus the gene remains under
exclusive control of the cell-specific promoter and is not influenced
by intrinsic viral promoters.
Functionally, we obtained a fourfold increase in ATPdependent
calcium uptake over endogenous SERCA ATPase levels in chick cardiac
embryonic myocytes, after SERCA1 gene transfer using recombinant adenovirus vectors under control of the cell-specific promoter. This is
quite a bit higher than that obtained previously by means of transgenic
expression of heterologous SERCA2a under control of constitutive viral
promoters in cultured myocytes of neonatal rats (11, 13) and in
transgenic mouse hearts (15). Most importantly, our experiments suggest
that there is an upper limit for the ability of the myocytes to express
functional SERCA protein, a limit that can be reached either under
control of the cell-specific or the constitutive viral promoter.
Finally, we find that transgenic SERCA1 expression under control of the
cTnT promoter affects contractile dynamics and
[Ca2+]i
transients in situ just as much as transgenic expression under control
of the viral promoter.
Our findings raise the possibility of manipulating
Ca2+ homeostasis and
Ca2+-dependent functions
specifically in cardiac myocytes within heterogeneous cell populations
by means of gene transfer under control of cell-specific promoters.
Furthermore, our observations may be helpful in designing suitable
constructs for cell-specific transgenic targeting in whole cardiac
muscle by means of recombinant adenovirus vectors (8).
 |
ACKNOWLEDGEMENTS |
This work was partially supported by National Heart, Lung, and
Blood Institute Grants P01-HL-27867 (to G. Inesi) and HL-43821 (to C. P. Ordahl).
 |
FOOTNOTES |
Address for reprint requests: G. Inesi, Dept. of Biochemistry and
Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201-1503.
Received 18 August 1997; accepted in final form 14 November 1997.
 |
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