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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 pDelta 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 (beta -galactosidase) and EGFP reporter genes, obtained from Clontech (Palo Alto, CA), were also subcloned into the pDelta 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(beta -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
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Abstract
Introduction
Methods
Results
Discussion
References

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

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.

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 beta -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.

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.

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 (black-triangle, triangle ) recombinant adenovirus, with PFU levels of 100 (+), 3 (black-triangle), or 10 (triangle ) PFU/seeded cell. Myocytes were collected for measurements of ATP-dependent Ca2+ uptake 48 h after infection. Control cultures (bullet ) were subjected to gene transfer procedure without cDNA. Note that Ca2+ uptake was totally inhibited by 1 µM thapsigargin (black-square), 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.

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.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
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Abstract
Introduction
Methods
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Discussion
References

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