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(Circulation Research. 1996;78:971-977.)
© 1996 American Heart Association, Inc.

Articles

Novel Adenovirus Component System That Transfects Cultured Cardiac Cells With High Efficiency

Trudy A. Kohout, Jeffrey J. O'Brian, Shirley T. Gaa, W. Jonathan Lederer, Terry B. Rogers

From the Department of Biochemistry and Molecular Biology (T.A.K., S.T.G., T.B.R.) and the Department of Physiology (T.A.K., W.J.L.), University of Maryland School of Medicine, Baltimore, and Dupont/Merck (J.J.O.), Research and Development, Glenholden Laboratory, Glenholden, Pa.

Correspondence to Dr Terry B. Rogers, Department of Biological Chemistry, University of Maryland School of Medicine, 108 N Greene St, Baltimore, MD 21201. E-mail trogers@umabnet.ab.umd.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Although it is clear that gene transfection is a potentially valuable approach in the study of cardiac cell function and differentiation, classic transfection methods are limited by their poor efficiencies in cardiac cells. Recent studies show that recombinant replication-defective human adenovirus can transfect primary cardiac cultures with near 100% efficiency. Since such recombinants are time consuming to prepare, the goal of this study was to develop a plasmid/viral transfection system that would capitalize on the advantages of adenovirus. We have found that a "component system" formed by preincubation of Ad5dl312 adenovirus, poly-L-lysine, and an expression plasmid (lacZ reporter gene under control of the human cytomegalovirus (HCMV) major immediate early promoter) can transfect cultured cardiac cells. Optimal conditions were determined by quantifying ß-galactosidase expression. Histochemical analysis of cultures revealed that the component system transfected 70% of the cells under these conditions. LacZ-positive myocytes could be identified in intact myocytes with the fluorescent substrate C₁₂-fluorescein di-ß-galactopyranoside. Functional studies with such cells indicated that contractile behavior was maintained in transfected cardiocytes. Furthermore, the component system was used to transfect a DNA vector expressing a physiologically relevant protein, protein kinase C. In summary, this powerful and simple approach can promote the expression of heterologous genes that can be studied at the biochemical and cellular level in cardiac cells.

Key Words: transfection • adenovirus • myocytes • gene expression

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The efficient introduction of foreign DNA into heart cells is an important goal of gene therapy research. Such techniques also show great promise in the molecular characterization of cardiac tissue function and development. However, since there are no stable cardiac myocyte cell lines, studies have been restricted to transient transfections. Several classic transfection methods, including cationic lipid/plasmid DNA complexes, incubations with "naked" plasmid DNA, and calcium phosphate precipitation, have been used to transfect cultured neonatal cardiac myocytes and adult cardiac cells. All of these methods achieve very low transfection efficiencies in cardiac cells, generally no more than a few percent.¹ Although these techniques have provided valuable information on cardiac gene regulation,² their utility in the expression and characterization of functional cardiac proteins is limited.

Recently, replication-defective recombinant adenoviruses that overcome many of the limitations of the classic DNA transfection methods have been introduced.³ In fact, recombinant adenovirus constructs have been shown to transfect adult rat and cultured neonatal ventricular myocytes with virtually 100% efficiency because of the unique properties of the adenovirion.^{4 5} Adenovirus enters the host cell by attachment to _v-ß₃/ß₅ integrin receptors of host cells. The virion-receptor complex is then internalized into endosomal vesicles, which are disrupted by the viral capsid proteins, releasing the virion into the cytosol.⁶ Thus, the endosomolytic properties of adenovirus enhance the delivery of plasmid DNA into the cell. Although the recombinant vectors are a major advancement for cardiac transfections, experimental design is limited by the size of the DNA that can be incorporated into the viral genome, 7 kb or smaller, and by the effort required to produce new recombinant constructs.

Thus, the goal of the present study was to develop a simple cardiac transfection system that combines the convenience of plasmid DNA with the unique targeting properties of adenovirus vectors. We report the development of an adenovirus/polylysine/plasmid DNA "component system" that has an efficiency that approaches that observed with recombinant adenovirus vectors. Furthermore, by performing cotransfections with appropriate reporters, one can select transfected cells for functional studies. Finally, the value of this system has been underscored by the characterization of the expression of epitope-tagged PKC in cultured cardiac cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
The serum-free chemically defined culture medium, PC1, was purchased from Hycor Biomedical Inc. The HEK 293 cell line was purchased from American Type Culture Collection. The ß-galactosidase substrates, C₁₂FDG (ImaGene Green kit) and X-gal, were purchased from Molecular Probes and GIBCO-BRL, respectively. Lipofectin reagent, a 1:1 cationic lipid formulation of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoyl phosphatidylethanolamine, was purchased from GIBCO-BRL. The polyclonal anti-PKC antibody, directed against a PKC peptide, was obtained from GIBCO-BRL. Nitrocellulose membranes, Duralose-UV, were purchased from Stratagene, and the ECL detection kit was from Amersham Life Sciences. All other reagents used, including poly-L-lysine (molecular weight, 34 000 to 48 000), were obtained from Sigma Chemical Co.

Neonatal Rat Ventricular Myocyte Culture
Cultured neonatal rat ventricular myocytes were prepared from 1-day-old Sprague-Dawley rats and grown in serum-free PC1 medium, as previously described.⁷ The cultures were exposed to -irradiation (35 Gy) 24 hours after plating, which, as previously described, effectively limits the proliferation of nonmyocytes and maintains myocyte levels to >80% over time.⁸ The medium was then changed to a 1:4 dilution of PC1/DMEM supplemented with 5 mmol/L glutamine and 1% gentamicin. Transfections were performed on 1- to 2-day-old cultures.

Vectors for Transfections
The vector pHCMVßgal, used to determine transfection efficiencies, was constructed from the pBEX1 vector (British Biotechnology Ltd) and contains the Escherichia coli lacZ gene immediately downstream from the human cytomegalovirus major immediate-early promoter. A replication-deficient recombinant adenovirus, Ad/CMV-lacZ, was constructed by recombining a lacZ expression cassette into the E1 region of Ad5dl324. The dl324 mutant contains deletions of the E1 and E3 regions. The expression cassette contains the HCMV major immediate-early promoter, an E coli lac Z gene modified for mammalian expression, and the SV40 polyadenylation signal. The PKC epitope–tagged PKC expression vector was generously provided by Peter Blumberg and Zoltán Szállási (National Institutes of Health, Bethesda, Md). The vector was constructed by subcloning the wild-type PKC into the mammalian expression vector with a metallothionein promoter upstream from the PKC epitope sequence, MTH.⁹

Transfection Using Component System
Transfections were performed with the replication-deficient human adenovirus type 5 mutant, Ad5dl312, generously provided by Thomas Shenk (Princeton University, Princeton, NJ).¹⁰ Ad5dl312 adenovirus was propagated in the complementing human embryonal kidney cell line, HEK 293,¹¹ as described previously.² Briefly, virus was harvested from HEK 293 cells 24 hours after infection. The cells were lysed with five cycles of freezing and thawing to release virus. The virus was then purified by density gradient centrifugation with two consecutive CsCl gradients (step gradients with 1.2 and 1.45 g/mL). The viral band was collected and dialyzed (Spectra/Por; MWCO, 12 000 to 14 000) against 10 mmol/L Tris-HCl, pH 7.4, 1 mmol/L MgCl₂, and 10% glycerol for 4 hours at 4°C.¹² The viral stock solution was stored in aliquots at -70°C. Virion concentration was determined using the following relationship: one absorbance unit at 260 nm is equal to 10¹² viral particles per milliliter.¹³ Plaque assays on HEK 293 cells with purified virus were performed to determine the titer of each preparation.¹² The particle–to–plaque forming unit ratio of the Ad5dl312 preparation used in this study was 32:1.

Pilot studies revealed that a combination of Ad5dl312, poly-L-lysine (molecular weight, 34 000 to 48 000), and plasmid DNA was an effective gene delivery mixture in cultured heart cells. Optimal concentrations of the components were 2x10¹⁰ viral particles per milliliter with 2.5 µg/mL polylysine and 2.5 µg/mL plasmid DNA. The transfection mixture was prepared as a twofold concentrated stock in the following manner. An aliquot of the Ad5dl312 virus stock solution was combined with 12.5 µL of polylysine-concentrated solution (33 µg/mL in PC1/DMEM) in a final volume of 125 µL of PC1/DMEM (1:4), such that the final concentration of virus was 4x10¹⁰ particles per milliliter. After an incubation of 30 minutes at room temperature, an aliquot of plasmid DNA (1 mg/mL in 10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.4) was added to the mixture at a final concentration of 5 µg/mL. After a second 30-minute incubation at room temperature, 6 µL of polylysine stock was added to the mixture, which was then incubated for 10 minutes. The gene delivery mixture solution was diluted with an equal volume of PC1/DMEM (1:4) medium, and the transfection was initiated by replacing the culture medium with 250 µL of this transfection mixture per 17-mm-diameter well (seeded at 6x10⁵ cells per well 24 to 48 hours before transfection). After an incubation of 90 minutes at 37°C, the reaction was terminated by diluting the transfection mixture by the addition of 750 µL of PC1/DMEM (1:4). The cells were maintained in culture in this medium for 48 hours before further analysis.

ß-Galactosidase Expression
The transfection efficiencies of the various methods were assessed by measuring ß-galactosidase activity by histochemical staining of fixed cells, by a colorimetric enzyme assay with cell extracts, and by a cell-permeant fluorescent substrate, C₁₂FDG (Molecular Probes), with intact myocytes.¹⁴ For the histochemical analyses, the cells were fixed with 0.5% glutaraldehyde and stained as previously described.¹⁵ Transfection efficiency was evaluated by the percentage of cells that were stained blue.

ß-Galactosidase activity was measured in cell lysates by a method adapted from a previous procedure.¹⁶ Lysates were prepared by removing the monolayers with a scraper in 0.25 mol/L Tris-HCl and 5 mmol/L dithiothreitol, pH 7.8. The suspensions were then homogenized in microfuge tubes with disposable pestles. Cell lysates (2.5 µg cell protein) were incubated in 300 µL of 40 mmol/L Na₂HPO₄, 27 mmol/L NaH₂PO₄, 33 µmol/L KCl, 3.3 µmol/L MgCl₂, 166 µmol/L ß-mercaptoethanol, and 1.3 mg/mL o-nitrophenyl ß-D-galactopyranoside at 37°C until a visible yellow color was achieved, usually by 15 minutes.¹⁶ The reaction was stopped by adding 0.5 mL of 1 mol/L Na₂CO₃, and the product was quantified by measuring the absorbance at 420 nm. The ß-galactosidase activity was normalized to total cell protein for each reaction using the Bradford method (Bio-Rad Laboratories Inc) and was corrected for endogenous ß-galactosidase using extracts from nontransfected cells. The resulting ß-galactosidase activity was reported as the OD₄₂₀ per milligram of protein per hour.

Visualization of ß-galactosidase expression in intact cells was accomplished by incubating cultures with 33 µmol/L C₁₂FDG substrate in normal growth media or PBS for 30 minutes at 37°C.¹⁴ Transfected cells were selected as intensely fluorescent cells when illuminated with light at 488 nm and detected with epifluorescent optics. Control experiments indicated that the fluorescence of nontransfected cells was negligible under these conditions. However, background labeling became significant in incubations of >2 hours.

Transfections with PKC Expression Vector
Since the expressed PKC was tagged with a PKC epitope, transfected gene expression was quantified by Western blot analysis using an antibody against PKC. Cells were lysed with 20 mmol/L Tris-HCl, pH 7.5, 2.0 mmol/L EDTA, 0.5 mmol/L EGTA, 0.25 mol/L sucrose, 25 µg/mL leupeptin, 20 µg/mL phenylmethylsulfonyl fluoride, 10 mmol/L ß-mercaptoethanol, and 1% Triton X-100. Cell lysates were centrifuged, and the supernatants were removed and concentrated in Centricon 10 microconcentrators (Amicon Inc) for 2 hours at 4°C. The samples were electrophoresed on an 8% SDS Laemmli gel and then transferred onto a Duralose-UV membrane (Stratagene). After the membranes were blocked with 5% nonfat powdered milk/0.1% Tween 20 in PBS, they were incubated with the anti-PKC primary antibody (GIBCO-BRL) diluted 1:1000 in 0.83% nonfat milk/0.1% Tween 20 in PBS overnight at 4°C. The protein bands were visualized by chemiluminescence using the ECL kit (Amersham Life Sciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is clear that the adenovirus construct is a promising gene delivery vector in cardiac cells.¹⁷ However, recombinant constructs are complicated and time consuming to produce. Initial results in the present study revealed that a simple adsorptive ternary complex of adenovirus, polylysine, and plasmid DNA can effectively transfect cardiac cells.

In order to critically assess the properties of this new DNA delivery system, neonatal rat cardiac myocytes were transfected with a DNA plasmid encoding the reporter gene, lacZ. The level of transfection was quantified by measuring ß-galactosidase activity in total cell extracts from the cultures. Fig 1A shows the effects of polylysine on ß-galactosidase expression. In the absence of polylysine, there was no reporter gene expression in incubations with a binary complex of Ad5dl312 and lacZ expression plasmid. Expression was markedly dependent on polylysine concentration, with maximal levels of ß-galactosidase activity at 2.5 µg/mL polylysine. At higher polylysine concentrations, expression was reduced by 90%, in part because these doses were toxic to the cells. Fig 1B illustrates that efficient transfection was defined by a narrow range of plasmid DNA concentrations as well, with an optimal ratio of DNA molecules to polylysine observed at 1:100. At concentrations of >2.5 µg/mL DNA, the transfection efficiency rapidly declines, with no measurable ß-galactosidase expression observed at three times the optimal DNA concentration. In this case, however, based on cell morphology, the decline is not due to toxic effects but may be due to an increase in negative charge (see "Discussion"). These observations demonstrate that the plasmid DNA–to–polylysine ratio is a critical factor for the production of an efficient DNA delivery vehicle.

View larger version (11K): [in this window] [in a new window]   Figure 1. Optimization of the components of the transfection complex. Neonatal rat ventricular myocytes were transfected with various concentrations of polylysine (A), pHCMVßgal DNA vector (B), and Ad5dl312 virus (C). In panel A, the polylysine concentration added to the myocytes (in micrograms per milliliter) is plotted against the resulting ß-galactosidase activity measured in OD420 per milligram protein per hour incubation, as described in "Materials and Methods." The pHCMVßgal vector and the Ad5dl312 virus concentrations were held constant at 1 µg/mL and 2x1010 particles per milliliter, respectively. Parallel experiments were performed in panel B; the pHCMVßgal vector concentrations were varied as indicated on the x axis, and the polylysine and the Ad5dl312 virus concentrations were held constant at 2.5 µg/mL and 2x1010 particles per milliliter, respectively. In panel C, the expressed ß-galactosidase activities were measured after transfections with various Ad5dl312 virus concentrations. The transfection complexes also contained 2.5 µg/mL polylysine and 1 µg/mL pHCMVßgal vector. Data points are the mean±SEM of at least three experiments.

Fig 1C shows the optimization of the Ad5dl312 required for transfection. The DNA plasmid/polylysine complex alone yields no ß-galactosidase activity; thus, the adenovirus component is strictly required for lacZ expression. The data in Fig 1C indicate that concentrations of Ad5dl312 of 2x10¹⁰ particles per milliliter and above yield similar transfection rates when polylysine and DNA are fixed. However, since the ß-galactosidase assay is normalized to cell protein, possible toxic effects of virus on the heart cells could go undetected in this assay. Complementary histochemical analyses were performed to further evaluate the transfection efficiency (Fig. 2). Fig 2B shows results with optimal conditions for transfection efficiency and cell viability. Quantification by counting cells in five different ocular fields from four different transfections revealed that 68±2% of myocytes expressed ß-galactosidase. Although the transfection efficiencies with virus concentrations of 2x10¹⁰ particles per milliliter and above are similar (in agreement with Fig 1C), toxic effects are seen with higher concentrations, as displayed by the poor morphology and the loss of cells from the culture plates (Fig 2C and 2D). The toxic effects are attributed to adenovirus, since addition of the plasmid/polylysine complex alone did not result in cellular toxicity. Also, addition of Ad5dl312 alone to the cells produces toxic effects similar to those seen with the component system. Furthermore, concentrations of Ad5dl312 >2x10¹⁰ particles per milliliter compromised spontaneous beating behavior. In summary, when polylysine, plasmid DNA, and Ad5dl312 are added directly to cultures, there is no DNA transfection. However, by preforming a ternary complex, within well-defined concentration ranges for these components, one can prepare a remarkably effective DNA transfection system.

View larger version (142K): [in this window] [in a new window]   Figure 2. Expression of lacZ in cardiac myocytes transfected with various concentrations of Ad5dl312 virus. Representative fields are shown of myocytes transfected with the component system with various concentrations of Ad5dl312 virus, 2.5 µg/mL polylysine, and 1 µg/mL pHCMVßgal vector as described in "Materials and Methods." Concentrations of Ad5dl312 used were 1x1010 particles per milliliter (A), 2x1010 particles per milliliter (B), 4x1010 particles per milliliter (C), and 10x1010 particles per milliliter (D). Myocytes were fixed with 0.5% glutaraldehyde and stained for ß-galactosidase activity with X-gal 48 hours after transfection.

Since recombinant adenovirus vectors containing lacZ have been shown to be extremely efficient DNA delivery vectors in cardiac cells,⁵ the efficacy of this component system was directly compared with such a construct. A comparison of the absolute levels of lacZ expression in transfected cells under optimal conditions for both methods shows that myocytes transfected with the component system express 70% of the ß-galactosidase activity of the recombinant adenovirus-transfected myocytes (Fig 3). This value is in agreement with the fraction of the cells transfected, as seen in histochemical analyses described above. In contrast, transfection with a charged lipid DNA delivery system yielded only 1.6% of the ß-galactosidase activity found in the Ad/CMV-lacZ–transfected myocytes (Fig 3). Efficiency of gene transfer can also be analyzed in terms of the molecules of plasmid DNA that need to be added per cell to yield optimum expression. By such an analysis, adenovirus-transducing chromosomes are far more efficient. The component system requires an addition of 75 000 molecules of DNA per cell for optimum transfection, whereas in the recombinant adenovirus, only 170 produce maximal ß-galactosidase expression. In fact, the recombinant adenovirus would appear even more efficient if concentrations were decreased to produce ß-galactosidase expression equivalent to that seen with the component system. However, both systems are far more efficient when compared with the lipofectin-mediated transfections, where 750 000 molecules of DNA per cell produced the low responses depicted in Fig 3. However, taken together, these data reveal that in terms of protein expression levels, an important strategy in many studies, the ternary component system compares very favorably with the very effective recombinant adenovirus constructs.

View larger version (23K): [in this window] [in a new window]   Figure 3. Comparison of Ad/CMV-lacZ–mediated, component system–mediated, and lipofectin-mediated transfections. The bar on the left shows myocytes transfected with 4x108 particles of Ad/CMV-lacZ per milliliter in 250 µL PC1:DMEM (1:4) medium for 90 minutes. A parallel transfection was performed (middle bar) with the component system consisting of 2x1010 particles of Ad5dl312 per milliliter, 2.5 µg/mL polylysine, and 1 µg/mL pHCMVßgal. The bar on the right shows the transfection of myocytes with 10 µg/mL of lipofectin reagent and 5 µg/mL pHCMVßgal. Myocytes were incubated with the lipofectin-vector complex for 10 hours at 37°C. Then, the transfection mixture was removed, and fresh medium was added. After 48 hours, the transfected myocytes were harvested, and the ß-galactosidase activity was measured as described in "Materials and Methods." The error bars show the standard error of the mean of at least three experiments. To compare the transfection efficiencies of the three methods, the ß-galactosidase activity measured with the Ad/CMV-lacZ–mediated transfection was taken as 100% transfection efficiency.

Although the component system achieves a very high transfection rate, it is clear that not all the cells are transfected (Fig 2B). Thus, it is crucial to develop methods to identify transfected cells from the general population. We exploited a method that images ß-galactosidase expression in intact cells with the use of a fluorescent cell-permeable ß-galactosidase substrate, C₁₂FDG.¹⁴ As shown in the photomicrographs of Fig 4, transfected myocytes can indeed be identified by fluorescence microscopy. Under the incubation conditions used, there is no background labeling of the cells (Fig 4C).

View larger version (133K): [in this window] [in a new window]   Figure 4. Imaging of transfected myocytes with the ß-galactosidase substrate C12FDG. Panels A and B show myocytes transfected with the component system containing 2x1010 particles of Ad5dl312/mL, 2.5 µg/mL polylysine, and 2.5 µg/mL pHCMVßgal vector. After 48 hours, the myocytes were incubated with 33 µmol/L C12FDG in PBS for 30 minutes. To stop the reaction, the substrate was removed, and the medium was replaced with PBS. Panel A is the fluorescence image that corresponds to the transmitted light image in panel B. The fluorescence image was obtained by exciting the cells at 488 nm and collecting the emitted light at 560 nm. Panels C and D show nontransfected myocytes incubated with 33 µmol/L C12FDG in PBS for 30 minutes. Panel C is the fluorescence image taken at the same light intensity, brightness, and contrast as the image in panel A. Panel D is the corresponding transmitted light image.

This ß-galactosidase imaging system was used to assess the contractile behavior of transfected cardiac cells. After identification of a transfected cell with fluorescent optics, the cell was field-stimulated, and the resulting contractile behavior was recorded by video edge detector as previously described.¹⁸ The contractile response of the transfected myocyte (Fig 5A) was identical to that of a control cell in a nontransfected culture (Fig 5B). Taken together, these data indicate that neither the transfection, the induced ß-galactosidase activity, nor the C₁₂FDG substrate alters the contractile function of the myocytes.

View larger version (32K): [in this window] [in a new window]   Figure 5. Comparison of contractile behavior of transfected and nontransfected myocytes. Myocytes were mounted in a superfusion chamber with constant superfusion with DMEM with 25 mmol/L HEPES, and contractile behavior was monitored using a video edge detector. Panel A shows the contractile behavior of a control (nontransfected) myocyte. Parallel cultures were exposed to the component adenovirus system, and a transfected cell was identified using the C12FDG substrate as described in Fig 4. The optics were changed to phase-contrast transmitted light, and the contractile behavior was recorded as shown in panel B.

Since detection assays for ß-galactosidase expression are very sensitive, the levels of expressed protein produced with the component system could be very low. Yet, if the component system is to be broadly useful, it must be able to promote the expression of relevant proteins at biological levels. For this purpose, we transfected an expression plasmid containing the wild-type PKC, which contained an epitope tag derived from the immunoreactive peptide sequence of PKC. Since the molecular weights of PKC and PKC are different, this epitope tag allowed for a direct comparison of the levels of exogenous PKC expression with those for native PKC through Western blot analyses.⁹ As shown in Fig 6A, the levels of PKC expression (lane 1, 70-kD band) were comparable to those of endogenous PKC (lane 1, 90-kD band). Thus, the component system directed the expression of PKC to levels nearly identical to that of a major PKC isoform of heart cells, PKC.^{19 20 21} In related experiments, the myocytes that were cotransfected with both vectors (Fig 6B, lane 3) express at least as much ß-galactosidase activity as the cultures that were transfected with lacZ alone (lane 2). Therefore, the cotransfection of PKC and lacZ does not compromise the level of expression of either construct. In summary, the data in the present study demonstrate that the component system is flexible and is broadly useful for many plasmid cotransfection applications.

View larger version (65K): [in this window] [in a new window]   Figure 6. Cotransfection of PKC and ß-galactosidase into cardiac myocytes. (A), Western blot analysis of transfected myocytes with 2.5 µg/mL PKC vector (lane 1), 2.5 µg/mL pHCMVßgal vector (lane 2), and PKC and pHCMVßgal vectors, 2.5 µg/mL of each (lane 3). Cells were transfected with the component system, with vector concentrations stated above plus 2x1010 particles per milliliter of Ad5dl312 and 5 µg/mL polylysine. In lane 4 are control nontransfected cells. Transfection cultures were incubated for 48 hours before analysis for expression of vectors. Whole-cell lysates were prepared from the transfected cells, and the proteins were resolved on an 8% SDS Laemmli gel and then transferred onto a Duralose-UV membrane. The blot was probed with anti-PKC antibody as described in "Materials and Methods." (B), Parallel transfections performed to measure the corresponding ß-galactosidase activities under the same conditions as above. The first three bars show the ß-galactosidase activities measured from cells transfected with the PKC vector, the pHCMVßgal vector, and the pHCMVßgal and the PKC vectors together. The bar on the right shows the ß-galactosidase activity of nontransfected (Cont) myocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gene expression studies in cultured cardiac myocytes that use classic plasmid DNA transfection methods, including those with cationic lipids, calcium phosphate, or DEAE dextran, have been limited by their very low yield of transfected cells. Thus, there is a need for further development of convenient, efficient, and flexible gene delivery systems for cardiac tissue. An important advancement was the discovery that recombinant adenovirus constructs were excellent vectors for heterologous gene expression in cardiac tissue.^{3 4 12 17 22} From what is known of the infective pathway for adenovirus,¹⁷ the remarkable efficiency of the adenovirus systems in nondividing cardiac myocytes resides, in part, from the avid binding of adenovirus to _v-ß₃/ß₅ integrin receptors, which are found in high levels on such cells. After receptor-mediated endocytosis, the ability of adenovirus to lyse and escape from endosomes and to deliver DNA to the nucleus underlies the efficient infective pathway of recombinant Ad5 vectors in this setting.^{5 17}

Several ingenious transfection/plasmid DNA complexes have been devised that capitalize on this unique infective pathway. Chemically modified adenovirus, consisting of polylysine covalently linked to adenoviral coat proteins, has been used to adsorb plasmid DNA and transferrin/polylysine reagent onto the virion surface.²³ Such transferrin-targeted complexes direct protein expression in hepatocytes with impressive efficiency. Another covalent-conjugated complex of asialogylcoprotein/polylysine/adenovirus was also a very effective vector that delivered adsorbed plasmid DNA into several cell lines.²⁴

The main finding in the present study was that a far simpler, noncovalent adsorptive component complex of polylysine/plasmid DNA/adenovirus was a remarkably effective system that directed heterologous gene expression in cultured cardiac cells. In fact, levels of protein expression approached those seen with the very powerful, but complicated to produce, recombinant adenovirus constructs.^{3 17} Furthermore, in terms of the DNA required for optimum gene expression, the component system is surprisingly efficient when compared with other targeting complexes. Under optimal conditions, the component system requires only 7.5% of the DNA molecules per cell required for the transferrin/adenovirus-targeted conjugates.²³ Thus, these considerations underscore the utility of the ternary component system developed in the present study for cardiac cell transfections.

A remarkable finding was the importance of the DNA-to-polylysine ratio in the action of the component system. For example, at constant levels of polylysine, DNA concentrations at one half the optimal level could produce only 40% of the maximal protein, whereas at DNA levels threefold higher than optimal, no reporter gene expression is seen. These data suggest that the net charge of the complex is an important factor. These data also imply that the ability of polylysine to condense plasmid DNA into small 80- to 100-nm structures, as previously reported,²⁵ is an important element in the effective gene delivery vector.

The adenovirus component transfection system has been designed with several important advantages. First, the component system is simple and rapid to prepare when compared with either covalent conjugates or recombinant adenovirus vectors. Furthermore, there is great flexibility, in that combinations of plasmids can be used for the concomitant expression of multiple proteins. There is no size limitation to the DNA that can be used, since expression does not include viral packaging constraints. It is important to note that the component system levels of transfection efficiencies are 70-fold greater than those for lipofectin-mediated transfections of plasmid DNA. However, the component system may not be broadly useful in many cell types. For example, adult isolated skeletal muscle cells, which express low levels of integrin receptors, are not transfected (data not shown and Reference 26²⁶ ). Another potential concern is that other charged molecules in the medium might adsorb on the component complex and be transported into the cell as well. However, we have not seen any deleterious side effects related to this possibility.

Taken together, these results reveal that the component system is a very efficient method of gene delivery that should be broadly useful for cells that are permissive to adenoviral binding. It will be interesting to determine if this system can be used in vivo.

	Selected Abbreviations and Acronyms

 

C12FDG = C12-fluorescein di-ß-D-galactopyranoside OD420 = optical density at 420 nm PKC = protein kinase C X-gal = 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside

	Acknowledgments

 
This study was supported by National Institutes of Health grant P01 HL-27867, a grant from the American Heart Association, Maryland Affiliate, Inc (Dr Rogers), and a Minority Postdoctoral Supplement Fellowship from the National Institutes of Health (Dr Kohout).

Received December 11, 1995; accepted March 25, 1996.

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