Tight Control of Exogenous SERCA Expression Is Required to Obtain Acceleration of Calcium Transients With Minimal Cytotoxic Effects in Cardiac Myocytes
Abstract—Collateral effects of exogenous sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) expression were characterized in neonatal rat and chicken embryo cardiac myocytes, and the conditions required to produce acceleration of Ca2+ transients with minimal toxicity were established. Cultured myocytes were infected with adenovirus vector carrying the cDNA of wild-type SERCA1, an inactive SERCA1 mutant, or enhanced green fluorescence protein under control of the cytomegalovirus promoter. Controls were exposed to empty virus vector. Each group was tested with and without phenylephrine (PHE) treatment. Under conditions of limited calf-serum exposure, the infected rat myocytes manifested a more rapid increase in size, protein content, and rate of protein synthesis relative to noninfected controls. These changes were not accompanied by reversal to fetal transcriptional pattern (as observed in hypertrophy triggered by PHE) and may be attributable to facilitated exchange with serum factors. SERCA virus titers >5 to 6 plaque-forming units per cell produced overcrowding of ATPase molecules on intracellular membranes, followed by apoptotic death of a significant number of rat but not chicken myocytes. Enhanced green fluorescence protein virus and empty virus also produced cytotoxic effects but at higher titers than SERCA. Expression of exogenous SERCA and enhancement of Ca2+ transient kinetics could be obtained with minimal cell damage in rat myocytes if the SERCA virus titer were maintained within 1 to 4 plaque-forming units per cell. Expression of endogenous SERCA was unchanged, but expression of exogenous SERCA was higher in myocytes rendered hypertrophic by treatment with PHE than in nontreated controls.
The sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol back into the sarcoplasmic reticulum (SR) after myocardial contraction, thereby coordinating contractile tension and relaxation kinetics. Ca2+ uptake by the SR has been reported to be inadequate in failing human heart1 2 as a consequence of reduced SERCA activity,3 SERCA protein expression,4 5 and SERCA mRNA levels.6 On the other hand, it was shown in experimental models that uptake of cytosolic Ca2+ by the SR can be accelerated by expression of exogenous SERCA genes and consequent increase of the ATPase copy number in cardiac myocytes.7 8 9 In fact, isolated failing human cardiac myocytes have shown improved performance after overexpression of exogenous SERCA.10
Recombinant adenovirus has proven to be a very effective vector for delivery of exogenous SERCA cDNA into cardiomyocytes,11 with 100% efficiency of infection compared with 5% to 10% efficiency by other transfection methods.12 The positive benefits of exogenous SERCA expression on Ca2+ homeostasis continues to be characterized by several laboratories, whereas collateral effects of gene expression have received little attention. We have observed important side effects that are much more evident in neonatal rat than in chicken embryo cardiac myocytes. In this study, we made comparative observations on cells infected with empty virus, with viral vectors carrying wild-type or inactive SERCA, or with enhanced green fluorescence protein (EGFP) cDNA. We describe here the effects of these procedures on protein synthesis, cell viability, and calcium handling in controls and hypertrophic (treated with phenylephrine [PHE]) myocytes. We then define restricted conditions under which the level of exogenous SERCA gene expression and improvement of cytosolic Ca2+ control can be obtained in rat myocytes with minimal cell damage.
Materials and Methods
DNA Constructs and Vectors
EGFP or wild-type or mutant chicken SERCA113 cDNA was subcloned into pAdlox14 or pΔE1sp1A15 plasmid. The cDNA was preceded by the cytomegalovirus (CMV) promoter and followed by simian virus polyadenylation signal. Recombinant adenovirus with EGFP or SERCA1 cDNA was obtained as previously described.7 16 The recombinant products were selected by plaque purification in HEK293 cells and band-purified by centrifugation in cesium gradients to yield concentrations of the order of 109 to1011 plaque-forming units (pfu) per milliliter.
Preparation and Treatment of Neonatal Rat Myocytes
Chicken embryo cardiac myocytes were prepared and cultured as previously described.7 Neonatal rat cardiac myocytes were prepared and cultured as follows.
Primary cultures were obtained from 1-day-old Sprague-Dawley rats, as previously described,16 17 and cultured in MEM (GibcoBRL) containing Hanks’ salts, 5% calf serum, vitamin B12, and 0.1 mol/L bromodeoxyuridine at 37°C, 350 cells/mm2, in the presence of 1% CO2. The culture medium was replaced every 12 hours throughout the experiment.
The cells were washed, and the medium was replaced with half the original volume of serum-free medium (identical to that previously described except for the absence of serum). The cells were infected with adenovirus vector (0 to 20 pfu/cell) containing wild-type SERCA1, EGFP, or mutant cDNA encoding inactive SERCA1. Controls were exposed to empty virus. Infections were obtained by exposing the cells to the viral vectors for 1 hour. At this time, the medium was diluted 1:1 with medium containing serum to yield a final 5% concentration.
Cell-culture medium was removed and replaced with a defined MEM containing 10 μg/mL transferrin, 10 μg/mL insulin, 0.1% BSA, 0.1 mol/L bromo deoxyuridine, 100 μmol/L vitamin C, and no serum. Two sets of plates (infected or noninfected) from day 2 were treated with 20 μmol/L PHE to induce hypertrophy.17 Two sets of alternative plates were not treated with PHE.
Multiple-phase contrast images of cell populations were taken from each plate to establish cell counts and viability. Both attached cells and dead cells (ie, floaters) were counted.
Cells were harvested for Western and Northern blots, [14C]phenyl-alanine experiments, or fluorescence measurements of cytosolic calcium.
Cell Death and Protein Synthesis
The number of dead cells was estimated by counting attached cells and floaters in culture dishes. Total protein was measured by bicinchoninic acid assay (Pierce) after counting the cells in culture. Protein synthesis rate was measured using radioactive amino acid as described by Simpson.17 To this aim, the culture media was brought to 0.1 μCi/mL [14C]phenylalanine on day 3, and on day 5 [14C]phenylalanine incorporation into the total cell protein was determined by scintillation counting.
Western and Northern Blot Analysis
SERCA protein content was determined by Western blots, as previously described.12 16 Wild SERCA1 and inactive SERCA1 mutant were detected using primary antibodies CaF3-5C313 and Myc1-9E10 for the c-myc tag.18 Endogenous SERCA2a was detected using MA3-919 antibody (Affinity Bioreagents). Atrial natriuretic factor (ANF), skeletal α-actin, and 18S mRNA levels were determined by Northern blots as described by Sumbilla et al.12 cDNA probes for rat ANF and skeletal α-actin were radiolabeled with α[32P]-dCTP using a random priming labeling kit (Amersham). The synthetic oligonucleotide probe for 18S mRNA was radiolabeled by terminal deoxynucleotide transferase with α[32P]-dCTP.19 Values for ANF and actin mRNA were normalized to endogenous 18S mRNA. In situ immunofluorescence staining of SERCA1 was performed as previously described.7
Intracellular Calcium Measurements
As described previously,16 cell cultures were loaded with the Ca2+ indicator dye Fluo-4, mounted on an Olympus 1X70 inverted microscope, and superfused with Ringer’s buffer solution at 30±2°C. The cells were field-stimulated, and cell fluorescence was recorded, corrected for background signal, and plotted as ΔF/Fo. Some experiments were conducted with cells loaded with the indicator dye Fura-2.16
Apoptosis was assessed by microscopic visualization of condensed nuclei and electrophoretic demonstration of fragmented DNA patterns. For nuclear visualization, the cells were fixed with 4% paraformaldehyde (Sigma) and stained with 10 μg/mL Hoechst 33258 (Sigma B2283) in the presence of 0.1% Triton X-100 overnight in the dark at 4°C as previously described.20 The stained cells were then visualized using an ultraviolet light (365 nm) on a Zeiss inverted microscope.
For demonstration of fragmented DNA pattern, DNA was isolated by phenol extraction and ethanol precipitation21 and run on a 1% agarose gel. DNA patterns were compared with the classic fragmentation of DNA isolated from myocytes treated with staurosporin to induce apoptosis.22
Experiments were done in triplicate. Data set comparisons were performed with Student’s unpaired, 2-tailed t-test. Difference in mean values were considered statistically significant at P<0.05.
Efficiency of Exogenous Gene Transfer in Neonatal Rat Cardiac Myocytes
In infections with adenovirus vectors, an important variable is the number of viral particles per cultured cell. We characterized this variable in our experiments by exposing neonatal rat cardiac myocytes to increasing titers (0 to 20 pfu/cell) of recombinant adenovirus vectors. It is important to realize that we indicate here viral titer with reference to pfu per attached, rather than seeded, cell to circumvent the variability of seeding rate in various experiments. Fluorescence images of cell cultures infected with EGFP virus revealed that 100% infection of rat myocytes is obtained with a viral titer of 5 pfu/cell (Figure 1A⇓). Western blot analysis of SERCA1 expression revealed similar titer requirements. This pattern is different from that observed in chicken myocytes,12 in which the titer required for 100% infection is 10 pfu/cell (Figure 1B⇓).
Viral Infection and Cell Viability
Exogenous SERCA expression in neonatal rat cardiac myocytes decreases cell viability, resulting in detachment of a significant number of cells (floaters) even at viral titers as low as 5 pfu/cell (Figure 1A⇑). This effect is observed to a much lesser extent in chicken-embryo cardiac myocytes (Figure 1B⇑). It should be pointed out that an identical cytotoxic effect is produced by expression of wild-type SERCA1 or inactive SERCA mutant. Similar toxicity was observed after expression of SERCA2 (cDNA from either chicken or rabbit) in rat myocytes (not shown). EGFP virus (Figure 1A⇑) and empty virus (not shown) also produce toxic effects. However, these effects are observed at significantly higher titer. It is clear that the range of viral titer between induction of protein expression and production of toxicity is narrower for SERCA than for EGFP (Figure 1A⇑).
Although we only use viral vectors derived from the first large-scale amplification of purified plaques, we considered whether the observed cytotoxic effects may be attributable to the presence of E1A and consequent replication of adenovirus in the infected cells. To rule out this possibility, we conducted parallel infections (1 pfu/cell) of A549 cells (able to amplify only replication-competent and not replication-defective virus) and HEK293 cells (E1A-transformed, used to amplify replication-defective virus) with SERCA1 adenovirus. We found no significant cell death in the A549 cells 3 days after infection, whereas 100% of the HEK293 cells were detached and obviously dead (Figure 2⇓). Furthermore, plaque assays showed a 3 order of magnitude increase of plaque density in HEK293 cells and no increase in A549 cells and rat cardiac myocytes. This demonstrates that our stock of adenovirus vector does not contain E1A or replication-competent contaminants.
In situ immunofluorescence staining with antibodies specific for the exogenous SERCA1 reveals very dense packing of ATPase molecules within intracellular membranes even in seemingly healthy cells (Figure 3⇓). Drastic structural changes are apparent in cells undergoing cytotoxic effects (Figure 3⇓). It is noteworthy that cytotoxic effects are produced by wild-type SERCA and inactive SERCA mutant as well (Figure 1⇑). It is likely that SERCA targeting of intracellular membranes and dense packing of ATPase molecules within a rather limited membrane space (Figure 3⇓) produce perturbation of membrane structure and function and consequent alteration of calcium homeostasis. Furthermore, the occurrence of nuclear condensation and DNA fragmentation (Figure 4⇓) suggests that apoptotic mechanisms, rather then necrosis, are involved in the cytotoxic effects of SERCA expression. The apoptotic index (percentage of nuclei exhibiting condensation) was 7% in myocytes infected with 2 pfu/cell and 31% in myocytes infected with 10 pfu/cell. It is noteworthy that similar apoptotic effects (Figure 4B⇓) are also produced by higher titers of EGFP or empty virus.
Cell Growth and Synthesis of Total and Specific SERCA Protein
An unexpected finding was that, under certain conditions, infection with adenovirus vector promotes increase in cell size and total protein synthesis. The magnitude of this effect is comparable to that of PHE. Figure 5⇓ presents images of neonatal rat cardiac myocytes maintained with a defined medium in the absence of serum (Figures 5A⇓ and 5D⇓), exposed to serum for 1 day (Figures 5B⇓ and 5E⇓), or exposed continuously to serum (Figures 5C⇓ and 5F⇓). Cells in Figures 5C⇓ through 5E were infected with SERCA1 virus. The cells were observed by phase-contrast microscopy 4 days after seeding. It is clear that the cells exposed continuously (Figures 5C⇓ and 5F⇓) to serum are larger than the cells not exposed to serum (Figures 5A⇓ and 5D⇓), independent of whether they were infected or not. This increase in cell size is comparable to that observed in cells treated with PHE (not shown). On the other hand, under conditions of limited exposure to serum, the size of infected cells is larger than that of noninfected cells (compare Figures 5B⇓ and 5E⇓).
We next investigated whether the transcriptional pattern of cell growth and protein synthesis triggered by exogenous gene transfer was the same as that triggered by PHE. To this aim, we tested specific markers of α-adrenoceptor–mediated hypertrophy,23 24 25 such as ANF and skeletal α-actin mRNA. We found these markers increased in myocytes treated with PHE but not at all increased in infected myocytes undergoing increase in size in the absence of PHE (Figure 6⇓).
In all cases, the observed changes in cell size were matched by increased total protein content per cell (not shown) and rates of protein synthesis as revealed by radioactive phenylalanine incorporation (Figure 7A⇓). In agreement with earlier studies,17 we found that treatment with PHE for 48 hours resulted in a 70% increase in [14C]phenylalanine incorporation, independent of viral infection. A similar increase was observed in the infected cells undergoing increase in size (compare control and infected cells in the absence of PHE, Figure 7A⇓).
With regard to specific synthesis of SERCA, we found that endogenous SERCA2a is produced at the same level (per total protein unit weight) in control myocytes and myocytes undergoing PHE hypertrophy (Figure 7B⇑). The endogenous SERCA2a level, however, is significantly reduced after infection with SERCA1 adenovirus (Figure 7B⇑). This is likely attributable to competition with exogenous SERCA expression and membrane occupancy. In fact, we have previously shown16 that even in noninfected myocytes, endogenous SERCA2a is quite densely spaced in the sarcoplasmic reticulum membrane. The total Ca2+-dependent (thapsigargin-sensitive) ATPase (per total protein unit weight) is increased more than 3-fold in infected myocytes as a consequence of exogenous SERCA1 expression (Figure 7C⇑). In fact, the rate of exogenous gene expression was higher in cells treated with PHE, as revealed by EGFP fluorescence and SERCA Western blots (results not shown). Consistent with these findings, the cytotoxic effect of SERCA expression was observed at lower titers in PHE-treated myocytes than in nontreated myocytes.
Exogenous SERCA Expression and Ca2+ Transients
The experiments reported above emphasize the importance of establishing conditions that limit exogenous SERCA expression to levels producing minimal cell damage while still improving the kinetics of Ca2+ transients. In a preliminary set of experiments, neonatal rat cardiac myocytes were infected with viral titers producing minimal cell damage (ie, 4 and 2 pfu/cell in the absence and presence of PHE, respectively). Ca2+ transients were then measured using the fluorescent Ca2+ indicator dye fluo-4 after a voltage stimulus. The data shown in Figure 8⇓ were averaged from transients obtained from several cells selected at random (n=15 to 30). They demonstrate that a faster decay to baseline can be obtained after limited overexpression of exogenous SERCA in cells incubated either in the absence (Figure 8A⇓) or in the presence (Figure 8B⇓) of PHE. It is of interest that development of PHE-induced hypertrophy by itself does not significantly affect the Ca2+ transients (compare control and PHE transients in Figures 8A⇓ and 8B⇓, respectively).
A series of measurements were also made with Fluo-4 in myocytes exposed to various viral levels, ranging between 0 and 10 pfu/seeded cell. Ca2+ transients were then obtained from several cells selected randomly in each plate. The time constants of decay and one-half width of the transients were averaged with the understanding that at viral titer <1 pfu/cell, the average values derive in part from infected and in part from noninfected cells. Nevertheless, it is clear from Figure 8C⇑ that the average decay constant of the entire cell population is significantly shortened after infection with 2 pfu/cell. This later titer produces minimal toxicity. Most importantly, Figure 8⇑ shows that no additional reduction of the time constants is obtained by raising the viral titer above 2 pfu/cell.
We also obtained measurements with the indicator fura-2 and noted a modest reduction of the resting Ca2+ concentration in the cytosol of infected cells (24±5 nmol/L versus 42±7 nmol/L). On the other hand, no significant change in the peak Ca2+ concentration on stimulation after adequate rest was observed.16 However, these measurements were obtained from healthy cells. Dead cells are supercontracted and often floating and are not suited to cytosolic calcium measurements.
This study identifies important collateral effects of adenovirus vectors and gene transfer in neonatal rat cardiac myocytes as they relate to specific SERCA expression and modification of cytosolic Ca2+ transients. Cytotoxic effects, resulting in cell death, are most important collateral effects and are more prominently observed in neonatal rat than in chicken embryo myocytes. The difference in titer requirements may be a function of viral receptor density (related to species or tissue) as well as cell proliferation and density. Whereas the neonatal rat myocytes display limited cell division, the chicken embryonic myocytes proliferate significantly over the same period of time. We find that the entire population of rat myocytes is effectively infected with a viral titer of 2 to 5 pfu/attached cell, resulting in expression of SERCA levels that are sufficient to modify significantly the cytosolic Ca2+ transients. On the other hand, a significant percentage of rat myocytes receiving more than 2 to 4 pfu/cell undergo cytotoxicity and end up as floaters. The nuclear condensation and DNA fragmentation observed in our experiments are consistent with apoptotic death.26 It is noteworthy that DNA fragmentation was also noted27 in oncotic myocytes of infarct areas.
Because interference with cardiac gene transcription28 and apoptotic effects29 30 can be produced by adenovirus E1A, we made special efforts to exclude the presence of E1A and replication-competent virus in our preparation (Figure 2⇑). Furthermore, the cytotoxic effects produced by EGFP or empty virus require a much higher titer than those produced by SERCA virus (Figure 1⇑). It is likely that excessive expression and dense packing of membrane-bound SERCA molecules (Figure 3⇑) damage the structural integrity of intracellular membranes. The consequent perturbation of membrane structure and function interferes then with intracellular calcium homeostasis. It is noteworthy that toxic effects are also observed with EGFP virus or empty virus if titers significantly higher are used.
Another collateral effect of viral infection is the increase in cell size and total protein synthesis observed under conditions of limited exposure to calf serum. This effect is in some cases of magnitude comparable to that of PHE hypertrophy but does not involve reversal to the fetal transcription pattern. The growth stimulus is evidently attributable to serum growth factors and is likely related to facilitated access from the medium to the cytosol in the infected myocytes. No effect of infection on growth is observed in the absence of serum, and maximal growth is observed independent of infection when the myocytes are continually exposed to serum. Awareness of this effect is likely to be helpful in studies of viral vectors and exogenous gene expression.
The optimal level of exogenous SERCA expression is clearly the viral titer that produces minimal toxic effects while achieving the desired functional response. This limit is 2 to 4 pfu/cell in neonatal rat cardiac myocytes expressing exogenous SERCA gene under control of the CMV promoter. This titer yields a 3-fold increase in SERCA activity and a pronounced kinetic effect on the cytosolic Ca2+ transients attributable to faster Ca2+ uptake by the sarcoplasmic reticulum. At higher viral titers, we observed no additional acceleration of Ca2+ transients but apoptotic death of a significant number of myocytes. It should be noted that we used the SERCA1 rather than the SERCA2 isoform, because SERCA1 has a higher turnover and can influence calcium transients with lower (and less toxic) levels of expression compared with SERCA2.16
An interesting alternative to our experiments of expression under control of the strong CMV promoter is the use of weaker promoters. We found that in this case, a higher viral titer is required to obtain SERCA expression levels that are effective on calcium transients. Consequently, no significant improvement in cytotoxicity is realized. Additional studies with promoters that may have the advantage of cell specificity as well as suitable strength are being conducted in our laboratory.
It is of interest that myocytes rendered hypertrophic by treatment with PHE increase their production of endogenous SERCA2a in proportion to total protein and retain unchanged Ca2+ transients. On the other hand, they react to adenovirus infection with faster expression of exogenous gene. Thereby, expression of exogenous SERCA and acceleration of Ca2+ transients, as well as cytotoxicity, are obtained at lower viral titers.
In conclusion, attempts to influence Ca2+ homeostasis by exogenous SERCA gene expression require careful control of protein expression levels and characterization of associated cell functions. Failure to optimize conditions for adenovirus vector delivery and define collateral effects in various cell types is likely to create unwanted interference with progress in the experimental, and possibly therapeutic, use of this procedure.
This work was supported by the National Institutes of Health (NIH PO1-HL27867) and the University of Maryland Interdisciplinary Training Program in Muscle Biology (NIH 5T32-AR07592). J.M.O. received additional support from the Mid-Atlantic American Heart Association (Beginning Grant-in-Aid 0060286U).
Original received August 24, 2000; resubmission received December 12, 2000; accepted January 5, 2001.
- © 2001 American Heart Association, Inc.
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