© 2001 American Heart Association, Inc.
Cellular Biology |
Tight Control of Exogenous SERCA Expression Is Required to Obtain Acceleration of Calcium Transients With Minimal Cytotoxic Effects in Cardiac Myocytes
From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Md.
Correspondence to Giuseppe Inesi, University of Maryland, 108 N Greene St, Baltimore, MD 21201-1503. E-mail ginesi{at}umaryland.edu
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Abstract |
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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.
Key Words: SERCA • gene therapy • heart • adenovirus • calcium transients
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Introduction |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA Constructs and Vectors
EGFP or wild-type or mutant chicken SERCA113 cDNA was subcloned into pAdlox14 or p
E1sp1A15 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.
Day 1
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.
Day 2
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.
Day 3
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.
Day 5
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 
-actin, and 18S mRNA levels were
determined by Northern blots as described by Sumbilla et
al.12 cDNA probes for rat
ANF and skeletal -actin were radiolabeled with
[32P]-dCTP using a random priming
labeling kit (Amersham). The synthetic oligonucleotide probe for 18S
mRNA was radiolabeled by terminal deoxynucleotide transferase with
[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 F/Fo. Some experiments were
conducted with cells loaded with the indicator dye
Fura-2.16
Apoptosis
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
Statistical Analysis
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.
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Results |
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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).
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, levels of wild-type SERCA1 expression; 
, percentage of cell death after expression of SERCA1; 
, inactive SERCA1 mutant; 
, EGFP. EGFP was visualized by fluorescence microscopy, and SERCA protein was visualized by reaction with specific antibodies. Number of dead cells was determined by counting detached cells (floaters).
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.
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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.
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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).
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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 -adrenoceptor–mediated
hypertrophy,23 24 25
such as ANF and skeletal -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).
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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).
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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).
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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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).
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Footnotes |
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Original received August 24, 2000; resubmission received December 12, 2000; accepted January 5, 2001.
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  J. M. O'Donnell, A. Fields, X. Xu, S. A. K. Chowdhury, D. L. Geenen, and J. Bi Limited functional and metabolic improvements in hypertrophic and healthy rat heart overexpressing the skeletal muscle isoform of SERCA1 by adenoviral gene transfer in vivo Am J Physiol Heart Circ Physiol, December 1, 2008; 295(6): H2483 - H2494. [Abstract] [Full Text] [PDF]
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H. Ma, C. M. Sumbilla, I. K. G. Farrance, M. G. Klein, and G. Inesi Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes Am J Physiol Cell Physiol, March 1, 2004; 286(3): C556 - C564. [Abstract] [Full Text]
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Z. Kassiri, C. Zobel, T.-T. T. Nguyen, J. D. Molkentin, and P. H. Backx Reduction of Ito Causes Hypertrophy in Neonatal Rat Ventricular Myocytes Circ. Res., March 22, 2002; 90(5): 578 - 585. [Abstract] [Full Text] [PDF]
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F. del Monte, R. J. Hajjar, S. E. Harding, and G. Inesi Overwhelming Evidence of the Beneficial Effects of SERCA Gene Transfer in Heart Failure Response Circ. Res., June 8, 2001; 88 (11): e66 - e67. [Full Text] [PDF]
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M. Periasamy Adenoviral-Mediated SERCA Gene Transfer Into Cardiac Myocytes : How Much Is Too Much? Circ. Res., March 2, 2001; 88(4): 373 - 375. [Full Text] [PDF]
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Z. Kassiri, C. Zobel, T.-T. T. Nguyen, J. D. Molkentin, and P. H. Backx Reduction of Ito Causes Hypertrophy in Neonatal Rat Ventricular Myocytes Circ. Res., March 22, 2002; 90(5): 578 - 585. [Abstract] [Full Text] [PDF]
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