© 1996 American Heart Association, Inc.
Articles |
High and Low Molecular Weight Fibroblast Growth Factor-2 Increase Proliferation of Neonatal Rat Cardiac Myocytes but Have Differential Effects on Binucleation and Nuclear Morphology
Evidence for Both Paracrine and Intracrine Actions of Fibroblast GrowthFactor-2
From the Department of Physiology (K.B.S.P., P.A.C.), University of Manitoba, Winnipeg, Canada, and the Departments of Anatomy and Physiology (E.K.), Division of Cardiovascular Sciences, St Boniface Hospital Research Centre, Winnipeg, Canada.
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Abstract |
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Abstract Basic fibroblast growth factor (FGF-2) plays a vital role in the growth and differentiation of cardiac myocytes. It exists in high and low molecular weight forms because of the use of alternative initiation codons in the same mRNA. Higher levels of high molecular weight forms (molecular mass of 22 and 21.5 kD) are present in the rat heart during the neonatal stage, whereas the low molecular weight form (molecular mass of 18 kD) is predominant in the adult heart, suggesting different roles in development. Rat FGF-2 cDNAs that can preferentially express high or low molecular weight forms were introduced into neonatal rat ventricular myocyte cultures. Significant and comparable increases in overall cardiac myocyte DNA synthesis and proliferation were seen with 22/21.5- and 18-kD FGF-2 expression. A significantly higher mitotic index was seen in the vicinity of cardiac myocytes overexpressing high or low molecular weight forms of FGF-2 compared with nonoverexpressing cells. This increase was inhibited in the presence of neutralizing antibodies to FGF-2, pointing to a proximity-dependent paracrine effect of 22/21.5- and 18-kD FGF-2 on mitosis. By contrast, overexpression of high but not low molecular weight FGF-2 was associated with a significant increase in binucleation (
36% of cardiac myocytes
overexpressing 22/21.5-kD FGF-2 were binucleated compared with 9% of
cardiac myocytes overexpressing 18-kD FGF-2), which was not affected by
neutralizing antibodies to FGF-2. These results suggest that 22/21.5-kD
FGF-2 and 18-kD FGF-2 have similar paracrine effects on proliferation
but that 22-21.5-kD FGF-2 exerts a distinct intracrine effect on
binucleation.
Key Words: fibroblast growth factor • cardiac myocytes • binucleation • proliferation
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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FGF-2 is a multifunctional regulator of cell proliferation, migration, differentiation, and survival1 2 and plays a vital role in the growth and differentiation of cardiac myocytes.3 4 5 6 7 FGF-2 mediates its biological effects through binding to two classes of cell surface receptors, specifically, high-affinity tyrosine kinase receptors and low-affinity heparan sulfate proteoglycans.8 FGFR-1 is the only high-affinity receptor type present in the embryonic and adult heart.9 10 11 12 13 FGF-2 exists in multiple forms because of the initiation of translation from alternative codons (leucine versus methionine) in the same mRNA,14 alternative splicing of RNA,3 and proteolysis of high molecular weight forms.15 16 In gene-transfer studies, high molecular weight forms arising from leucine (CUG) codons of rat FGF-2 mRNA preferentially localize to the nucleus of embryonic chicken cardiac myocytes, whereas the low molecular weight form resulting from a methionine (AUG) codon localizes to both the nucleus and cytoplasm.17 There is a switch in the relative levels of high and low molecular weight forms of FGF-2 during the development of the rat heart. Higher levels of high molecular weight forms (referred to as 22/21.5-kD FGF-2) are present in the rat heart during the neonatal stage, whereas the low molecular weight form (referred to as 18-kD FGF-2) is predominant in the adult.18
Although the biological significance of FGF-2 nuclear localization is
unclear, it could be linked to the dramatic changes in nuclear events
that occur during cardiac development. These include a reduction in the
proliferative potential of rat cardiac myocytes as they develop through
embryonic (dividing), neonatal (transitional), and adult (nondividing)
stages.19 20 In addition, entry into a nondividing
and
hypertrophic state is accompanied by binucleation in the
rat.20 In the adult rat heart, 82% of
ventricular myocytes are reported to be binucleated, and
5% contain three or more nuclei (multinucleated).21
Previously, we expressed high and low molecular weight rat FGF-2 in
embryonic (dividing) chicken ventricular myocytes.
Increases in DNA synthesis and proliferation were observed with both
high and low molecular weight FGF-2, and overexpression of 22/21.5-kD
but not 18-kD FGF-2 was associated with clumping of the
DNA.17 The significance of these effects, particularly the
distinct effect of 22/21.5-kD FGF-2 on DNA clumping, was not considered
established, since they might have resulted from the heterologous
system used (rat proteins in chicken cells), rendering potential
physiological implications uncertain. Thus, we
reexamined the effects of high and low molecular weight FGF-2
overexpression on DNA synthesis, mitosis, and cell proliferation in
neonatal (transitional) rat cardiac myocytes. We show that the
stimulatory effects of high and low molecular weight FGF-2 on neonatal
rat cardiac myocytes are of a magnitude similar to that observed in
embryonic chicken cells and that the effect of 22/21.5-kD FGF-2 on DNA
clumping is also seen in the rat. Therefore, these effects are not a
peculiarity of the heterologous system used previously. In addition, we
provide evidence that the stimulation of mitosis by 22/21.5- or 18-kD
FGF-2 likely represents a proximity-dependent paracrine
effect. Furthermore, in contrast to the effects on hyperplastic growth,
which were similar for high and low molecular weight FGF-2, we show
that overexpression of high but not low molecular weight FGF-2 is
associated with an increase in cardiac myocyte binucleation. The
presence of nuclear furrows and of nuclei of various sizes indicates
that amitotic nuclear division might contribute to the binucleation
observed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Plasmid Constructs
Expression vectors containing the wild-type and modified rat FGF-2 cDNAs were described previously.17 The
metFGF
cDNA has the potential to generate only high molecular weight
(22/21.5-kD) FGF-2 species from upstream leucine (CTG) sites, because
of an insertional mutation of the methionine (ATG) codon responsible
for initiating the 18-kD form in the wild-type FGF-2
cDNA.22 The metFGF cDNA is deleted from all sequences of
more than five amino acids upstream from the ATG site responsible for
initiating the 18-kD form, including the two CTG start sites.
Wild-type (FGF) as well as modified FGF-2 sequences (metFGF and
metFGF) were cloned in between the RSV promoter (indicated by p) and
SV40 polyadenylation signal to obtain RSVp.FGF, RSVp.metFGF, and
RSVp.metFGF, respectively. Hybrid FGF-2 cDNA gene plasmid preparations
were tested routinely in kidney COS-1 cells because they are easy to
transfect and have a low level of endogenous FGF-2
expression (Fig 1
). The expression vector without any
FGF-2 sequence (RSVp.CONT) was used as a control. A bacterial gene
coding for ß-gal was also cloned in the identical expression
vector to obtain RSVp.ß-gal and used to assess transfection
efficiency.
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Cell Culture and Gene Transfer
African green monkey kidney
COS-1 cells and FBHE cells were
obtained from the American Type Culture Collection and grown in
monolayer culture in 10% (vol/vol) FBS in DMEM. Cardiac
ventricular myocytes were isolated from newborn
Sprague-Dawley rat (1-day) hearts by enzymatic disaggregation
with 0.1% (wt/vol) trypsin (Sigma Chemical Co) by use of a
temperature-regulated (35°C) spinner flask, with or without
subsequent fractionation on a Percoll gradient.23 In the
absence of Percoll fractionation, cells were preplated for 1.5 hours
without collagen to allow nonmuscle cells to attach. The remaining
unattached cardiac myocytes were obtained by low-speed
centrifugation and used for our experiments. Cells were
counted with a hemocytometer and plated on collagen-coated dishes
in Ham's F-10 medium containing 10% (vol/vol) FBS, 10% (vol/vol)
horse serum, and 140 µg/mL (wt/vol) calcium chloride.
For gene transfer, COS-1 cells were plated at a density of 0.5x106 cells per 100-mm dish, and cardiac myocytes were plated at a density of 1.5 to 2.0x106 per 60-mm dish or 0.7x106 per 35-mm dish. Cells were transfected by the calcium phosphate/DNA precipitation method essentially as described previously.24 Briefly, cells were transfected 24 to 48 hours after plating with 10 µg of test plasmid DNA per 5 mL of 10% FBS-DMEM. After 24 hours, cells were refed with growth medium and maintained for a further 48 to 72 hours before processing.
Immunoblotting
Transfected cultures were lysed in 1 mol/L
sodium chloride
solution (1.0 mL per 100-mm plate) containing 5 µg/mL
leupeptin, 5 µg/mL pepstatin, 5 µg/mL aprotinin, and 1 mmol/L
phenylmethylsulfonyl fluoride. Cell lysates were fractionated
by using heparin-Sepharose beads to evaluate levels of different forms
of FGF-2 in the total extracts, as described earlier.25
Protein concentrations of all the extracts were determined by using the
Bradford protein assay,26 and starting concentrations (2
mg of total cellular lysate) were normalized in all the treatments
before heparin-Sepharose adsorption. Heparin-Sepharose bound protein
was resolved in a 12.5% gel by SDS-PAGE and transferred onto Immobilon
P membrane (Millipore). Blots were blocked with 1% (wt/vol) gelatin in
calcium- and magnesium-free PBS for 30 minutes at room temperature
and probed with rabbit polyclonal anti–FGF-2 antibodies25
(at a dilution of 1:5000) for 18 hours at 4°C, and FGF-2 was
visualized by incubation with [125I]protein A (75
µCi/mL, Amersham Corp) as described previously.17 FGF-2
levels were quantified by densitometry. Samples of recombinant human
FGF-2 (Upstate Biotechnology Inc) as well as prestained SDS-PAGE
standards (low range, Bio-Rad) were used as molecular weight
markers.
Immunofluorescence Microscopy
(Subcellular Localization)
Transfected cells on collagen-coated 60-mm
dishes
(containing three coverslips, 22 mm in diameter) were fixed 48 hours
after transfections with 1% paraformaldehyde for 15
minutes and then permeabilized with 0.1% (vol/vol)
Triton X-100 in PBS for 15 minutes at 4°C. Coverslips were incubated
with rabbit FGF-2 antiserum (1:1000) in 1% (wt/vol) BSA in PBS and
then with biotinylated anti-rabbit immunoglobulins (1:20, Amersham
Corp), followed by fluorescein conjugated to streptavidin
(1:20, Amersham Corp). Rabbit FGF-2 antibodies used in the present
study were raised against the amino terminal residues 1 to 24 of bovine
FGF-2 and have been extensively characterized.25 27
Labeling for myosin was performed by using monoclonal antibodies
against striated myosin (1:2000; a generous gift from Dr R. Zak,
University of Illinois) in 1% (wt/vol) BSA in PBS, followed by
visualization with Texas red–conjugated anti-mouse
immunoglobulin (1:20, Amersham). Cellular DNA was stained with Hoechst
dye 33342 (Calbiochem-Behring) in PBS (10 µg/mL) for 30 seconds, as
described previously.28 Coverslips were mounted, examined,
and then photographed with a Nikon Diaphot microscope equipped with
epifluorescence optics.
ß-Gal Assay
Neonatal rat cardiac myocytes were
transfected with
RSVp.ß-gal or RSVp.CONT to determine the transfection efficiency.
For ß-gal activity, transfected cardiac myocyte cultures were
rinsed with PBS and then lifted with trypsin-EDTA (GIBCO-BRL). The
cells were pelleted at 1250g for 2 minutes, resuspended in
1.0 mL of X-gal solution containing 1 mmol/L magnesium chloride, 3.3
mmol/L potassium ferrocyanide, 3.3 mmol/L potassium ferricyanide, 150
mmol/L sodium chloride, 10 mmol/L sodium phosphate buffer (pH 7.0), and
0.2% (wt/vol) X-gal,29 and incubated for 20 hours at
37°C. Cells were assessed by using a hemocytometer, and the
percentage of stained cells was determined.
LI and Cardiac Myocyte Number
Rat ventricular myocytes in
60-mm
(collagen-coated) dishes containing two square collagen-coated
coverslips (22x22 mm) were transfected for 24 hours in 10% FBS-DMEM,
refed with 10% FBS–Ham's F-10 medium for 24 hours, and then pulsed
with [3H]thymidine (10 µCi/mL) in fresh medium for a
further 24 hours at 37°C. Cells were rinsed with PBS and fixed with
formyl-alcohol (9:1, 37% formaldehyde and 95% ethanol) at room
temperature for 15 minutes. Myocytes on coverslips were identified by
staining histochemically for glycogen by PAS (Sigma) stain.
Subsequently, [3H]thymidine uptake in myocyte cultures
was visualized by autoradiography using Kodak NTB
emulsion as described previously.17 A total of 800
radiolabeled or unlabeled PAS+ cells were scored per each treatment
(n=4). LI equals the proportion of radiolabeled PAS+ cells
(LI=radiolabeled PAS+ cells per total number of PAS+ cells)
and was
expressed as a percentage.5 Further, we also scored PAS+
cells in 16 random fields per each treatment (n=4) to estimate cardiac
myocyte number.
BrdU Labeling
For BrdU labeling, rat ventricular myocytes on
collagen-coated coverslips were transfected for 24 hours,
maintained for 24 hours, and incubated with 3 µg/mL (wt/vol) BrdU
(Sigma) for a further 24 hours. Myocyte cultures were fixed with 1%
paraformaldehyde for 15 minutes and then with 70%
ethanol for 30 minutes at room temperature and
permeabilized with 0.1% (vol/vol) Triton X-100 in PBS
for 15 minutes at 4°C.
Simultaneous labeling for myosin and/or BrdU in ventricular myocytes was performed by using monoclonal antibodies against striated myosin (1:2000) and BrdU (1:2, Amersham; 1:7, Becton Dickinson) in 1% (wt/vol) BSA in PBS. For BrdU labeling, fixed coverslips were treated with 70 mmol/L sodium hydroxide for 2 minutes and then rinsed with PBS before the addition of primary antibodies. Both myosin and BrdU were visualized with Texas red–conjugated anti-mouse immunoglobulin (1:20, Amersham).
For
quantification, 2500 cardiac myocytes from each culture
transfected with RSVp.CONT, RSVp.FGF, RSVp.metFGF, or RSVp.metFGF
were assessed from 11 randomly selected fields on four separate
coverslips, representing two independent transfection
experiments. The fraction of nuclei staining for BrdU was determined,
and the results are expressed as the fold difference relative to
RSVp.CONT, which was arbitrarily set to 1.0.
MI and Assessment of Binucleation
MI (defined here as the
fraction of cardiac myocytes in
metaphase, anaphase, or telophase) and the degree of binucleation were
determined for cultures transfected with RSVp.CONT, RSVp.FGF,
RSVp.metFGF, or RSVp.metFGF. Cardiac myocytes in various stages of
mitosis or containing two nuclei were identified by
fluorescence microscopy and a combination of DNA (Hoechst dye
33342) and antimyosin staining. About 3000 to 3500 cardiac myocytes
from each transfected culture were assessed from 25 to 35 randomly
selected fields on eight separate coverslips, representing
three independent transfection experiments. The results are expressed
as the fold difference relative to the levels seen with RSVp.CONT,
which were arbitrarily set to 1.0. In addition, we assessed the effect
of individual cardiac myocytes overexpressing FGF-2 (metFGF and
metFGF) on mitosis in surrounding cells in the presence of either 10
µg/mL anti-bovine basic FGF type 1 (mouse monoclonal IgG1k,
Upstate Biotechnology Inc) or 10 µg/mL NM Ab (Sigma). The
anti-bovine basic FGF type 1 preparation contains neutralizing
FGF-2 antibodies, which were used successfully to block an
FGF-2–mediated growth response.30 After transfection,
cells were refed growth medium containing FGF-2 antibodies or NM Ab for
48 hours. The fraction of mitotic cells in an area (radius, 125
µm) surrounding or not containing a myocyte overexpressing FGF-2 was
determined in the same culture (15 areas from three coverslips). Under
the culture conditions used, 125 µm corresponded to a five-cell
radius. "Background" values were also determined from cultures
transfected with RSVp.CONT and maintained in the presence of antibodies
to FGF-2 (15 areas from three coverslips) or NM Ab (20 areas from three
coverslips). The effect of neutralizing FGF-2 antibodies on
binucleation was also assessed (30 areas from three coverslips). To
ensure that sufficient levels of neutralizing antibodies were
present, at the end of the experiment conditioned medium was used
to perform a growth assay on FBHE cells, which require FGF-2 for growth
and survival. FBHE cells were plated at a density of 3x104
cells per 35-mm dish in DMEM with 10% FBS and 3 ng/mL human
recombinant FGF-2 (Upstate Biotechnology Inc). The use of conditioned
medium from each experimental dish, initially containing 10 µg/mL
FGF-2 antibodies, resulted in a total inhibition of FGF-2–dependent
growth of FBHE cells in 5 days as assessed by microscopy and a Coulter
counter. This negative effect on growth was not observed with
conditioned medium containing 10 µg/mL NM Ab.
In addition, the
level of binucleation was determined in the
population of cardiac myocytes overexpressing FGF-2. Approximately 400
cardiac myocytes overexpressing FGF-2 were assessed from cultures
transfected with RSVp.FGF, RSVp.metFGF, or RSVp.metFGF and
stained with Hoechst dye as well as antibodies to FGF-2 and myosin.
Statistical Analysis
Data presented in the text and figures
are mean±SEM.
Statistical analysis of the data was performed by a one-way
ANOVA and the Bonferroni multiple-comparison post hoc test. The
results were accepted if Bartlett's test for homogeneity of variances
indicated that the difference between standard deviations from each
test group was not significant. When this difference was shown to be
significant (level of binucleation in population of cardiac myocytes
overexpressing FGF-2), analysis was performed by using the
Mann-Whitney test (nonparametric). In all cases, a value
was considered statistically significant at P<.05.
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Results |
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Subcellular Localization of FGF-2 in the Transfected Neonatal Cardiac Myocytes
Wild-type and modified FGF-2 cDNAs, used previously to transfect embryonic chicken ventricular myocytes, were introduced into neonatal rat ventricular myocyte cultures derived from newborn (1- to 2-day-old) rat hearts. All cultures were processed for simultaneous localization of FGF-2, striated muscle myosin, and nuclei by using triple fluorescence labeling. Cardiac myocyte cultures transfected with RSVp.CONT as well as nontransfected cultures displayed low levels of FGF-2 staining (endogenous expression) under the experimental conditions described. By contrast, cells staining intensely for FGF-2, indicating overexpression, were observed exclusively in cultures transfected with wild-type or modified FGF-2 constructs. Estimates for transfection efficiency were determined by RSVp.ß-gal transfections of neonatal rat ventricular myocyte cultures. Staining for ß-gal was detected in 10% to 30% (18±4%, n=6) of cells in cultures transfected with RSVp.ß-gal compared with cultures transfected with the control plasmid (RSVp.CONT), where no staining was observed. Similar levels of transfection efficiency were obtained consistently when the percentage of cardiac myocytes overexpressing FGF-2 (after transfection with FGF-2 cDNAs) was determined by immunofluorescence microscopy.
Subcellular localization
of different forms of FGF-2 was examined
in overexpressing cardiac myocytes. Myocytes transfected with RSVp.FGF
and RSVp.metFGF displayed identical staining patterns. FGF-2 was
localized predominantly to the nucleus or nuclei of mononucleated and
binucleated cardiac myocytes, respectively, a finding that was
confirmed by counterstaining DNA with Hoechst 33342 (Fig 2). In
contrast, cardiac myocytes transfected with
RSVp.metFGF (which can generate only 18-kD FGF-2) displayed high levels
of cytoplasmic and nuclear staining with a range of patterns including
more intense nuclear or cytoplasmic staining (Fig 3).
However, the majority of cells displayed both cytoplasmic as well as
nuclear staining of comparable intensity. Control cultures transfected
with RSVp.CONT (Fig 2) or nontransfected cultures (not shown)
did not
display the intense anti–FGF-2 staining of nuclei seen after
overexpression of high or low molecular weight FGF-2 (Figs 2
and 3).
Clumping of the DNA, resembling chromatin condensation and prophase
nuclei, was seen in 20% of cardiac myocytes overexpressing high but
not low molecular weight FGF-2. These "clumps" were observed in
both mononucleated and binucleated cardiac myocytes (Fig 4).
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Effect of FGF-2 on DNA Synthesis
Two methods were used to
assess DNA synthesis in cardiac
myocytes. In the first, a combination of in situ
autoradiography ([3H]thymidine
incorporation) and PAS staining of glycogen/myocytes was used to obtain
an LI. The LI is the proportion of PAS+/glycogen-containing cells
(myocytes) that also show the presence of [3H]thymidine.
The results are shown in Fig 5 and are presented
as fold differences relative to the control (RSVp.CONT) value
(15.1±2.0%, n=8), which was arbitrarily set to 1.0. A
significant
increase in LI was observed in cultures transfected with RSVp.FGF
(2.3-fold), RSVp.metFGF (2.0-fold), and RSVp.metFGF (2.3-fold)
compared with cells transfected with RSVp.CONT (P<.001).
There was no significant difference between the effects of
overexpression of high and low molecular weight FGF-2 on LI.
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In a
second approach, immunofluorescence staining
with monoclonal antibodies to myosin and BrdU was used to confirm
myocyte identity and assess the proportion of myocytes undergoing
active DNA synthesis (S-phase nuclei) in cultures transfected with FGF
hybrid genes or control plasmid. Anti-BrdU staining was confined to the
nucleus, whereas anti-myosin staining was exclusively cytoplasmic
in the rat cardiac myocytes. The results are shown in Fig 5
and
are
presented as fold differences relative to the control
(RSVp.CONT) value (18.8±1.1%, n=11), which was arbitrarily set
to
1.0. There was a significant increase in the number of myocyte nuclei
staining for BrdU in cultures transfected with RSVp.FGF (2.3-fold),
RSVp.metFGF (1.9-fold), and RSVp.metFGF (2.7-fold) compared with
cells transfected with RSVp.CONT (P<.001). The difference
between the effects of overexpression of high molecular weight (FGF,
P<.05; metFGF, P<.001) versus low molecular
weight FGF-2 on BrdU staining was small but statistically
significant.
Effect of FGF-2 on Cell Number and MI
To determine the
effects of overexpression of high or low
molecular weight forms of FGF-2 on cardiac myocyte proliferation, we
scored PAS+ cells (myocytes) in random fields from cultures
transfected with FGF-2 cDNAs and RSVp.CONT. The results are shown in
Fig 6 and are presented as fold
differences relative to the control (RSVp.CONT) value, which was
arbitrarily set to 1.0. There was a significant increase in PAS+ cells
in cultures transfected with RSVp.FGF (2.1-fold), RSVp.metFGF
(1.8-fold), and RSVp.metFGF (2.1-fold) compared with cultures
transfected with RSVp.CONT (P<.001). There was no
significant difference between the effects of overexpression of high
and low molecular weight FGF-2 on the number of PAS+ cells.
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We
also determined an MI as a further indicator of proliferative
potential. The fraction of cardiac myocytes in three readily
identifiable stages of mitosis (metaphase, anaphase, and telophase) was
assessed in random fields from cultures transfected with FGF-2
cDNAs and RSVp.CONT. The chromosomes were easily identified by
a combination of Hoechst staining for DNA and
immunofluorescence microscopy. The results are
shown in Fig 6 and are presented as fold differences relative
to the control (RSVp.CONT) value (4.6±0.5%, n=13), which was
arbitrarily set to 1.0. There was a significant increase in MI of
cultures transfected with RSVp.FGF (2.2-fold), RSVp.metFGF
(2.1-fold), and RSVp.metFGF (2.4-fold) compared with cultures
transfected with RSVp.CONT (P<.001). There was no
significant difference between the effects of overexpression of high
and low molecular weight FGF-2 on overall MI.
In the process of
assessing MI, it was observed that cardiac myocytes
undergoing mitosis were often found in proximity to cardiac myocytes
overexpressing FGF-2 in cultures transfected with high or low molecular
weight FGF-2. In a field corresponding to a five-cell radius
centered around a cardiac myocyte overexpressing high molecular weight
(metFGF) or low molecular weight (metFGF) FGF-2, there was a
significant 3.9-fold increase in cells visibly undergoing mitosis
compared with an identical field in the same culture but lacking a cell
visibly overexpressing FGF-2 (Fig 7; see values obtained
in the presence of NM Ab). This apparent proximity-dependent effect
on mitosis was inhibited in the presence of FGF-2 antibodies (Fig
7).
There was no significant difference between the incidence of mitosis
near an FGF-2–overexpressing and –nonoverexpressing myocyte in the
presence of neutralizing FGF-2 antibodies. Further, this level was
similar to background levels obtained from cultures transfected with
RSVp.CONT and maintained with either NM Ab or FGF-2 antibodies (Fig
7).
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Effect of FGF-2 on Binucleation
The fraction of binucleated
cardiac myocytes was determined
in random fields from cultures transfected with FGF-2 cDNAs and
RSVp.CONT. The nuclei of cardiac myocytes were easily identified
by a combination of Hoechst staining for DNA, antibodies to
myosin, and immunofluorescence microscopy. The
results are shown in Fig 8 and are presented as
fold differences relative to the control (RSVp.CONT) value (6.6±0.6%,
n=33), which was arbitrarily set to 1.0. There was a significant
increase in binucleation in cultures transfected with RSVp.FGF
(2.2-fold, P<.001) and RSVp.metFGF (2.0-fold,
P<.001) but not RSVp.metFGF compared with cultures
transfected with RSVp.CONT.
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Triple staining for myosin, FGF-2, and DNA
suggested that a
significant proportion of cardiac myocytes overexpressing FGF-2 was
binucleated. We assessed the level of binucleation as a percentage of
cardiac myocytes overexpressing FGF-2 in cultures transfected with
RSVp.FGF, RSVp.metFGF, or RSVp.metFGF (Fig 8).
Binucleation was seen
in 33% and 40% of overexpressing cardiac myocytes in cultures
transfected with RSVp.FGF and RSVp.metFGF, respectively; the
difference between these results is not considered significant. In
contrast, only 9% of cardiac myocytes overexpressing RSVp.metFGF were
binucleated, which is significantly different from the value obtained
with either RSVp.FGF (P<.0001) or RSVp.metFGF
(P<.0001).
Since antibodies to FGF-2 were able to inhibit
the effect of high as
well as low molecular weight FGF-2 on mitosis, we assessed whether
there was a similar effect on binucleation. Percentage binucleation was
assessed in cultures expressing high or low molecular weight FGF-2 and
treated with NM Ab or neutralizing antibodies to FGF-2 (Fig 9).
There was no significant effect of FGF-2 antibodies
on the stimulation of binucleation observed with overexpression of high
molecular weight FGF-2 (FGF or metFGF, Fig 9). Further,
the
percentage level of binucleation of cardiac myocytes overexpressing
FGF, metFGF, or metFGF was unaffected by the presence of
neutralizing FGF-2 antibodies (not shown).
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A small number of cardiac
myocytes (<1%) overexpressing high
but not low molecular weight FGF-2 contained what appeared to be
multiple nuclei of varying sizes (Fig 10). These
multiple nuclei as well as the DNA clumping described previously were
still apparent in the presence of neutralizing antibodies to FGF-2. In
addition, a line of nuclear cleavage was observed by FGF-2 and DNA
staining in 5% of cardiac myocytes overexpressing high molecular
weight FGF-2 (Fig 11) compared with <0.1% of cells
overexpressing 18-kD FGF-2 or transfected with RSVp.CONT. The cleavage
line appeared to divide the nucleus symmetrically or
asymmetrically.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Previously, we compared the effects of high versus low molecular weight rat FGF-2 overexpression on proliferation and nuclear morphology in embryonic chicken cardiac ventricular myocytes. In the present study, we used the same rat FGF-2 cDNAs to extend these observations in homologous rat neonatal cardiac ventricular myocyte cultures. The subcellular distribution of FGF-2 in the overexpressing rat cardiac myocyte cultures was identical to that observed with embryonic chicken myocytes.17 The staining was predominantly nuclear in cultures transfected with FGF or
metFGF
(Fig 2), whereas staining of the nucleus as well as cytoplasm
was seen
with metFGF (Fig 3). This pattern of FGF-2 staining was also
obtained
when noncardiac (kidney) COS-1 cells were transfected with the FGF
cDNAs and assessed by immunofluorescence staining
(not shown). This, in turn, is consistent with the identical
patterns of FGF-2 expression seen in these three cell types by
immunoblotting. Modified rat FGF-2 cDNAs, metFGF and
metFGF, express high molecular weight (22- and 21-kD) or low molecular
weight (18-kD) species, respectively.17 Overexpression of
wild-type FGF-2 cDNA (FGF) generates a pattern identical to that
seen with metFGF, and only the 22- and 21.5-kD forms are observed.
The level of 18-kD FGF-2 after overexpression of metFGF was
consistently fivefold greater than 22- and 21.5-kD FGF-2
generated from metFGF and FGF cDNAs (determined by densitometry,
n=8). This is in agreement with the observation that translation from
the methionine codon is repressed by upstream sequences containing the
leucine start sites.17 31
Overexpression of both high and low molecular weight FGF-2 in neonatal
rat cardiac myocyte cultures resulted in a significant overall
2.1-fold stimulation of proliferative potential, which compares well
with the 2.6-fold effect seen previously with embryonic chicken
ventricular myocytes.17 Any differences in the
potency of high versus low molecular weight FGF-2 may have been masked
by the higher levels of 18-kD FGF-2 expression. The LI for rat cardiac
myocytes is reported to decline from day 15 of gestation and approaches
zero by the end of the third week after birth.32 The LI
(15.1%) obtained with neonatal rat cardiac control cultures
(transfected with RSVp.CONT) in the present study is in agreement
with LI values reported previously for neonatal rat hearts
(15.4%)20 as well as cardiac myocyte cultures
(12.5%).32 Similarly, the LI for neonatal rat myocyte
cultures transfected with rat FGF-2 cDNAs (43.1%), is comparable to
the LI values of 32.5% and 30.6% determined for embryonic cardiac
myocytes in vitro and in vivo, respectively.32 33
Thus,
these results suggest that the overexpression of both high and low
molecular weight forms of rat FGF-2 in neonatal cardiac myocyte
cultures can increase basal levels of DNA synthesis to those levels
associated with the embryonic phenotype.
When determining the MI, it became apparent that these mitotic cells
were often located in proximity (five-cell radius) to cardiac
myocytes overexpressing FGF-2. There was no significant increase in
mitosis above background levels in adjacent areas where no
overexpressing cell was apparent. Further, this proximity-dependent
increase in mitosis seen with both high or low molecular weight FGF-2
was blocked in the presence of neutralizing FGF-2 antibodies (Fig
7).
Thus, the 2.2-fold increase in MI seen in cultures overexpressing
22/21.5- and 18-kD FGF-2 (Fig 6) is consistent with the release
of FGF-2 from overexpressing cells stimulating mitosis in a paracrine
and proximity-dependent manner. Presumably, this occurs through a
receptor-mediated pathway. Although FGF-2 lacks a signal sequence
for its secretion, there is evidence to suggest that it can be actively
released and bind to the immediate extracellular matrix.34
It is also possible that a contribution to the effects on DNA synthesis
and cell division occurs through the release of FGF-2 from damaged
cells. Regardless, these effects are mediated by FGF-2 as opposed to
the transfection process, since results were significantly different
from those obtained with control cultures transfected with
RSVp.CONT.
The stimulatory effects of high molecular weight (2.3- and 2.0-fold for
FGF and metFGF, respectively) and low molecular weight (2.3-fold for
metFGF) forms of FGF-2 on DNA synthesis as determined by
[3H]thymidine incorporation and PAS staining were
comparable (Fig 5). However, assessment of DNA synthesis by a
combination of BrdU incorporation and anti-myosin staining revealed
a small but significant difference in the degree of stimulation by
22/21.5-kD (2.3- and 1.9-fold for FGF and metFGF, respectively)
versus 18-kD (2.7-fold) FGF-2. The reason for the discrepancy in the
results obtained with thymidine versus BrdU incorporation is unclear
but is possibly related to the different methods used to identify
myocytes. Regardless, we observed no significant difference between the
stimulatory effects of high and low molecular weight forms of FGF-2 on
cell number and MI.
By contrast, a significant (2.0-fold) increase in total binucleation
was observed in neonatal ventricular myocyte cultures
transfected with high molecular weight (FGF or metFGF) but not low
molecular weight (metFGF) FGF-2 (Fig 8). Unlike the effect of
high (or
low) molecular weight FGF-2 on cell division as measured by mitosis
(Fig 7), stimulation of binucleation in cardiac myocytes by
22/21.5-kD
FGF-2 was not blocked by neutralizing antibodies to FGF-2 added to the
culture medium (Fig 9). Thus, in contrast to the paracrine
(cell
surface receptor–mediated) pathway indicated for hyperplastic
growth, the results are consistent with a distinct
intracellular effect of high molecular weight FGF-2 on binucleation in
rat neonatal cardiac myocytes in culture. Further evidence for an
intracellular effect of high molecular weight FGF-2 was reported
recently on the basis of phenotypic changes occurring in mouse 3T3
cells overexpressing dominant negative FGFR-1.35
Binucleation is considered to be an early marker of cardiac myocyte
growth hypertrophy.20 Rat cardiac myocytes are
mononucleated during fetal and early neonatal development,
representing a period of hyperplastic growth. This is
followed by a slow transition into hypertrophic growth, during which
85% of cardiac myocytes become binucleated by the third week after
birth.20 Binucleation maintains the normal nuclear versus
cytoplasmic ratio in cardiac myocytes during their
physiological hypertrophic
growth.36 37 However, it is also possible that some
of
these binucleated cardiac myocytes may serve as potential sources of
"new" cells in pathological hypertrophy. It was
proposed that some binucleated cardiac myocytes might divide into two
by formation of new intercalated disks.38 In addition,
binucleation in hepatocytes decreases during regenerative,
hyperplastic, and neoplastic growth.39 Binucleation in
cardiac myocytes is believed to result from karyokinesis without
cytokinesis, although binucleation as a result of amitotic
division has been
described.20 33 36 40 There
is no
information about the factors responsible for the binucleation
resulting from failure of cytokinesis or amitosis. Our results
show that there is a strong correlation between overexpression of
22/21.5-kD FGF-2 and an increase in the incidence of binucleation in
neonatal rat cardiac myocyte cultures. More than a third of neonatal
rat cardiac myocytes overexpressing 22/21.5-kD FGF-2 were binucleated
(Fig 8). Our data do not rule out karyokinesis without
cytokinesis or amitosis as mechanisms for binucleation in
cardiac myocytes overexpressing FGF-2. With regard to karyokinesis, a
background level of mitosis still occurs, even in the presence of
neutralizing FGF-2 antibodies (Fig 7). This would be induced,
presumably, by factors other than extracellular FGF-2. However, we also
observed lines of symmetric and asymmetric nuclear cleavage in 5%
of cardiac myocytes overexpressing the high molecular weight forms,
suggesting that they were undergoing amitotic division. If this is the
case, this percentage would be an underestimate of the overall extent
of amitosis, since it represents a "snapshot" of the
whole process and does not include cells with fully separated nuclei;
unless binucleated cells are the product of a clearly asymmetric
nuclear division (ie, containing two nuclei of different sizes), it
would not be possible to differentiate between karyokinesis and
amitosis. Amitosis could represent a culture phenomenon;
however, a few nuclear divisions in cardiac myocytes were reported to
occur during postnatal cardiomyogenesis through amitosis with patterns
suggestive of nuclear partitions, fragmentation, paired nuclei, and
nuclear chains.33 Cardiac myocytes containing three or
more nuclei (multinucleated) were reported to constitute 5% of
ventricular myocytes in the adult rat heart,21
and examples were also seen in the adult human heart.38
These patterns were evident in our cultures. Chains of nuclei of
varying sizes were observed in cardiac myocytes overexpressing high but
not low molecular weight FGF-2 (Fig 10). This is consistent
with the notion that 22/21.5-kD FGF-2 mediates its effect on nucleation
of cardiac myocytes through amitosis, presumably, in an intracrine
manner. It is also possible that the DNA clumping seen in mononucleated
and binucleated cardiac myocytes overexpressing 22/21.5-kD FGF-2
(Fig 4)17 represents an aspect of this process;
however, chromatin condensation and nuclear fragmentation are also
features of apoptosis. Apoptosis has been linked to the
control of the primitive myocardial cell overgrowth associated with
cardiac rhabdomyoma as well as removal of damaged cardiac myocytes
after reperfusion injury.41 42 FGF-2 is known to bind
to
chromatin43 and is capable of modifying gene transcription
in vitro.44 The high molecular weight species would be
expected to associate with chromatin at a higher affinity than 18-kD
FGF-2 because of the additional basic amino acids present in the
amino terminal extension.45
In summary, overexpression of both 22/21.5- and 18-kD FGF-2 can stimulate hyperplastic growth of neonatal rat cardiac myocytes. This is mediated, at least in part, through a proximity-dependent paracrine effect on adjacent cardiac myocytes and is of a similar magnitude for both forms of FGF-2. In contrast, overexpression of high but not low molecular weight FGF-2 leads to a significant increase in binucleation and changes in nuclear morphology, even in the presence of neutralizing antibodies, suggesting an intracrine pathway. Although the mechanism by which 22/21.5-kD FGF-2 induces binucleation remains to be elucidated, nuclear amitotic cleavage might contribute to this process.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by the Medical Research Council of Canada. Dr Pasumarthi is the recipient of a Heart and Stroke Foundation of Canada Studentship, Dr Kardami is the recipient of a Medical Research Council of Canada Cardiovascular Group Award, and Dr Cattini is the recipient of a Medical Research Council of Canada Scientist Award. The authors would like to thank M.E. Bock, Y. Chen, and R.R. Fandrich for excellent technical assistance.
|
Footnotes |
|---|
Reprint requests to Dr Peter A. Cattini, Department of Physiology, University of Manitoba, 730 William Ave, Winnipeg, Manitoba, R3E 3J7, Canada.
Received April 6, 1995; accepted September 12, 1995.
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References |
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