Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 1996;78:126-136

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pasumarthi, K. B.S.
Right arrow Articles by Cattini, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pasumarthi, K. B.S.
Right arrow Articles by Cattini, P. A.
(Circulation Research. 1996;78:126-136.)
© 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

Kishore B.S. Pasumarthi, Elissavet Kardami, Peter A. Cattini

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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 ({approx}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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, {approx}82% of ventricular myocytes are reported to be binucleated, and {approx}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.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Plasmid Constructs
Expression vectors containing the wild-type and modified rat FGF-2 cDNAs were described previously.17 The {Delta}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 ({Delta}metFGF and metFGF) were cloned in between the RSV promoter (indicated by p) and SV40 polyadenylation signal to obtain RSVp.FGF, RSVp.{Delta}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 1Down). 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.



View larger version (74K):
[in this window]
[in a new window]
  		Figure 1. Schematic representation of hybrid FGF-2 genes and their pattern of expression after transient gene transfer. Wild-type (FGF [CTG and ATG sites]) as well as modified FGF-2 sequences coding for high molecular weight ({Delta}metFGF [modified ATG site]) or low molecular weight (metFGF [deleted CTG sites]) species were introduced between the RSV promoter (RSVp) and SV40 polyadenylation signal to obtain RSVp.FGF, RSVp.{Delta}metFGF, and RSVp.metFGF, respectively. These hybrid genes, as well as the RSVp vector without any FGF-2 sequence (RSVp.CONT), were used to transfect COS-1 cells. Heparin-Sepharose–purified COS-1 cell lysates were fractionated in a 12.5% SDS-PAGE gel, immunoblotted, probed with rabbit FGF-2 antibodies, and visualized with [125I]protein A. The mobilities of 22-, 21.5-, and 18-kD FGF-2 are indicated.

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 {approx}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, {approx}2500 cardiac myocytes from each culture transfected with RSVp.CONT, RSVp.FGF, RSVp.{Delta}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.{Delta}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 ({Delta}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, {approx}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.{Delta}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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
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.{Delta}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 2Down). 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 3Down). However, the majority of cells displayed both cytoplasmic as well as nuclear staining of comparable intensity. Control cultures transfected with RSVp.CONT (Fig 2Down) 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 2Down and 3Down). 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 4Down).



View larger version (112K):
[in this window]
[in a new window]
  		Figure 2. Localization of FGF-2 in cardiac myocytes transfected with RSVp.{Delta}FGF (a through f) and RSVp.CONT (g through i). Cells were stained for FGF-2 (a, d, and g), DNA (b, e, and h), and myosin (c, f, and i). Predominant nuclear localization of FGF-2 was observed in mononucleated (a through c) and binucleated (d through f) cardiac myocytes. An identical pattern of staining was seen in cultures transfected with RSVp.FGF. Bar=50 µm.



View larger version (93K):
[in this window]
[in a new window]
  		Figure 3. Localization of FGF-2 in cardiac myocytes transfected with RSVp.metFGF. Cells were stained for FGF-2 (a and d), DNA (b and e), and myosin (c and f). Both nuclear and cytoplasmic localization of FGF-2 was observed in mononucleated (a through c) and binucleated (d through f) cardiac myocytes. Note the range of nuclear and cytoplasmic staining observed with metFGF overexpression. Bar=20 µm (a through c) and 50 µm (d through f).



View larger version (113K):
[in this window]
[in a new window]
  		Figure 4. Overexpression of 22/21-5 kD FGF-2 in neonatal rat cardiac myocyte cultures is associated with DNA clumps. Light micrographs show paired examples of mononucleated and binucleated cardiac myocytes stained for FGF-2 (a and b) and DNA (c and d) and displaying DNA clumping. The intensity and pattern of DNA staining is suggestive of chromatin condensation. Bar=20 µm.

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 5Down 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.{Delta}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.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 5. Overexpression of 22/21.5-and 18-kD FGF-2 in cardiac myocyte cultures stimulates DNA synthesis. DNA synthesis in transfected cardiac myocytes was assessed by [3H]thymidine and BrdU incorporation. LI (radiolabeled PAS+ cells per total number of PAS+ cells) was determined (n=8) by using a combination of in situ autoradiography and histochemistry. BrdU labeling was determined (n=11) by immunofluorescence staining with monoclonal antibodies to BrdU and myosin. The results are presented as fold differences relative to the control (RSVp.CONT) value, which was arbitrarily set to 1.0. Error bars are SEM. There are significant increases of LI and BrdU labeling in cultures transfected with RSVp.{Delta}metFGF, RSVp.metFGF, and RSVp.FGF compared with cultures transfected with the RSVp.CONT. There was no significant difference between the effects of high versus low molecular weight FGF-2 overexpression on LI. There was, however, a small statistically significant difference between the effects of overexpression of high molecular weight (FGF, P<.05; {Delta}metFGF, P<.001) versus low molecular weight FGF-2 on BrdU staining.

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 5Up 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.{Delta}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; {Delta}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 6Down 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.{Delta}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.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 6. Overexpression of 22/21.5- and 18-kD FGF-2 in neonatal rat cardiac myocyte cultures is associated with increases in cell number and MI. Cardiac myocyte proliferation was determined by scoring PAS+ cell number as well as by readily visible mitotic figures (by Hoechst staining) in random fields (n=8 to 13) in cultures transfected with FGF-2 cDNAs and RSVp.CONT. The results are presented as fold differences relative to the control (RSVp.CONT) value, which was arbitrarily set to 1.0. Error bars are SEM. There are significant increases in PAS+ cell number (P<.001) and MI (P<.001) of cultures transfected with RSVp.{Delta}metFGF, RSVp.metFGF, and RSVp.FGF. There is no significant difference between the effects of high versus low molecular weight FGF-2 overexpression on PAS+ cell number and MI.

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 6Up 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.{Delta}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 ({Delta}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 7Down; 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 7Down). 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 7Down).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 7. A proximity-dependent increase in mitosis seen with both high molecular weight ({Delta}metFGF) and low molecular weight (metFGF) FGF-2 is inhibited in the presence of neutralizing antibodies (Abs) to FGF-2. MI values for cardiac myocytes were determined by scoring the percentage number of cells with readily visible mitotic figures (by Hoechst staining) in areas corresponding to a five-cell radius (n=15) containing or not containing an overexpressing cell, from the same culture, transfected with either RSVp.{Delta}metFGF or RSVp.metFGF. This experiment was performed with cells maintained in the presence of NM Ab or FGF-2 Ab. Cultures transfected with RSVp.CONT were also used to determine background control levels (n=15 to 20). A significant increase was observed in cells visibly undergoing mitosis in the presence versus absence of an FGF-2–overexpressing cardiac myocyte in the same culture, whether transfected with high molecular weight ({Delta}metFGF, P<.02) or low molecular weight (metFGF, P<.0001) FGF-2. This proximity-dependent stimulation of mitosis was inhibited when cardiac myocytes were maintained with FGF-2 antibodies after transfection.

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 8Down 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.{Delta}metFGF (2.0-fold, P<.001) but not RSVp.metFGF compared with cultures transfected with RSVp.CONT.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 8. Overexpression of 22/21.5- but not 18-kD FGF-2 in neonatal rat cardiac myocyte cultures stimulates binucleation. Cardiac myocyte binucleation was determined by scoring binucleated cells (by myosin and Hoechst staining) in random fields (n=25 to 35) in cultures transfected with FGF-2 cDNAs and RSVp.CONT. The results 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. Error bars are SEM. There were significant increases in overall binucleation of cultures transfected with RSVp.{Delta}metFGF (P<.001) and RSVp.FGF (P<.001) but not RSVp.metFGF. Also, the level (presented as a percentage) of binucleation in cardiac myocytes overexpressing FGF-2 after transfection with RSVp.FGF, RSVp.{Delta}metFGF, or RSVp.metFGF is shown. Error bars represent SEM. There is a significant difference between the percentage of binucleation seen in cardiac myocytes overexpressing 18-kD FGF-2 (RSVp.metFGF) versus 22/21.5-kD FGF-2 with either RSVp.FGF (P<.0001) or RSVp.{Delta}metFGF (P<.0001).

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.{Delta}metFGF, or RSVp.metFGF (Fig 8Up). Binucleation was seen in 33% and 40% of overexpressing cardiac myocytes in cultures transfected with RSVp.FGF and RSVp.{Delta}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.{Delta}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 9Down). 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 {Delta}metFGF, Fig 9Down). Further, the percentage level of binucleation of cardiac myocytes overexpressing FGF, {Delta}metFGF, or metFGF was unaffected by the presence of neutralizing FGF-2 antibodies (not shown).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 9. Stimulation of binucleation by 22/21.5-kD FGF-2 is not inhibited in the presence of neutralizing antibodies (Abs) to FGF-2. Cardiac myocyte binucleation was determined by scoring binucleated cells (by myosin and Hoechst staining) in random fields (n=30) in cultures transfected with FGF-2 cDNAs and RSVp.CONT and then maintained in the presence of NM Ab or neutralizing FGF-2 Ab. There was no significant effect of FGF-2 Abs on the stimulation of binucleation observed with overexpression of high molecular weight FGF-2 (FGF or {Delta}metFGF).

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 10Down). 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 {approx}5% of cardiac myocytes overexpressing high molecular weight FGF-2 (Fig 11Down) 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.



View larger version (57K):
[in this window]
[in a new window]
  		Figure 10. Cardiac myocyte containing nuclei of varying sizes in cultures overexpressing 22/21.5-kD FGF-2. Triple staining was done for FGF-2 (a), DNA (b), and myosin (c). Light micrographs show an example of a cardiac myocyte overexpressing FGF-2 and containing multiple nuclei of different sizes in a culture transfected with RSVp.{Delta}metFGF. Multiple nuclei were also seen in cultures overexpressing RSVp.FGF. Bar=20 µm.



View larger version (66K):
[in this window]
[in a new window]
  		Figure 11. Nuclear cleavage in cardiac myocytes overexpressing 22/21.5-kD FGF-2. Triple staining was done for FGF-2 (a and d), DNA (b and e), and myosin (c and f). Light micrographs show different stages of nuclear cleavage. Nuclear furrowing is visible by FGF-2 or DNA staining (a and b), and traces of myosin can be detected in the furrow (c). After what appears to be cleavage, the nuclei continue to stain intensely for FGF-2 (d and e), and myosin staining between nuclei, in this case of different sizes, is evident (f). Arrows indicate plane of cleavage furrow. Bar=10 µm (a through c) or 15 µm (d through f).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
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 {Delta}metFGF (Fig 2Up), whereas staining of the nucleus as well as cytoplasm was seen with metFGF (Fig 3Up). 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, {Delta}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 {Delta}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 {Delta}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 {approx}2.1-fold stimulation of proliferative potential, which compares well with the {approx}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 ({approx}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 7Up). Thus, the {approx}2.2-fold increase in MI seen in cultures overexpressing 22/21.5- and 18-kD FGF-2 (Fig 6Up) 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 {Delta}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 5Up). 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 {Delta}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 {Delta}metFGF) but not low molecular weight (metFGF) FGF-2 (Fig 8Up). Unlike the effect of high (or low) molecular weight FGF-2 on cell division as measured by mitosis (Fig 7Up), 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 9Up). 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 {approx}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 8Up). 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 7Up). This would be induced, presumably, by factors other than extracellular FGF-2. However, we also observed lines of symmetric and asymmetric nuclear cleavage in {approx}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 {approx}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 10Up). 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 4Up)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
 
ß-gal = ß-galactosidase
BrdU = bromodeoxyuridine
FBHE cell = fetal bovine heart endothelial cell
FGF = fibroblast growth factor
FGFR-1 = FGF receptor-1
LI = labeling index
MI = mitotic index
NM Ab = normal mouse antibody (IgG)
PAS = periodic acid–Schiff
PAS+ cell = PAS-positive cell
RSV = Rous sarcoma virus
SV40 = simian virus 40
X-gal = 5-bromo-4-chloro-3-indolyl-ß-D-galactoside


*    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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 

  1. Baird A. Fibroblast growth factors, activities and significance of non-neurotrophin neurotrophic growth factors. Curr Opin Neurobiol. 1994;4:78-86. [Medline] [Order article via Infotrieve]
  2. Mason IJ. The ins and outs of fibroblast growth factors. Cell. 1994;78:547-552. [Medline] [Order article via Infotrieve]
  3. Borja AZM, Meijers C, Zeller R. Expression of alternatively spliced bFGF first coding exons and antisense mRNAs during chicken embryogenesis. Dev Biol. 1993;157:110-118. [Medline] [Order article via Infotrieve]
  4. Claycomb WC, Moses RL. Growth factors and TPA stimulate DNA synthesis and alter the morphology of cultured terminally differentiated adult rat cardiac muscle cells. Dev Biol. 1988;127:257-265. [Medline] [Order article via Infotrieve]
  5. Kardami E. Stimulation and inhibition of cardiac myocyte proliferation in vitro. Mol Cell Biochem. 1990;92:129-135. [Medline] [Order article via Infotrieve]
  6. Sugi Y, Sasse J, Lough J. Inhibition of precardiac mesoderm cell proliferation by antisense oligodeoxynucleotide complementary to fibroblast growth factor-2 (FGF-2). Dev Biol. 1993;157:28-37. [Medline] [Order article via Infotrieve]
  7. Ueno H, Perryman MB, Roberts R, Schneider MD. Differentiation of cardiac myocytes after mitogen withdrawal exhibits three sequential states of the ventricular growth response. J Cell Biol. 1988;107:1911-1918. [Abstract/Free Full Text]
  8. Jaye M, Schlessinger J, Dionne CA. Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction. Biochim Biophys Acta. 1992;1135:185-199. [Medline] [Order article via Infotrieve]
  9. Engelmann GL, Dionne CA, Jaye MC. Acidic fibroblast growth factor and heart development: role in myocyte proliferation and capillary angiogenesis. Circ Res. 1993;72:7-19. [Abstract/Free Full Text]
  10. Jin Y, Pasumarthi KBS, Bock ME, Lytras A, Kardami E, Cattini PA. Cloning and expression of fibroblast growth factor receptor-I isoforms in the mouse heart: evidence for isoform switching during heart development. J Mol Cell Cardiol. 1994;26:1449-1459. [Medline] [Order article via Infotrieve]
  11. Liu L, Pasumarthi KBS, Padua RA, Massaeli H, Fandrich RR Pierce GN, Cattini PA, Kardami E. Adult cardiomyocytes express functional high-affinity receptors for basic fibroblast growth factor. Am J Physiol. 1995;268:H1927-H1938. [Abstract/Free Full Text]
  12. Peters KG, Werner S, Chen G, Williams LT. Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development. 1992;114:233-243. [Abstract]
  13. Wanaka A, Milbrandt J, Johnson EM. Expression of FGF receptor gene in rat development. Development. 1991;111:455-468. [Abstract]
  14. Florkiewicz RZ, Sommer A. Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons. Proc Natl Acad Sci U S A. 1989;86:3978-3981. [Abstract/Free Full Text]
  15. Doble BW, Fandrich RR, Liu L, Kardami E. Calcium protects pituitary basic FGF from proteolysis by co-purifying protease. Biochem Biophys Res Commun. 1991;173:1116-1122.
  16. Klagsbrun M, Smith S, Sullivan R, Shing Y, Davidson S, Smith JA, Sasse J. Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases. Proc Natl Acad Sci U S A. 1987;84:1839-1843. [Abstract/Free Full Text]
  17. Pasumarthi KBS, Doble BW, Kardami E, Cattini PA. Over-expression of CUG- or AUG-initiated forms of basic fibroblast growth factor in cardiac myocytes results in similar effects on mitosis and protein synthesis but distinct nuclear morphologies. J Mol Cell Cardiol. 1994;26:1045-1060.[Medline] [Order article via Infotrieve]
  18. Liu L, Doble BW, Kardami E. Perinatal phenotype and hypothyroidism are associated with elevated levels of 21.5- to 22-kDa basic fibroblast growth factor in cardiac ventricles. Dev Biol. 1993;157:507-516. [Medline] [Order article via Infotrieve]
  19. Zak R. Factors controlling cardiac growth. In: Zak R, ed. Growth of the Heart in Health and Disease. New York, NY: Raven Press Publishers; 1984:165-185.
  20. Clubb FJ, Bishop SP. Formation of binucleated myocardial cells in the neonatal rat: an index for growth hypertrophy. Lab Invest. 1984;50:571-577. [Medline] [Order article via Infotrieve]
  21. Kellerman S, Moore JA, Zierhut W, Zimmer HG, Campbell J, Geredes AM. Nuclear DNA content and nucleation patterns in rat cardiac myocytes from different models of cardiac hypertrophy. J Mol Cell Cardiol. 1992;24:497-505. [Medline] [Order article via Infotrieve]
  22. Shimasaki S, Emoto N, Koba A, Mercado M, Shibata F, Cooksey K, Baird A, Ling N. Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA. Biochem Biophys Res Commun. 1988;157:256-263. [Medline] [Order article via Infotrieve]
  23. Iwaki K, Sukhatme VP, Shubeita HE, Chien KR. {alpha}- and ß-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells. J Biol Chem. 1990;265:13809-13817. [Abstract/Free Full Text]
  24. Cattini PA, Eberhardt NL. Regulated expression of chimaeric genes containing the 5' flanking region of human growth hormone related genes in transiently transfected rat pituitary tumor cells. Nucleic Acids Res. 1987;15:1297-1309. [Abstract/Free Full Text]
  25. Kardami E, Stoski RM, Doble B, Yamamoto T, Hertzberg EL, Nagy JI. Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions. J Biol Chem. 1991;266:19551-19557. [Abstract/Free Full Text]
  26. Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
  27. Cattini PA, Nickel B, Bock M, Kardami E. Immunolocalization of basic fibroblast growth factor (bFGF) in growing and growth-inhibited placental cells: a possible role for bFGF in placental cell development. Placenta. 1991;12:341-352. [Medline] [Order article via Infotrieve]
  28. Kardami E, Fandrich RR. Basic fibroblast growth factor in atria and ventricles of the vertebrate heart. J Cell Biol. 1989;109:1865-1875. [Abstract/Free Full Text]
  29. Xu H, Miller J, Liang BT. High-efficiency gene transfer into cardiac myocytes. Nucleic Acids Res. 1992;20:6425-6426. [Free Full Text]
  30. Matsuzaki K, Yoshitake Y, Matuo Y, Sasaki H, Nishikawa K. Monoclonal antibodies against heparin-binding growth factor II/basic fibroblast growth factor that block its biological activity: invalidity of the antibodies for tumor angiogenesis. Proc Natl Acad Sci U S A. 1989;86:9911-9915. [Abstract/Free Full Text]
  31. Prats AC, Vagner S, Prats H, Amalric F. cis-Acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA. Mol Cell Biol. 1992;12:4796-4805. [Abstract/Free Full Text]
  32. Marino TA, Haldar S, Williamson EC, Beaverson K, Walter RA, Marino DR, Beatty C, Lipson KE. Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells. Circ Res. 1991;69:1353-1360. [Abstract/Free Full Text]
  33. Rumyantsev PP. Reproduction of cardiac myocytes developing in vivo and its relation to processes of differentiation. In: Carlson BM, ed. Growth and Hyperplasia of Cardiac Muscle Cells. New York, NY: Harwood Academic Publishers; 1991:70-159.
  34. D'Amore PA. Modes of FGF release in vivo and in vitro. Cancer Metastasis Rev. 1990;9:227-238. [Medline] [Order article via Infotrieve]
  35. Bikfalvi A, Klein S, Pintucci G, Quarto N, Mignatti P, Rifkin DB. Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms. J Cell Biol. 1995;129:233-243. [Abstract/Free Full Text]
  36. Brodsky VYA. Cell ploidy in the mammalian heart. In: Oberpriller JO, Oberpriller JC, Mauro A, eds. The Development and Regenerative Potential of Cardiac Muscle. New York, NY: Harwood Academic Publishers; 1991:253-292.
  37. Engelmann GL, Vitullo JC, Gerity RG. Age-related changes in ploidy levels and biochemical parameters in cardiac myocytes isolated from spontaneously hypertensive rats. Circ Res. 1986;58:137-147. [Abstract/Free Full Text]
  38. Shozawa T, Okada E, Kawamura K, Sageshima M, Masuda H. Development of binucleated myocytes in normal and hypertrophied human hearts. Am J Cardiovasc Pathol. 1990;3:27-36. [Medline] [Order article via Infotrieve]
  39. Gerlyng P, Stokke T, Huitfeldt HS, Stenersen T, Danielsen HE, Grotmol T, Seglen PO. Analytical methods for the study of liver cell proliferation. Cytometry. 1992;13:404-415. [Medline] [Order article via Infotrieve]
  40. Katzberg AA, Farmer BB, Harris RA. The predominance of binucleation in isolated rat heart myocytes. Am J Anat. 1977;149:489-500. [Medline] [Order article via Infotrieve]
  41. Medioni D, Pages A, Sarda P. Natural history of cardiac rhabdomyoma: presentation of 2 cases with immunohistochemical study and review of the literature. Arch Anat Cytol Pathol. 1994;42:29-34. [Medline] [Order article via Infotrieve]
  42. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest. 1994;94:1621-1628.
  43. Gualandris A, Coltrini D, Bergonzoni L, Isacchi A, Tenca S, Ginelli B, Presta M. The NH2-terminal extension of high molecular weight forms of basic fibroblast growth factor (bFGF) is not essential for the binding of bFGF to nuclear chromatin in transfected NIH 3T3 cells. Growth Factors. 1992;8:49-60.
  44. Nakanishi Y, Kihara K, Mizuno K, Masamune Y, Yoshitake Y, Nishikawa K. Direct effect of basic fibroblast growth factor on gene transcription in a cell-free system. Proc Natl Acad Sci U S A.. 1992;89:5216-5220. [Abstract/Free Full Text]
  45. Brigstock DR, Klagsbrun M, Sasse J, Farber PA, Iberg N. Species-specific high molecular weight forms of basic fibroblast growth factor. Growth Factors. 1990;4:45-52.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
S. Y. Lu, D. P. Sontag, K. A. Detillieux, and P. A. Cattini
FGF-16 is released from neonatal cardiac myocytes and alters growth-related signaling: a possible role in postnatal development
Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1242 - C1249.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
P. Ahuja, P. Sdek, and W. R. MacLellan
Cardiac Myocyte Cell Cycle Control in Development, Disease, and Regeneration
Physiol Rev, April 1, 2007; 87(2): 521 - 544.
[Abstract]