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Cardiovascular Research 2004 64(3):516-525; doi:10.1016/j.cardiores.2004.08.009
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Copyright © 2004, European Society of Cardiology

Inhibition of TGFβ signaling potentiates the FGF-2-induced stimulation of cardiomyocyte DNA synthesis

Farah Sheikha,1, Cheryl J.A. Hirsta,b, Yan Jina, Margaret E. Bocka, Robert R. Fandrichb, Barbara E. Nickelb, Brad W. Doblea,b, Elissavet Kardamia,b,c and Peter A. Cattinia,*

aDepartment of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7
bInstitute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6
cDepartment of Human Anatomy and Cell Science, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7

* Corresponding author. Tel.: +1 204 789 3696; fax: +1 204 789 3934. Email address: peter_cattini{at}umanitoba.ca

Received 24 April 2004; revised 29 July 2004; accepted 16 August 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 References
 
Objective:: Added transforming growth factor beta (TGFβ) inhibits the proliferation of immature cardiomyocytes. We have now examined the hypothesis that suppression of endogenous TGFβ signaling will boost the proliferative response (DNA synthesis) of cardiac myocytes to serum and/or to the mitogenic factor fibroblast growth factor-2 (FGF-2).

Methods and results:: Overexpression of a kinase-deficient TGFβ type II receptor (TGFβRII{Delta}KD) resulted in a 2.8-fold increase in cardiomyocyte DNA synthesis in serum-rich cultures, an effect requiring active FGFR-1 since it was not observed in the presence of excess kinase-deficient FGFR-1. This finding suggested that endogenous TGFβ–TGFβRII suppressed endogenous FGFR-1-mediated signals that stimulate or are permissive for DNA synthesis. TGFβ had no effect, however, on the FGF-2-induced acute stimulation of extracellular signal regulated kinase1/2. FGF-2, added in the absence or presence of TGFβ inhibition, elicited a 3- or a 13-fold stimulation of DNA synthesis, respectively, pointing to a synergistic effect.

Conclusion:: Inhibition of TGFβRII-transduced signaling upregulates the proliferative response of cardiomyocytes to serum, and greatly potentiates the stimulatory effect of FGF-2. A combinatorial strategy including activation of FGF-2 and inhibition of TGFβ-triggered signal transduction may be required for maximal stimulation of immature cardiomyocyte DNA synthesis.

KEYWORDS Cardiomyocytes; DNA synthesis; Receptor crosstalk; Fibroblast growth factor-2; Transforming growth factor β; Dominant negative receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Experimental procedures
 3. Results
 4. Discussion
 References
 
The notion that the heart does not regenerate due to ‘irreversible’ withdrawal of cardiomyocytes from the cell cycle is currently being reevaluated. To a large degree, this is due to seminal work by Anversa and co-workers [1–3], who, in several elegant studies, documented the presence of a small number of mitotic cardiomyocytes in human and animal hearts. Also, multiple groups have detected the presence of resident cardiac progenitor/stem-cell-like populations that can replace lost muscle in cardiac infarcts [4–6]. Thus, it has been proposed that the adult heart contains a small population of myocytes (derived from a local ‘renewal’ pool, similar to the satellite cells of skeletal muscle, or homing in from the bone marrow) that are capable of proliferating and, presumably, replacing lost tissue [3]. Fibroblast growth factor-2 (FGF-2) is a local mitogen that can be expected to play a major role in stimulating proliferation of these cells. FGF-2 has been demonstrated to stimulate the proliferation of embryonic and neonatal cardiomyocytes [7,8] as well as stem cell populations [9], including skeletal muscle satellite cells [10] and cardiac resident stem cells [6]. In addition, FGF-2 stimulated DNA synthesis of adult cardiomyocytes in long-term cultures [11,12], suggesting the possibility that even these cells may not be as ‘terminally’ differentiated as originally thought. The mitogenic effects of FGF-2 are mediated by binding to and activation of tyrosine kinase membrane receptors (FGFR), among which the FGFR-1 isoform is predominant in cardiomyocytes of all developmental stages [13]. The mitogenic effects of cardiac FGF-2 may be limited or antagonized by local inhibitory factors. One such factor is transforming growth factor beta (TGFβ), a ubiquitous and multifunctional protein that can cause cell cycle arrest [14,15]. In fact, TGFβ can inhibit the FGF-2 and serum-stimulated proliferation of immature cardiomyocytes in vitro [7]. It is conceivable that endogenous, cell associated or secreted TGFβ as well as TGFβ present in serum (and serum-containing media for in vitro studies), could act in a similar fashion and thus mask or reduce growth stimulation by FGF-2. In other words, TGFβ inhibition might be expected to increase the proliferative response of cardiomyocytes to FGF-2. To address this, we used a dominant negative approach [16] to suppress signaling by all TGFβ receptor isoforms and examined effects on serum- and FGF-2-induced stimulation of cardiomyocyte DNA synthesis. We also examined whether the upregulation of cardiomyocyte DNA synthesis that we observed with TGFβ inhibition required presence of active FGFR-1, again using a dominant negative approach. Our data show that: inhibition of TGFβ-TGFβRII stimulates DNA synthesis in an FGFR-1-dependent manner, and that simultaneous stimulation of FGF-2- and inhibition of TGFβ-signal transduction elicits maximal stimulation of immature cardiomyocyte DNA synthesis.


   2. Experimental procedures
Top
 Abstract
 1. Introduction
 2. Experimental procedures
3. Results
 4. Discussion
 References
 
2.1 Cell culture
Neonatal rat ventricular cardiac myocytes (NRCMs) were isolated from 1–2-day-old Sprague Dawley rats by enzymatic digestion with 0.085–0.1% (w/v) trypsin (Invitrogen, Burlington. ON) using a temperature regulated (35 °C) spinner flask, followed by fractionation on a Percoll gradient [17]. Cells were counted using a hemacytometer and plated on collagen-coated dishes in Ham's F10 medium containing 10% (v/v) fetal bovine serum albumin (FBS), 10% (v/v) horse serum and antibiotics.

Plates were coated using 0.1% (w/v) type I collagen stock solution (UBI, Lake Placid, NY). The investigation conforms with the current Guide To The Care and Use of Experimental Animals published by the Canadian Council on Animal Care and conforms to the NIH guidelines.

2.2 Adenoviral gene construction and generation of virus
For adenoviral gene construction of the kinase domain-deficient TGFβ type II receptor (TGFβRII{Delta}KD), the full length human TGFβRII cDNA (H2-3FF) was obtained as a 4.7-kb EcoRI fragment in pcDNAI [18]. The TGFβRII{Delta}KD fragment was generated by polymerase chain reaction (PCR) using specific primers with unique restriction sites generating a stop codon (forward primer: 5'-AGCCAGGCCTGCCATGGGTCGGGGGCTGC-3'; reverse primer: 5'-TCTCTCTAGATTATGTCTCAAACTGCTCTGAAGTGTTCTG-3') and using PCR amplification conditions as described previously [19]. The PCR fragment was subsequently digested by StuI/XbaI and subcloned into SmaI/XbaI sites of pBluescript II SK+ (pBS) to generate pBS.TGFβRII{Delta}KD. The pBS.TGFβRII{Delta}KD was then cut with EcoRI/XbaI to release the approximately 850 bp TGFβRII{Delta}KD fragment, which was then cloned into the EcoRI/XbaI sites of the pcDNA3.1 vector (Promega; Madison, WI) to generate the CMVp.TGFβRII{Delta}KD hybrid gene. The CMVp.TGFβRII{Delta}KD hybrid gene was digested with HindIII/XbaI to release the approximately 850-bp TGFβRII{Delta}KD fragment which was subsequently cloned into the HindIII/XbaI sites of the pShuttle-CMV adenoviral transfer vector (Quantum) to generate pSCMV.TGFβRII{Delta}KD. Generation of recombinant adenovirus expressing TGFβRII{Delta}KD (Ad.TGFβRII{Delta}KD) was performed as described by the manufacturer’s instructions (AdEasy system, Quantum).

For adenoviral gene construction of the kinase-deficient FGFR-1 (FGFR-1{Delta}KD), lysine 316 was mutated in the FGFR-1(S) cDNA (previously cloned from embryonic mouse heart) [19], to a stop codon, and the cDNA was then truncated at nucleotide 1058 (FspI) to delete the kinase domain. The modified cDNA was released from pBS by HindIII/FspI (FGFR-1{Delta}KD) and inserted into HindIII/EcoRV sites of pcDNA3.1 to generate CMVp.FGFR-1{Delta}KD. The approximately 1-kb FGFR-1{Delta}KD fragment was released from pcDNA3.1 by PmeI and inserted into the PmeI site of pQBI-AdCMV5 adenoviral transfer vector (Quantum) to generate pSCMV5.FGFR-1{Delta}KD. Generation of recombinant adenovirus expressing FGFR-1{Delta}KD (Ad.FGFR-1{Delta}KD) was performed as described by the manufacturer's instructions (Adenoquest system, Quantum). An adenovirus expressing β-galactosidase (Ad.CMV5.β-gal) was also generated using a recombinant Ad.CMV5.β-gal DNA supplied by the manufacturer (QBI-viral DNA) and following the manufacturer’s instructions (Adenoquest system, Quantum). Finally, an adenovirus expressing both high and low molecular weight forms of human FGF-2 (Ad.CMV.hFGF-2) as well as vector alone were described previously [20].

2.3 Adenoviral gene transfer
NRCMs, plated at a density of 0.75–0.9x106 cells per 35-mm collagen coated dish (or on collagen-coated glass coverslips contained within 35 mm dishes), in the presence of F12-DMEM medium supplemented with 10% fetal bovine serum (FBS) were infected with Ad.TGFβRII{Delta}KD, or Ad.β-gal, at multiplicity of infection (MOI) 50. The same batch of fetal bovine serum was used (Gibco) for all experiments. After 24 h, cells refed with growth medium were stimulated with recombinant FGF-2 (Upstate Biotechnology; Lake Placid, NY) at 1, 10 and 1000 ng/ml for a further 24 h before processing. For experiments involving FGFR-1{Delta}KD, cardiomyocytes were pre-infected with Ad.FGFR-1{Delta}KD at MOI 50 and 12 h later infected with Ad.CMV.hFGF-2 or Ad.TGFβRII{Delta}KD at an MOI 50. Cells were subsequently maintained for a further 24 h in growth medium before processing.

2.4 Protein isolation and Western blotting
To detect TGFβRII, total protein was isolated from cardiomyocyte cultures 1 day after infection by lysis in 1% sodium dodecyl sulfate (SDS), 50 mmol/L Tris–HCl (pH 7.4), 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, 2 µg/ml pepstatin, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) as described previously [17]. Immunodetection of TGFβRII was performed using specific rabbit polyclonal affinity purified antibodies raised against the carboxyl terminal residues 550–565 of the human TGFβRII (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), for 1 h at room temperature. TGFβRII antibodies can detect both full-length and kinase domain-deficient TGFβRII and have been shown not to cross-react with TGFβRI (Santa Cruz Biotechnology). They have been shown to be specific for detection via Western blotting and immunofluorescence microscopy [21,22].

Proteins were transferred to Immobilon-P membrane (Millipore) by electroblotting, and blocked for 1 h at room temperature or overnight at 4 °C with 10% skim milk powder in TBST (10 mM Tris–HCl pH8.0, 150 mM NaCl and 0.5% Tween-20). All rabbit polyclonal antibodies were followed by horseradish peroxidase-conjugated anti-rabbit Ig (1:10000; Bio-Rad Laboratories) for 1 h at room temperature. Antigen–antibody complexes were visualized using enhanced chemiluminescence and assessed by autoradiography and densitometry (Pierce, Rockford, IL). Equal protein loading was confirmed by staining blots with 0.2% S. Ponceau, (Sigma-Aldrich) in 3% trichloroacetic acid (TCA) prior to immunoblotting or staining with 0.1% amido black subsequent to immunoblotting.

2.5 Immunofluorescence microscopy
To assess TGFβRII protein and subcellular localization in NRCMs transfected with Ad.TGFβRII{Delta}KD or Ad.β-gal, cardiomyocytes were fixed 24 h after transfection using 1% paraformaldehyde for 15 min at 4 °C and permeabilized with 0.1% Triton X-100 for 15 min at 4 °C. Coverslips were first incubated with affinity purified rabbit TGFβRII antibodies (1:200; Santa Cruz Biotechnology) or rabbit nonimmune serum at the same dilution in 1% BSA in PBS for 16 h at 4 °C, then with biotinylated anti-rabbit immunoglobulins (Ig, 1:20; Amersham) for 1 h at room temperature, followed by incubation with fluorescein conjugated to streptavidin (Strep-FITC, 1:20, Amersham) for 1 h at room temperature. Myocytes were identified by counterstaining with mouse sarcomeric {alpha}-actinin (1:400, Sigma), which was visualized with Texas red-conjugated anti-mouse Ig (1:20; Amersham), and nuclei were identified by staining cellular DNA with 0.0125% Hoechst dye 33342 (Calbiochem-Behring). The mouse sarcomeric {alpha}-actinin antibody is specific for {alpha}-skeletal and {alpha}-cardiac muscle actinins, and labels the Z lines and dots in stress fibers of skeletal muscle in myotubes, but does not cross react with non-skeletal muscle elements (e.g., connective tissue, epithelium, nerves, smooth muscle) [23,24]. Coverslips were mounted in mounting medium for fluorescence (Crystal/Mount, Biomedia, Foster City, CA) and examined by epifluorescence and photographed using a Nikon ECLIPSE 800 microscope.

2.6 Tritiated thymidine, bromodeoxyuridine (BrdU) and phosphorylated (Phospho)-H1 labeling assays
Tritiated thymidine incorporation was determined after 8 h incubation with 1 µCi/ml of tritiated thymidine, as described [25]. BrdU or phospho-H1 Labeling Index (LI), representing the percentage of cardiomyocytes staining positive for BrdU or phospho-H1, respectively, over total number of myocytes was determined as described [8]. Briefly, 8 h prior to the termination of the various experiments, myocytes were incubated with 15 µg/ml BrdU. Cultures were subsequently fixed with 1% paraformaldehyde for 15 min at 4 °C, permeabilized with 0.1% Triton X-100 for 15 min at 4 °C followed by 0.07 M sodium hydroxide for 2 min at room temperature. Simultaneous labeling for sarcomeric {alpha}-actinin (non nuclear, to identify myocytes) and BrdU (nuclear) was done using monoclonal antibodies against mouse {alpha}-actinin and BrdU (1:1, Amersham) in 1% (w/v) BSA-PBS. To detect myocytes in early mitosis, we used specific and well-characterized antibodies to phosphorylated histone H1, a marker associated with mitotic condensation (1:250, UBI) [26]. Both {alpha}-actinin and BrdU were visualized with Texas red-conjugated anti-mouse Ig (1:20; Amersham), whereas phospho-H1 was visualized with biotinylated anti-rabbit Ig (1:20; Amersham), followed by incubation with fluorescein conjugated to streptavidin (Strep-FITC, 1:20, Amersham).

2.7 Determination of labeling index and data analysis
For each study, we scored not less than three coverslips per group. Fifteen random fields (~600 cells) were scored per coverslip, to determine the fraction of myocytes in S-phase (BrdU LI) or in early mitosis (phospho-H1 LI). Each complete study with TGFβRII{Delta}KD overexpression was repeated.

Data presented in the text and figures represent the means plus or minus standard error of the mean (S.E.M.) from n=3. Statistical analysis was done using the parametric Student's t or non-parametric Mann–Whitney tests. In all cases, a value was considered statistically significant if p was determined to be <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
4. Discussion
 References
 
3.1 Expression of TGFβRII{Delta}KD in cardiomyocytes
Expression and localization of TGFβRII were assessed by protein blotting and immunofluorescence microscopy of NRCMs transfected with the kinase-deficient TGFβRII (Ad.TGFβRII{Delta}KD) or with Ad.β-gal (as a control) for 24 h. Polyclonal antibodies recognizing the carboxyl terminus of the TGFβRII were used to detect endogenous and "truncated" forms of TGFβRII. A faint immunoreactive band of 62 kDa detected in control myocytes represented, presumably, endogenous TGFβRII (Fig. 1). This size is comparable to the reported 70-kDa size [27,28]. Cultures transfected with Ad.TGFβRII{Delta}KD (MOI 50) presented a major immunoreactive band at 43 kDa (Fig. 1), which was consistent with the expected size of the truncation mutant [28]. This protein was expressed in great excess (>50-fold) over the faint 62-kDa endogenous TGFβRII band and would be expected to overcome the function of the latter. Another less abundant, immunoreactive 52.5-kDa band was also present in these cultures, and may represent post-translational modifications of the TGFβRII{Delta}KD. Faint, smaller (<29 kDa) immunoreactive proteins were also present, likely representing degradation products of the TGFβRII{Delta}KD.


Figure 1
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  		Fig. 1 Detection of endogenous and kinase domain-deficient TGFβRII by protein blotting. Total protein was isolated from NRCMs transfected with either Ad.β-gal (MOI 50), used as a control, or Ad.TGFβRII{Delta}KD (MOI 50), analyzed by SDS-PAGE blotting and immunodetection with specific antibodies raised against the carboxyl terminus of human TGFβRII. Open and closed arrowheads denote bands corresponding to either endogenous or the major kinase domain-deficient TGFβRII protein, respectively. The mobility of molecular mass standards (kDa) is indicated.

 
Cultures were triple-labeled for {alpha}-actinin (striated-muscle specific isoform), BrdU and TGFβRII to specifically identify myocytes overexpressing TGFβRII{Delta}KD and synthesizing DNA. Endogenous anti-TGFβRII staining was weak and localized to the perinuclear region of myocytes (Fig. 2). Intense anti-TGFβRII staining was only observed in myocytes from cultures infected with Ad.TGFβRII{Delta}KD (Fig. 2), representing, presumably expression and localization of TGFβRII{Delta}KD. The majority of overexpressing cells displayed strong perinuclear, cytoplasmic as well as membrane staining (Fig. 2). TGFβRII{Delta}KD infected cultures incubated with non-immune serum showed no detectable TGFβRII staining (not shown), confirming the specificity of the TGFβRII antibodies.


Figure 2
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  		Fig. 2 Detection of endogenous and kinase domain-deficient TGFβRII in cardiomyocytes by immunofluorescence microscopy. NRCM cultures transfected with either Ad.β-gal (MOI 50), used as a control, or Ad.TGFβRII{Delta}KD (MOI 50) were simultaneously stained for TGFβRII, {alpha}-actinin, BrdU and DNA as indicated. White arrows denote cardiomyocytes expressing TGFβRII that have incorporated BrdU. Bar is equivalent to 75 µm.

 
3.2 Expression of TGFβRII{Delta}KD increases cardiomyocyte DNA synthesis
Studies have demonstrated that TGFβRII{Delta}KD acts in a dominant-negative fashion to inhibit TGFβ signaling in NRCMs [28]. Since TGFβ signaling results in inhibition of proliferative growth, we examined the effects of TGFβRII{Delta}KD expression on serum-supported cardiomyocyte DNA synthesis and entry into mitosis. Serum was utilized for these experiments to allow for healthy looking cells as well as a more potent growth response. As expected, anti-BrdU and anti-phospho-H1 staining was confined to the nucleus, whereas anti-{alpha}-actinin staining was exclusively cytoplasmic. Results from one complete study are shown in Fig. 3. There was a significant 2.8-fold increase in the fraction of myocyte nuclei incorporating BrdU in cultures transfected with Ad.TGFβRII{Delta}KD compared to control myocytes (p<0.05, n=3). Expression of TGFβRII{Delta}KD induced a significant 2.9-fold increase in the fraction of myocyte nuclei staining for phospho-H1 compared to controls (p<0.05, n=3) (Fig. 3). This study was repeated in its entirety producing similar statistically significant results (2.1- and 2.8-fold increases in BrdU and phospho-H1 LIs, respectively, in response to TGFβRII{Delta}KD overexpression; p<0.05, n=3).


Figure 3
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  		Fig. 3 Effect of TGFβRII{Delta}KD overexpression on cardiomyocyte DNA synthesis and early mitosis. The fraction of myocytes (cells staining positive for {alpha}-actinin) labeled for BrdU (BrdU LI) or phospho-H1 (phospho-H1 LI) was determined using immunofluorescence staining with monoclonal antibodies to BrdU and {alpha}-actinin as well as polyclonal antibodies to phospho-H1. In both cases, the BrdU LI and phospho-H1 LI are expressed as fold differences relative to control (Ad.β-gal) values (BrdU, 2.0±0.3; phospho-H1, 2.0±0.6), which were arbitrarily set to 1.0 (n=3). Bars represent standard error of the mean and *p<0.05.

 
3.3 TGFβRII{Delta}KD amplifies the effect of FGF-2 on cardiomyocyte DNA synthesis
We examined whether suppressing TGFβ signaling affected FGF-2-induced stimulation of cardiomyocyte DNA synthesis. Increasing doses of FGF-2 (to ensure a maximal effect) were added to cultures pre-infected with Ad.TGFβRII{Delta}KD or vector. In the vector-infected cultures, as expected, FGF-2 increased cardiomyocyte DNA synthesis significantly by 2–4 fold (p<0.05, n=3) in a dose-dependent manner compared to untreated cells (Fig. 4). The effects of 10 ng/ml FGF-2 on cardiomyocyte DNA synthesis were not significantly different from 1000 ng/ml FGF-2, suggesting that 10 ng/ml of FGF-2 was sufficient to achieve a maximal effect (Fig. 4). In TGFβRII{Delta}KD expressing cultures, FGF-2 resulted on average in a significant 13-fold increase in DNA synthesis, (p<0.01, n=3), compared to control cells (Fig. 4); all FGF-2 doses elicited comparable increases, suggesting that under these conditions, 1 ng/ml of added FGF-2 was sufficient for maximal effect. The 13-fold increase in DNA synthesis was significantly greater than that induced by TGFβRII{Delta}KD or FGF-2 alone, pointing to a synergistic effect. This study was repeated in its entirety producing similar and statistically significant results (3–6 and 13-fold increases in DNA synthesis with FGF-2 alone (p<0.05, n=3) and FGF-2 with TGFβRII{Delta}KD overexpression (p<0.005, n=3), respectively). Representative immunofluorescence images illustrating the effects of TGFβRII{Delta}KD on DNA synthesis, with and without FGF-2 treatment, are shown in Fig. 5.


Figure 4
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  		Fig. 4 Assessment of the effects of FGF-2 addition, and TGFβRII{Delta}KD overexpression on cardiomyocyte DNA synthesis. The BrdU LI for cardiomyocytes was determined for cultures transfected with Ad.β-gal and Ad.TGFβRII{Delta}KD, and maintained in the absence or presence of 1, 10 and 1000 ng/ml FGF-2 (n=3). The BrdU LI is expressed as fold differences relative to a control (Ad.β-gal in the absence of FGF-2) value, which was arbitrarily set to 1.0. Bars represent standard error of the mean, *p<0.05, **p<0.01, and ***p<0.005.

 

Figure 5
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  		Fig. 5 Detection of BrdU incorporation in NRCMs treated with FGF-2 and/or overexpression of a kinase domain-deficient TGFβRII (TGFβRII{Delta}KD) by immunofluorescence microscopy. NRCM cultures were transfected (MOI 50) and/or treated with either "control" Ad.β-gal (a,b), 1 ng/ml FGF-2 (c,d), Ad.TGFβRII{Delta}KD (e,f) or Ad.TGFβRII{Delta}KD and FGF-2 (g,h). Cultures were triple stained for {alpha}-actinin, and BrdU (a,c,e,g), and DNA (b,d,f,h). Examples of cardiomyocytes staining positive or negative for BrdU are indicated with an arrow or arrowhead, respectively. Bar is equivalent to 75 µm.

 
3.4 Inhibition of FGF-2 signaling by a kinase domain-deficient FGF-2 receptor prevents the TGFβRII{Delta}KD-induced increase in DNA synthesis
To examine whether the effects of TGFβRII{Delta}KD would be exerted independently of the FGF-2/FGFR-1 signaling pathway, it was necessary to interfere with the activity of FGFR-1. To this end use was made of an adenoviral vector expressing kinase domain-deficient FGFR-1. Expression of the FGFR-1{Delta}KD was confirmed by protein immunoblotting and immunolocalization; it distributed to plasma membrane, perinuclear and nuclear sites, in agreement with other reports [29,30] (data not shown). To verify that our construct was effective in preventing FGF-2 signal transduction, we tested its effect on FGF-2-stimulated DNA synthesis and ERK1/2 (Fig. 6). FGF-2 stimulation was achieved by adenoviral overexpression (Ad.CMVhFGF-2; MOI 50). We have documented previously that FGF-2 administered in this manner is released from cells and stimulates DNA synthesis in an autocrine/paracrine fashion [20]. In agreement, myocytes overexpressing FGF-2 showed significantly increased DNA synthesis compared to non-overexpressing cells (Fig. 6A). This increase was inhibited in cultures pre-treated with and expressing FGFR-1{Delta}KD (MOI 50). In the absence of FGF-2 overexpression, FGFR-1{Delta}KD did not significantly alter the ‘baseline’ serum stimulated DNA synthesis. In addition, morphological examination indicated that FGFR-1{Delta}KD-treated cultures did not result in increased cell death. FGFR-1{Delta}KD expression resulted in decreased acute activation of ERK1/2 by added FGF-2 (Fig. 6B). Total ERK1/2 levels were unchanged within the time frame, 15 min, of the experiment (not shown).


Figure 6
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  		Fig. 6 Overexpression of FGFR-1{Delta}KD inhibits FGFR1-1 signal transduction. NRCMs, maintained in the presence of 10% FBS, were infected with Ad.FGFR-1{Delta}KD or vector (MOI 50) 24 h after plating and then (A) infected with Ad.CMVhFGF-2 or vector (MOI 50) 12 h later. Incorporation of tritiated thymidine was determined 24 h later after being pulsed (1 µCi/ml) for 8 h; n=4. (B) Cardiomyocytes, infected with Ad.FGFR-1{Delta}KD or vector for 24 h, were stimulated with 10 ng/ml FGF-2 for 15 min. Active (phosphorylated) ERK1/2 protein (p42/44) levels were assessed by Western blotting using antibodies against dually phosphorylated (active) ERK1/2. The results were visualized by chemiluminescence and autoradiography, and values obtained by densitometry; n=3. Total levels of ERK1/2 (determined by probing an identical blot with antibodies recognizing total ERK1/2) were unchanged. Data shown are mean values and error bars represent standard error of the mean, *p<0.05, and **p<0.01.

 
We next examined whether the effects of TGFβRII{Delta}KD would be exerted independently of the FGF-2/FGFR-1 signaling pathway, through inhibition of the latter by expressing FGFR-1{Delta}KD. Cardiomyocyte cultures were pre-infected with Ad.FGFR-1{Delta}KD or vector (MOI 50), followed by infection with Ad.TGFβRII{Delta}KD (MOI 50). The fraction of BrdU-positive myocytes was determined 24 h later. Results from one complete study are shown in Fig. 7. As expected, expression of TGFβRII{Delta}KD induced a significant 2.8-fold increase in BrdU LI (p<0.05, n=3). Pretreatment with Ad.FGFR-1{Delta}KD, eliminated the TGFβRII{Delta}KD-induced stimulatory effect on LI. This study was repeated with similar statistically significant results (2.1-fold increase in BrdU LI with TGFβRII{Delta}KD overexpression (p<0.05, n=3) and elimination of this effect with Ad.FGFR-1{Delta}KD pretreatment).


Figure 7
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  		Fig. 7 The effect of FGFR-1 inhibition of the TGFβRII{Delta}KD-induced NRCM DNA synthesis. The BrdU LI was determined for cultures transfected with Ad.β-gal, Ad.TGFβRII{Delta}KD, and Ad.TGFβRII{Delta}KD pre-infected with Ad.FGFR-1{Delta}KD) (MOI 50) (n=3). Bars represent standard error of the mean, and *p<0.05.

 
To determine whether TGFβ interferes with the ability of FGF-2 to activate FGFR-1, we examined its effect on immediate (5 min) FGF-2/FGFR-1-dependent downstream signals, such as activation (dual phosphorylation) of ERK1/2. As shown in Fig. 8, FGF-2 activated ERK1/2 to the same extent, irrespectively of the presence of TGFβ.


Figure 8
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  		Fig. 8 The effect of TGFβ on FGF-2-FGFR-1-induced acute activation of ERK1/2. Lysates (20 µg protein) from NRCM stimulated with FGF-2 (10 ng/ml) for 5 min, in the absence or presence of pre-incubation (30 min) with TGFβ (10 ng/ml), were examined for activated (dually phosphorylated) ERK1/2 by Western blotting. C, F, T and F/T denote relative levels of activated ERK1/2 in untreated control, FGF-2-only, TGFβ-only, and FGF-2/TGFβ-treated cultures, respectively. FGF-2 elicited significant ERK1/2 stimulation (p<0.01; n=3), that remained unchanged in TGFβ-pretreated cells. Insets show typical Western blot images. Bars represent standard error of the mean. Total levels of ERK1/2 (determined by probing an identical blot with antibodies recognizing total ERK1/2) were unchanged (not shown). **p<0.01.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
References
 
Recent advances on heart repair/regeneration have indicated that the adult heart contains cardiomyocytes, likely in less differentiated state, which are still capable of a proliferative response [2,3]. Stimulating the proliferation of these cardiac cell populations would be expected to improve the regenerative response after myocardial infarction. The general objective of the present studies therefore was to determine ‘proof of principle’ conditions for boosting the proliferative response of immature cardiomyocytes, using proliferative neonatal cardiomyocyte cultures as our model. We have focused on FGF-2 and TGFβ, two endogenous factors that have opposing effects (stimulatory and inhibitory, respectively) on cardiomyocyte proliferation [7]. All our studies were conducted on cells grown in serum-rich media (maintaining the same batches of serum) to approximate conditions in vivo where cells are exposed to blood-born factors, and to maintain cells in vigorous health and good mitotic responsiveness. Furthermore, serum-rich media do not mask the stimulatory effect of FGF-2 [7,8], and thus allow the comparative studies presented here.

4.1 TGFβRII{Delta}KD stimulates DNA synthesis and early mitosis
Assessment of S phase (BrdU LI) and G2/M phase (phospho-H1 LI) in NRCM cultures clearly demonstrated that suppression of TGFβRII-mediated signals increases serum supported DNA synthesis and entry of cells into early mitosis. Endogenous and/or serum-derived TGFβ is, presumably, responsible for the activation of these signals in our cultures. Serum and culture conditioned media contains mitogens, including FGF-2, that would be expected to contribute to stimulation of cardiomyocyte DNA synthesis. Thus, the increased DNA synthesis observed through TGFβRII inhibition suggests that these mitogens were, as a result, "permitted" to exert their full stimulatory effect on cells. It is intriguing that active FGFR-1 was required for this stimulation to manifest itself, since it was abolished in cells overexpressing FGFR-1 (KD). This implies that serum and/or cell-derived FGF-2 (or members of the FGF-family that can activate FGFR-1) were likely responsible for the observed stimulation. FGFR-1, however, is activated downstream of several different pathways, triggered by growth factors, cytokines and hormones that can promote nuclear translocation of plasma membrane or intracellular FGFR-1 and subsequent FGFR-1-dependent gene expression [30]. It is therefore possible that, in addition to FGF-2, other serum mitogens (for example epidermal growth factor, insulin like growth factor, platelet derived growth factor) participate in the serum-supported stimulation of DNA synthesis upon TGFβRII inhibition. This latter possibility could explain the apparent synergistic effect of TGFβ inhibition and FGF-2 stimulation on DNA synthesis discussed below.

Overall very little is known about how TGFβ prevents the FGF-2-induced mitotic stimulation. TGFβ binds to TGFβRII (a constitutively active serine/threonine kinase), an event that enables it to interact and activate TGFβRI [31]. This leads to Smad protein-mediated gene transcription [31] that includes genes mediating cell cycle arrest such as Cdk inhibitors [32]. Because FGF-2 stimulates expression of genes that promote entry and progression through the cell cycle [33,34], it is expected that, in the long run, the net outcome from TGFβ- and FGF-2-triggered gene expression will determine whether the cell will progress through the cell cycle. Since however TGFβRII{Delta}KD-stimulation of DNA synthesis was mediated by activated FGFR-1, we asked whether the converse may also be true. That is whether TGFβ/TGFβRII somehow ‘inactivated’ FGFR-1, preventing early FGF-2-FGFR-1 signal transduction and thus mitotic stimulation. This did not appear to be the case, at least for ERK1/2 activation. Dually phosphorylated (activated) ERK1/2 is an early ‘marker’ of FGFR-1 activation; this kinase is also implicated in the proliferative effect of FGF-2 as well as other mitogens [35,36]. TGFβ signaling did not prevent acute ERK1/2 activation by FGF-2 (Fig. 8), demonstrating that FGFR-1 does not become ‘inactivated’ in the presence of TGFβ, and that inhibition of FGF-2 action by TGFβ must occur at a different level. These data also indicated that acute ERK1/2 activation is not sufficient for the mitotic effect of FGF-2. It should be mentioned that FGF-2/FGFR-1 activates several pathways in addition to the ras-raf-ERK1/2 pathway and that the effect of TGFβ-TGFβRII on these pathways remains to be determined.

4.2 Maximal stimulation of DNA synthesis is achieved by simultaneous inhibition of TGFβ-TGFβRII and stimulation of FGF-2-FGFR1 signal transduction
The 3.5-fold increase in cardiac myocyte DNA synthesis observed by adding FGF-2 alone, without suppressing TGFβ signals, was consistent with our previous studies [7,8]. The 13-fold increase in DNA synthesis elicited by FGF-2 in a background of suppressed TGFβ signaling is, to our knowledge, the highest level of immature cardiomyocyte DNA synthesis stimulation reported so far, and implies that, under appropriate conditions, cardiomyocytes are capable of a strong mitotic response. The mechanism underlying this potent (compared to modest) stimulation remains to be determined. It is also important to determine whether a similar upregulation of cardiomyocyte proliferative potential can occur in vivo and in the context of cardiac injury-repair. TGFβ is present in the heart, and the increased TGFβ levels detected at the infarct border [37,38] could be limiting the response of cardiomyocytes to local or administered mitogens such as FGF-2. A combination strategy aiming to suppress TGFβ-TGFβRII while stimulating the FGF-2-FGFR-1 pathway at the site of infarction could result in an improved regenerative response, and has potential clinical applications.


   Acknowledgements
 
This work was funded by grants from Canadian Institutes of Health Research MT-12303 (FS, YJ, MEB, EK, PAC) and FRN-15256 (CJAH, RRF, BEN, BWD, EK).


   Notes
 
1 Current address: Institute of Molecular Medicine, School of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Back

Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 References
 

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