©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Basic Fibroblast Growth Factor Activates Calcium Channels in Neonatal Rat Cardiomyocytes (*)

(Received for publication, February 21, 1995; and in revised form, May 9, 1995)

Pierre-Laurent Merle (1)(§), Jean-Jacques Feige (2), Jean Verdetti (1)

From the  (1)Groupe d'Electrophysiologie Moléculaire, Centre de Physiologie et Physiophathologie Cellulaire, Université Joseph Fourier, BP 53X, F-38041 Grenoble Cedex, France and (2)INSERM Unité 244, Commissariat l'Energie Atomique, Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucleaires, F-38054 Grenoble Cedex 9, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Basic fibroblast growth factor (bFGF) is a potent mitogen for many cell lineages including fetal cardiomyocytes. Furthermore, bFGF has been shown to modify gene expression, in vitro, in adult nonproliferative ventricular myocytes. This effect is suspected to be partly responsible for the genetic modifications that occur in vivo under pathophysiological conditions such as ischemia or pressure overload and that lead to myocardial hypertrophy. However, little is known about the first steps of the molecular mechanisms that take place soon after cell activation by bFGF. In this study, using biochemical and electrophysiological approaches, we have established, on cardiomyocytes cultured from neonatal rat ventricles, that (i) differentiated beating cells express at least two classes of bFGF-receptors having high and low affinity (K = 10 ± 2 pM and 1 ± 0.5 nM); (ii) the stimulation of these bFGF receptors promotes an increase in the beating frequencies of cultured cardiomyocytes (40 ± 10%); (iii) bFGF provokes the activation of poorly specific and voltage-independent calcium channels (12pS); (iv) inositol 1,4,5-trisphosphate enhances similar bFGF-induced Ca currents and is therefore suspected to be a second messenger triggering this activation. These results support the presence, in cultured cardiomyocytes, of new calcium channels whose activation after bFGF binding may be partly responsible for the cell response to this growth factor.


INTRODUCTION

Basic fibroblast growth factor (bFGF),()the second member of the heparin-binding growth factor family(1) , has been found to be a powerful mitogen for a wide variety of cells(2) . Moreover, bFGF has been shown to be implicated in the maturation and activation of many differentiated cells, thus conferring to this growth factor a broad spectrum of activities(3) . All of these effects are known to be mediated by interaction with different types of receptors (FGFRs), among which some belong to a family of transmembrane high affinity binding proteins possessing a cytoplasmic tyrosine kinase activity (FGFR1 to FGFR4)(4, 5) . Via different activation pathways, these receptors initiate cascades of reactions that lead to the expression of nuclear oncogenes and finally regulate either DNA replication or gene expression(6) . One of the tyrosine kinase activation pathways has been shown, on different cell models, to affect ionic distribution that includes a transient increase of cytoplasmic calcium ([Ca]) from internal stores or influxes through channels of the plasma membrane(7, 8) . In many cases, this [Ca] increase appears to be an important step in the signal transduction that, in association with biochemical events, leads to the activation of oncogenes(9, 10) .

Such changes in gene expression induced by growth factors have become the subject of many investigations in cardiac cells, particularly concerning bFGF. Indeed, in the heart of vertebrates, bFGF is known to play fundamental roles in the normal and the pathological organ development. At the embryonic stage, bFGF, synthesized by different cardiac fetal cell lineages, participates in the proliferation and the differentiation of vascular and contractile cells(11) . In the adult heart, bFGF is detected at a relatively high level in the atria and the ventricles(12) , but its role in the adult cell metabolism remains unclear(13, 14) . An interesting hypothesis states that, in adult heart, bFGF may be implicated in the cardiac response to different physiopathological situations (hemodynamic overload or genetic diseases) leading to the development of hypertrophy(15, 16) . In particular, bFGF has been shown to activate isolated cardiomyocytes and to promote the reexpression of fetal proteins similar to those present in cells of hypertrophied heart(17) . Although a number of gene expression studies have been carried out, the total picture of bFGF-mediated cell activation remains to be built.

The objectives of this work were to investigate the presence of FGFRs on cardiomyocytes isolated from neonatal rat ventricles and to study the action of bFGF on both the cell contractility and the membrane permeability. Our experiments show that cultured cardiomyocytes express at least two classes of bFGF receptors. Their activation generates an increase in the spontaneous cell beating rate. Patch-clamp experiments allowed us to isolate a novel Ca channel activated in the presence of bFGF. In addition, inside-out patch-clamp experiments provide evidence indicating that the bFGF-induced Ca channel activation could be mediated by the second messenger inositol 1,4,5-trisphosphate (IP).


EXPERIMENTAL PROCEDURES

Materials

Bovine recombinant bFGF was purchased from Boehringer Mannheim (Meylan, France); trypsin was from Difco; NaI was from Amersham Corp.; Ham's F-10 and fetal calf serum were from Life Technologies, Inc. Genistein, tyrphostin 51, IP, and the other chemical products were from Sigma. Cell observations were achieved under a phase-contrast microscope, Nikon TMS (Nikon, Inc., NY). Patch-clamp signals were measured with an RK-300 amplifier and stored using a digital audio tape recorder 1200 (Biologic, Claix, France). After acquisition with an IEEE-N interface, data were analyzed with the Bio-Patch software (Biologic, Claix, France).

Cell Culture

Neonatal rat cardiac myocytes were trypsin-dissociated and grown according to an established technique (adapted from (18) ). Briefly, myocardial cells were isolated from ventricles of 1-2-day-old newborn Wistar rats by enzymatic digestion (0.1% trypsin 1/250) and gentle mechanical desegregation (stirred water bath at 37 °C). Cardiomyocytes were separated from the total cell population by centrifugation (at 1800 rpm for 10 min) on a discontinuous Percoll gradient (1.06/1.086 g/ml) followed by a 90-min preplating (in order to eliminate the few non-myocyte cells remaining after Percoll purification), which yielded cell cultures with >95% myocytes(19) . Culture medium (Ham's F-10, 20% heat-inactivated fetal calf serum) was changed every 48 h. After 2-3 days of culture under a 5% CO, 95% O atmosphere at 37 °C, myocytes reached a near confluent state corresponding to a terminal differentiation stage, after which myocyte proliferation no longer occurs. Electrophysiological properties of these spontaneously beating cardiomyocytes have been shown to be close to those of adult cells in vivo(20) . All experiments were performed 3 or 4 days after the cell preparation, when the degree of purity is maximum and 24 h after removal of serum from the medium, in order to avoid any interaction with growth factors from the fetal calf serum.

Electrical Recording

Membrane currents were recorded using the patch-clamp technique. For the cell-attached configuration, cells were placed in Tyrode's solution containing the following: 125 mM NaCl; 5.6 mM KCl; 2.4 mM CaCl; 1.2 mM MgCl; 10 mM HEPES; 11 mM glucose, pH 7.4. Before external application of bFGF (10 nM), bovine serum albumin (0.1%) was added in the medium to mask the nonspecific binding sites. For experiments with the inside-out configuration, IP (5 µM) was directly added to bath solution with the following composition: 150 mM KCl; 0.55 mM CaCl; 2 mM MgCl; 1.1 mM EGTA; 10 mM HEPES, pH 7.35 with KOH.

In both cases, pipettes, having resistances of 4-8 megaohms, were filled with high barium concentration solution (110 mM Ba(CHCOO); 10 mM HEPES, pH 7.4 adjusted with Ba(OH)). For some experiments, CaCl and BaCl were also used instead of Ba(CHCOO) at the same concentration.

Recording of Contraction Frequencies

Beating rates were determined by the means of a basic monitoring system using a photoconductive element placed on the screen of a TV monitor on which pictures of cultured cells were projected (adapted from (21) ). Changes in light intensity caused by contractions were locally detected (at the edge of the cells). Beating rates were computerized every 7 s, before and after the addition of drugs into Tyrode's solution, under thermostable conditions (at 35 °C). Tyrosine kinase inhibitors were dissolved in dimethyl sulfoxide so that the final concentration of the solvent was 0.1%. Cells were incubated with the corresponding inhibitor concentration for at least 20 min for genistein and 1 h for tyrphostin. All results are presented as mean ± S.E.

Identification of bFGF Receptors

The presence of bFGF-receptors on neonatal rat cardiomyocytes was detected according to the standard radioreceptor assay method(22) . Radioactive iodine was incorporated into basic FGF using the chloramine-T method (as described in (23) ). I-bFGF with a Specific Activity of 50,000 cpm/ng was routinely obtained. The binding assay, based on the experimental protocol of Moscatelli(24) , allowed the segregation of I-bFGF bound to different types of binding sites. Increasing concentrations of labeled bFGF were incubated (at least 2 h at 4 °C) with cardiomyocyte cultures. After removal of unbound I-bFGF, a first fraction of growth factor bound to low affinity receptors was collected by rinsing cells with a 2 M NaCl solution. The fraction of I-bFGF linked to high affinity receptors was extracted by the addition of 0.5% Triton X-100 and cell scrapping. The amount of labeled bFGF was determined by counting each fraction. These experiments were completed by cross-linking analysis(25) . Cardiomyocytes were incubated with radiolabeled bFGF (at least 2 h at 4 °C) in the absence or presence of different concentrations of unlabeled acidic or basic FGF. After phosphate-buffered saline washing, disuccinimidyl suberate was added (0.25 mM for 15 min at 4 °C) in order to covalently link bound I-bFGF to its high affinity receptors. Glycine (2 M) was added to stop the reaction, and cells were lysed (Triton X-100 and scrapping in phosphate-buffered saline containing protease inhibitors). The supernatants of the cell lysates were electrophoresed on a 7.5% SDS-polyacrylamide gel. The positions of the I-bFGF/high affinity receptor complexes were revealed either by autoradiography or by -imaging on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


RESULTS

Identification and Characterization of Basic FGF Binding Sites on Neonatal Rat Cardiomyocytes

In order to demonstrate the presence of bFGF receptors, cultures of cardiomyocytes, isolated from newborn rat ventricles, were incubated with increasing concentrations of I-bFGF. A Scatchard analysis of the data obtained in a representative experiment is shown in Fig. 1. The shape of this curve typically matches with a two binding site system. The low affinity receptors presented an affinity constant of about 1 nM (K = 1 ± 0.5 nM) and 50,000-160,000 sites/cell (n = 4). These values are similar to those described for the heparan sulfate proteoglycan receptors on other cell types(23, 24) . The K of the high affinity binding system (10 ± 2 pM) was also similar to that of tyrosine kinase receptors characterized in a variety of cell types(22, 23, 24) . However, the number of high affinity binding sites detected per cardiomyocyte (750 ± 200) is in lower range of the number of high affinity receptors in other cell lineages (1,000-80,000 sites/cell(24, 26) ). This seems to indicate that cardiomyocytes, under standard culture conditions, expressed FGFRs at a low level. Nonetheless, it is worth noticing that the real number of sites per cell may be more important than the calculated value. Indeed, the evaluation of the cell density was done by counting nuclei after crystal violet staining. Now, even under standard culture conditions, a certain percentage of cardiomyocytes are known to be binucleated(27) . This could lead to an overestimation of the number of cells/dish and thereby to an underestimation of the number of receptors/cell. The other technique of cell counting by trypsin dissociation was not used since cardiomyocytes are known to remain associated in groups of about 10 (because of their numerous cell to cell junctions).


Figure 1: Scatchard analysis of the binding of I-basic FGF to cardiomyocytes. Cardiomyocytes, isolated as described under ``Experimental Procedures,'' were incubated for 2 h at 4 °C with increasing concentrations of radiolabeled bFGF. The unbound ligand was removed, and cells were washed with a 2 M NaCl solution in order to release I-bFGF bound to low affinity receptors. The labeled bFGF bound to high affinity sites was extracted by membrane disruption in Triton X-100. The Scatchard analysis of data, representative of five independent experiments, showed the presence of two affinity binding systems (high affinity binding site: K = 12 pM, N = 700 sites/cell; low affinity binding site: K = 1.0 nM, N = 130,000 sites/cell). Cell density (5 10 cells/dish) was evaluated by nuclei counting, which leads to an underestimation of the site numbers since cardiomyocytes could present binucleation(27) . F, free I-bFGF; B, total bound I-bFGF.



In order to better characterize the presence of high affinity sites on cardiomyocytes, we performed cross-linking experiments. The molecular complexes formed between the I-bFGF and the high affinity receptors were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The results showed (Fig. 2, lane1) the presence of one major radiolabeled complex corresponding to a 102-kDa receptor and two minor complexes corresponding to 72- and a 152-kDa forms of receptors. Whether these receptors are distinct proteins or whether the smaller forms just represent degradation products from the larger one has not been determined. Competition experiments in which unlabeled basic or acidic FGF were added to the incubation medium showed a significant decrease of the detected radioactivity in every distinct form of receptor. Addition of 50 ng/ml of unlabeled bFGF in the previous incubation medium partially decreased the binding of I-bFGF (Fig. 2, lane2). The displacement was total at a concentration of 500 ng/ml bFGF (Fig. 2, lane 3). Similarly, unlabeled acidic FGF competed for the binding of I-bFGF (Fig. 2, lanes4 and 5), but some binding was still observed in the presence of 500 ng/ml aFGF. This indicates that the affinity of the cardiomyocyte FGFRs was stronger for bFGF than for aFGF. It is worth noting that cross-linking experiments have also been done with cell cultures having different degrees of purity (by reincorporating non-myocyte cells after Percoll separation). The results (data not shown) demonstrated that high affinity sites were detected in non-myocyte or poorly purified cell cultures but also in very pure cell preparations in which the presence of high affinity receptors could not be due to fibroblasts or any other non-myocyte cells.


Figure 2: Covalent cross-linking of I-bFGF to high affinity receptors in the presence or absence of unlabeled basic or acid FGF. Cardiomyocytes were incubated at 4 °C for at least 2 h with 20 ng/ml of I-bFGF (lane1). Cells were washed, and labeled FGF was covalently linked to the high affinity receptors by disuccinimidyl suberate as described under ``Experimental Procedures.'' Triton X-100 extracts of the cell lysates were electrophoresed on 7.5% SDS-polyacrylamide gels. For competition experiments, cells were incubated with the same concentration of I-bFGF in the presence of 50 ng/ml (lane2) or 500 ng/ml (lane3) of unlabeled basic FGF. Similarly, 50 and 500 ng/ml (lanes4 and 5) of unlabeled acidic FGF were added during the incubation period. After autoradiographic detection, the relative molecular weight of the different binding complexes was calculated by comparison with that of prestained size markers. Molecular mass of each isoform of the high affinity receptor could then be calculated after subtraction of the 18-kDa mass of the growth factor .



Effect of bFGF on the Mechanical Activity of Cardiomyocytes

The ability of bFGF to modify the contractile activity of cultured cardiomyocytes has been studied as described under ``Experimental Procedures.'' It can be seen from Fig. 3that, at a base line level, cells exhibited a constant and synchronized beating rate, even in absence of any electrical stimulation. Addition of bFGF (10 nM) to the medium led to a significant increase (40 ± 10%, n = 12) of the beating rate. After an average period of 5-15 min, contraction frequencies reached a maximum and remained at this top level for 30-90 min. This positive chronotropic effect caused by bFGF also led to arrhythmia and to a significant increase of the contraction amplitude. Complementary experiments have been achieved, using two tyrosine kinase inhibitors, genistein and tyrphostin 51. The obtained data are summarized in Table 1. When cells were incubated with low concentrations of genistein and tyrphostin (2 and 4 µM, respectively) a subtotal inhibition of the bFGF effect was observed since the bFGF-induced increase of the basal frequency was limited to 5 ± 2 and 7 ± 3% (n = 4). A total inhibition of the bFGF effect was obtained with 20 µM genistein and 40 µM tyrphostin. Under these conditions, the basal beating frequency (120 ± 15) was not modified during the incubation period and remained at the same basal level after the addition of bFGF in the medium (n = 12 and 10). Higher concentrations of inhibitors (200 and 400 µM) were found to have a certain cytotoxicity, which led to alterations of the basal beating rate, to unexpected beating stop and, at the end of the incubation period, to the presence of cytosolic granulations and alterations in cell shapes. These last doses were not used any more, since lower concentrations were revealed efficient to block the bFGF-induced effects. Isoproterenol (1 µM) was used as positive control, leading to an increase of the beating frequency of 100 ± 20% (n = 30).


Figure 3: Effect of bFGF on the beating rate of spontaneously active cardiomyocytes. Contractions were recorded as described under ``Experimental Procedures'' section, and beating rates were calculated every 7 s. This plot, representative of 12 experiments, shows that addition of bFGF (10 nM) in the medium rapidly provokes an increase of the base-line contraction frequency. The increase (40 ± 10%; n = 12) was maintained at the top level for 30-90 min. Arrhythmia, as well as an increase in the contraction amplitude, were also observed in response to bFGF. The beating frequency at base-line was 120 ± 15 (n = 30) contractions/min. Isoproterenol (1 µM) was used as a positive control, leading to a 100 ± 20% of increase of the basal rate (n = 30).





Effect of bFGF on Calcium Channel Activation

Patch-clamp experiments were performed in order to record possible calcium membrane permeability modifications at a single cell level. Fig. 4a shows a typical record obtained from a cell-attached patch held on cardiomyocytes, before and after bFGF (10 nM) addition to the bath solution, with a high concentration barium pipette solution (110 mM) and a holding potential of -30 mV. The current amplitude distribution of such bFGF-induced channels is shown on Fig. 4b, displaying a single inward current of 0.25 pA amplitude. This value varies linearly with holding potentials, which were converted to membrane potentials according to the diastolic resting potential of the cells assumed to -60 ± 5 mV (n = 10). The current-potential curve (Fig. 4c) displays a slope conductance of 12 picosiemens and a reversal potential of +20 mV. Since the reversal potential is lower than the theoretical value (calculated from the Nernst equation), the specificity of the observed channel either to calcium or to barium seems to be relatively weak. Barium was used as charge carrier since calcium channels are generally more permeable to this ion, which often permits a better resolution of differences in single-channel amplitudes. However, when barium was substituted for calcium ions, no significant modification of bFGF-induced current amplitude occurred. Moreover, current amplitudes were the same whether chloride or acetate was the anion of the pipette solution, which confirmed that the currents were carried by inward influx of cations (Ca or Ba), rather than by outward anion fluxes (which anyway would display, in the experimental conditions, negative reversal potentials). Potassium cannot be taken into account since this ion was not in the pipette solution and since high concentration of barium is known to inhibit K permeability. The histograms of distribution for the mean open and closed times were computerized as shown on Fig. 4, d and e; the open time constants were 1.5 and 14 ms and the closed time constants were 0.6 and 7 ms.


Figure 4: Effect of basic FGF on Ca channel activity. a, representative example of bFGF-induced Ca currents obtained from a cultured cardiomyocyte using the cell-attached patch-clamp configuration and a barium pipette solution. For an holding potential of -30 mV, no current was detected at a base-line level, but in minutes after the addition of basic FGF (10 nM, at the time pointed by the arrow) specific openings were observed. b, the probability density histogram of current amplitudes derived for the experiment shown in a reveals multiple step events corresponding to an inward unitary current of 0.25 pA. c, the current-voltage relation of single Ca channel current was plotted after conversion of holding potentials into membrane potentials (MP), the average diastolic resting potential being estimated at -60 mV. Each point represents the mean of three to six experiments. The resulting straightline defines a single channel conductance of 12 picosiemens with a reversal potential at +20 mV. d, distribution histogram of open times of Ca channel was fitted with the sum of two exponential curves. The corresponding time constants are 14 (slow constant) and 1.5 ms (fast constant). e, distribution histogram of closed times of Ca channel was fitted with the sum of two exponential curves, with time constants of 7 (slow constant) and 0.6 ms (fast constant).



No voltage dependence was detected, and inhibitors of voltage-dependent calcium channels such as dihydropyridines (nicardipine or nifedipine) or verapamil were without effect on the bFGF-activated currents. Besides, preliminary experiments have shown that epidermal growth factor and insulin-like growth factor 1 led to an increase of the calcium permeability of patched membranes, indicating that other growth factors could also prompt channel activations (data not shown). Finally, the fact that the presence of bFGF in the pipette was not necessary to stimulate the ion flux proves that a second messenger is required to convey the signal from FGFRs to calcium channels.

Role of IPin the bFGF-induced Ca Channel Activation, in Inside-out Configuration

With a success rate of around 50%, we found that inositol 1,4,5-trisphosphate promote an increase in channel openings of excised patches. As it can be seen in Fig. 5a, the addition of IP (5 µM) to the bath of an inside-out patch (held at +30 mV, corresponding to -30 mV of membrane potential) induced an activation of inward currents with elementary amplitude of 0.25 pA (Fig. 5b). Reversal potential (+15 mV) and conductance (12 pS) were deduced from the linear correlation of the current-potential curve (Fig. 5c). The mean open and closed time constants (Fig. 5, d and e) were also computed. When compared with the previous bFGF-induced currents (Table 2), these IP-activated openings displayed similar characteristics concerning the conductance, the reversal potential, and the kinetics constants. Thus, it could be hypothesized that the same type of channels was isolated under the patch pipette in both cases. This observation suggests that IP could be a second messenger participating in bFGF-activation of Ca channels. Other second messengers (cAMP, cGMP), used under the same conditions, failed to activate similar currents. Nevertheless, we cannot formally exclude the possibility that other molecular mechanisms would act in the regulation of the observed current. Failure to show opening inhibition by addition of calcium channel blockers (verapamil or nifedipine) suggest that the observed channels were insensitive to these agents. However, lanthanum has been found to reversibly inhibit the observed openings, which indicates that the currents were indeed carried by calcium (or barium) influxes (data not shown).


Figure 5: Effect of IP on Ca channel activity. a, representative example of IP-induced Ca currents obtained under inside-out configuration conditions. The holding potential of this trace was +30 mV, and only rare openings were detected. Thirty seconds after the addition of IP (5 µM, located by the arrow) an increase of the current activity was observed (record sampled at 1 kHz). b, the resulting density histogram of current amplitudes revealed multiple step events, which correspond to the sum of inward unitary currents of 0.25 pA. c, the current-voltage relation of single Ca channel current correspond to a straightline with a slope conductance of 12 picosiemens and a reversal potential of 15 mV (each point being the mean of five to nine experiments). d, distribution histogram of open times of Ca channel, fitted with the sum of two exponential curves. The corresponding time constants are 12 (slow constant) and 1.5 ms (fast constant). e, distribution histogram of closed times of Ca channel, fitted with the sum of two exponential curves, with time constants of 5 (slow constant) and 0.5 ms (fast constant).






DISCUSSION

Since their localization in the heart, it has been strongly hypothesized that fibroblast growth factors, produced by cardiac myocytes or endothelial cells, induce numerous biological effects relevant to the embryonic heart development and to the response of the mature organ to stimuli such as ischemia or pressure or volume overload (11, 12, 13) . Possible roles of bFGF in the regulation of proliferation, differentiation, and hypertrophy of cardiomyocytes have been suggested (15, 16, 17) . Since only little is known about the expression of fibroblast growth factor receptors and the precise mechanisms that link FGFR activation and cell response, we examined the mechanical and electrical effects of bFGF on cultured neonatal rat cardiomyocytes.

The presence of specific bFGF membrane receptors in cardiomyocytes is commonly admitted. Some authors even state, that modifications in the isoform expression of the different FGFR families explain the diverse roles of bFGF on proliferation, differentiation, and size growth of these cells. However no direct experiment has been done to control these statements. In the present work, performed on cardiomyocytes purified and cultured from newborn rat ventricles, we show the binding of bFGF to both low and high affinity receptors. The results of biochemical assays and cross-linking experiments indicate that these two binding sites are proteoglycan heparan sulfate and tyrosine kinase receptors described on other cell lineages(22, 23, 24) . Different isoforms of high affinity tyrosine kinase receptors are classically observed, but the molecular mass of the major complex determined in our cross-linking experiments (102 kDa) is in lower range of data presented in literature (about 145 kDa(25, 26, 27, 28) ). It is interesting to note that, very recently, the transfection and the expression of the tyrosine kinase FGFR1 gene (flg), cloned from mouse heart at adult stage, generated three isoform proteins. The major isoform of the adult cells was shown to have a molecular mass of 102 kDa, which is similar to the value observed in our model(29) . It is clear that more studies are required to determine the roles and the evolution of the expression level of the different classes and isoforms of FGFRs in cardiac myocytes under normal or pathological development. However, the obtained data provide evidence that FGFRs are expressed in the cardiomyocytes taken after 2 or 3 days of growth and placed for 1 day in a serum-free medium.

The activation of the FGFRs leads to an increase in the beating frequency and also in contraction amplitude of spontaneously active cardiomyocytes (Fig. 3). These observations corroborate previous experiments, showing that epidermal growth factor, insulin-like growth factor, and transforming growth factor have chronotropic or inotropic effects on cardiomyocytes(30, 31, 32) . This indicates that different growth factors, some acting through tyrosine kinase receptors, positively stimulate mechanical activity of these cells. In the present work, genistein and tyrphostin, two tyrosine kinase inhibitors, are found to suppress the bFGF-induced activation of the beating rate, providing evidence of the implication of the tyrosine kinase activation pathway. Obviously, such increases in contraction frequency as those observed in this in vitro model cannot be immediately extrapolated to the in vivo situation. However, these modifications of the beating rates and contraction amplitude reveal changes in the distribution of ions in the cardiomyocyte cytoplasm, indicating that bFGF triggers modifications in the ionic equilibriums, which partly results in an intracellular free Ca increase.

The major finding of these studies is the presence in cardiomyocyte membranes of channels activated in presence of bFGF and which generate, under patch-clamp experiment conditions, calcium influxes. These channels displayed low conductance and reversal potential, no voltage-dependent openings, and no dihydropyridine blocker sensitivity. Moreover, as it is mentioned in the result session, some other growth factors (epidermal growth factor and insulin-like growth factor) seems to stimulate some comparable currents, proving that the channel activation would not be specific to bFGF. These characteristics define a novel kind of channel, permeable to calcium and activated in the presence of bFGF, different from the other Ca channels previously identified in the cardiomyocyte membrane(33) . By contrast, the observed channel shares many common points with other growth factor-activated channels identified in the literature. Indeed, most of the currents, recorded on other cell models, show low specificity, low conductance, fast bursts, dihydropyridine insensitivity, and voltage independence(34, 35, 36) . Another point common to many of the growth factor-activated channels is their coupling to a second messenger generation system. In the present work, the possibility of an implication of IP was confirmed by inside-out experiments showing that this phosphoinositol could provoke similar channel activations. This result is consistent with the general activation pathways of the tyrosine kinase receptors. Indeed, similar to epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor, bFGF has been shown to stimulate phosphorylations of intracellular target proteins, among which is a subunit of phospholipase C-(5, 37) . The activation of this phospholipase C-, via the phosphatidyl metabolism, is known to stimulate the syntheses of diacylglycerol and IP, which both modify the distribution of several ions, particularly Ca and K(38, 39) . However, even if the pathway linking tyrosine kinase activation and inositol phospholipid synthesis is well known, the complete signal transduction cascade remains to be determined. In particular, little, if anything, is known about the mechanism of cell membrane channel opening triggered by IP. The present results corroborate previous findings stating that IP can directly enhance membrane permeability without interaction of cytosolic co-factors(34, 36) . However, the success rate of 50% in the observation of this phenomenon might be due to the fact that some agents are required together with the channel in the excised membrane.

In conclusion, the results presented in this article support the hypothesis that bFGF, by its binding to specific receptors, promotes, in part via phospholipid metabolism, the opening of calcium-permeable channels. The fact that some other growth factors could stimulate comparable currents may indicate that the channel activation could be a general mechanism implicated in the growth factor receptor activation. This raises the question of the implications of the recorded currents that, according to their activity (fast bursts and multiple step events), could modulate the rise in cytosolic calcium due to the release of calcium from intracellular stores(40) . The implication of such ionic fluxes are known to be a critical step in the cascade activation leading to oncogene expression in mitotic, quiescent, and differentiated cells(8, 40, 41) . Recently, it has been shown in neurons that the oncogene induction by bFGF was dependent on an increase in [Ca](10) . Moreover, some studies have strengthened the hypothesis that calcium fluxes through the plasma membrane are necessary for the nuclear activation of oncogene transcription by growth factors(42) . Consequently, the observed ionic fluxes may be sufficient to directly or indirectly affect cellular biosyntheses and in turn, may contribute to cardiomyocyte oncogene expression. Finally, the bFGF-induced channel activation, described in this work, may help in the understanding of the mechanism by which, during myocardium-increased work, bFGF and other growth factors can induce the expression of fetal-like contractile protein genes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-76-51-46-00 (ext. 36-50); Fax: 33-76-51-42-18.

The abbreviations used are: bFGF, basic fibroblast growth factor; bFGFRs, basic fibroblast growth factor receptors; [Ca], intracellular free calcium concentration; IP, inositol 1,4,5-trisphosphate; I-bFGF, radioactive iodine-labeled bFGF.


REFERENCES
  1. Basilico, C., and Moscatelli, D.(1992)Adv. Cancer Res. 59, 115-164 [Medline] [Order article via Infotrieve]
  2. Gospodarowicz, D., Ferrara, N., Schweigerer, L., and Neufeld, G.(1987) Endocrinol. Rev. 8, 95-114 [Medline] [Order article via Infotrieve]
  3. Burgess, W., and Maciag, T.(1988)Annu. Rev. Biochem. 58, 575-606 [CrossRef][Medline] [Order article via Infotrieve]
  4. Jaye, M., Schlessinger, J., and Dionne, C. A.(1992)Biochim. Biophys. Acta 1135, 185-199 [Medline] [Order article via Infotrieve]
  5. Coughlin, S. R., Barr, P. J., Cousens, L. S., Fretto, L. J., and Williams, L. T.(1988) J. Biol. Chem. 263, 988-993 [Abstract/Free Full Text]
  6. Ullrich, A., and Schlessinger, J.(1990)Cell 61, 203-212 [Medline] [Order article via Infotrieve]
  7. Sawyer, S. T., and Cohen, S.(1981)Biochemistry 20, 6280-6286 [Medline] [Order article via Infotrieve]
  8. Moolenar, W. H., Defize, L. H., and de Laat, S. W.(1986)J. Exp. Biol. 124, 359-373 [Abstract]
  9. Rozengurt, E. (1986)Science 234, 161-166 [Medline] [Order article via Infotrieve]
  10. Ferhat, L., Khrestchastisky, M., Roisin, M.-P., and Bardin, G.(1993) J. Neurochem. 61, 1105-1112 [Medline] [Order article via Infotrieve]
  11. Casscells, W., Speir, E., Sasse, J., Klagsbrun, M., Allen, P., Lee, M., Calvo, B., Chiba, M., Haggroth, L., Folkman, J., and Epstein, S. E.(1990) J. Clin. Invest. 85, 433-441 [Medline] [Order article via Infotrieve]
  12. Kardami, E., and Fandrich, R. R.(1989)J. Cell Biol. 109, 1865-1875 [Abstract]
  13. Speir, E., Tanner, V., Gonzales, A. M., Farris, J., Baird, A., and Casscells, W.(1992) Circ. Res. 71, 251-259 [Abstract]
  14. Spirito, P., Fu, Y.-M., Zu, Z.-X., Epstein, S. E., and Casscells, W.(1991) Circulation 84, 322-332 [Abstract]
  15. Cummins, P.(1993) Cardiovascular Res. 27, 1150-1154 [Medline] [Order article via Infotrieve]
  16. Parker, T. G., and Schneider, M. D.(1991)Annu. Rev. Physiol. 53, 179-200 [CrossRef][Medline] [Order article via Infotrieve]
  17. Schneider, M. D., McLellan, W. R., Black, F. M., and Parker, T. G.(1992) Basic Res. Cardiol.87,Suppl. 2, 33-48 [Medline] [Order article via Infotrieve]
  18. Pinson, A., Frelin, C., and Padieu, P.(1973)Biochimie (Paris)55,1261-1264 [Medline] [Order article via Infotrieve]
  19. Iwaki, K., Sukhatme, V. P., Shubeita, H. E., and Chien, K. R.(1990)J. Biol. Chem. 265, 13809-13817 [Abstract/Free Full Text]
  20. Athias, P., Frelin, C., Groz, B., Dumas, J. P., Klepping, J., and Padieu, P.(1979) Pathol. Biol. 27, 13-19 [Medline] [Order article via Infotrieve]
  21. Schanne, O. F. (1990)J. Appl. Physiol. 29, 892-893
  22. Kan, M., Shi, E. G., and McKeehan, W. L.(1991)Methods Enzymol. 198, 158-171 [Medline] [Order article via Infotrieve]
  23. Savona, C., Chambaz, E. M., and Feige, J.-J.(1991)Growth Factors 5, 273-282 [Medline] [Order article via Infotrieve]
  24. Moscatelli, D. (1987)J. Cell. Physiol. 131, 123-130 [Medline] [Order article via Infotrieve]
  25. Feige, J.-J., and Baird, A.(1988)J. Biol. Chem. 263, 14023-14029 [Abstract/Free Full Text]
  26. Walicke, P. A., Feige, J.-J., and Baird, A.(1989)J. Biol. Chem. 264, 4120-4126 [Abstract/Free Full Text]
  27. Katzberg, A. A., Farmer, B. B., and Harris, R. A.(1977)Am. J. Anat. 149, 489-500 [Medline] [Order article via Infotrieve]
  28. Neufeld, G., and Gospodarowicz, D.(1988)J. Cell. Physiol. 136, 537-542 [Medline] [Order article via Infotrieve]
  29. Jin, Y., Pasumarthi, K. B. S., Bock, M. E., Lytras, A., Kardami, E., and Cattini, P. A. (1994)J. Mol. Cell. Cardiol. 26, 1449-1459 [CrossRef][Medline] [Order article via Infotrieve]
  30. Vetter, U., Kupferschmid, C., Lang, D., and Pentz, S.(1988)Basic Res. Cardiol. 83, 647-654 [Medline] [Order article via Infotrieve]
  31. Rabkin, S. W., Sunga, P., and Myrdal, S.(1987)Biochem. Biophys. Res. Commun. 146, 889-897 [Medline] [Order article via Infotrieve]
  32. Roberts, A. B., Vodovotz, Z., Roche, N. S., Sporn, M. B., and Nathan, C. F. (1992)Mol. Endocrinol. 6, 1921-1930 [Abstract]
  33. Cohen, N. M., and Lederer, W. J.(1987)J. Physiol. (Lond.)391,169-191 [Abstract]
  34. Chapron, Y., Cochet, C., Crouzy, S., Jullien, T., Keramidas, M., and Verdetti, J.(1989) Biochem. Biophys. Res. Commun. 158, 527-533 [Medline] [Order article via Infotrieve]
  35. Peppelenbosch, M. P., Tertoolen, L. G. J., and de Laat, S. W.(1991)J. Biol. Chem. 266, 19938-19944 [Abstract/Free Full Text]
  36. Kuno, M., and Gardner, P.(1987)Nature 326, 301-304 [CrossRef][Medline] [Order article via Infotrieve]
  37. Bogoyevitch, M. A., Glennon, P. E., Andersson, M. B., Clerk, A., Lazou, A., Marshall, C. J., Parker, P. J., and Sugden, P. H.(1994)J. Biol. Chem. 269, 1110-1119 [Abstract/Free Full Text]
  38. Mohammadi, M., Honegger, A. M., Rotin, D., Fischer, R., Bellot, F., Li, W., Dionne, C. A., Jaye, M., Rubinstein, M., and Schlessinger, J.(1991)Mol. Cell. Biol. 11, 5068-5078 [Medline] [Order article via Infotrieve]
  39. Berridge, M. J., and Irvine, R. F.(1998)Nature 341, 197-205 [CrossRef]
  40. Soltoff, S. P., and Cantley, L. C.(1988)Annu. Rev. Physiol. 50, 207-223 [CrossRef][Medline] [Order article via Infotrieve]
  41. Mogami, H., and Kojima, I.(1993)Biochem. Biophys. Res. Commun. 196, 650-658 [CrossRef][Medline] [Order article via Infotrieve]
  42. Estacion, M., and Mordan, L. J.(1993)Cell Calcium 14, 439-454 [Medline] [Order article via Infotrieve]

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