© 1996 American Heart Association, Inc.
Articles |
Fibroblast Growth Factor-2 Decreases Metabolic Coupling and Stimulates Phosphorylation as Well as Masking of Connexin43 Epitopes in Cardiac Myocytes
the St. Boniface General Hospital Research Centre (B.W.D., Y.C., E.K.), Departments of Anatomy and Physiology, and the Manitoba Institute of Cell Biology (D.G.B., D.W.L.), Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Correspondence to E. Kardami, St. Boniface General Hospital Research Centre, Division of Cardiovascular Sciences, 351 Tache Ave, Winnipeg, Manitoba, Canada R2H 2A6. E-mail ekardami@salk.sbrc.umanitoba.ca.
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Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ–mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti–phosphotyrosine-specific antibodies in FGF-2–treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2–triggered and tyrosine phosphorylation–dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.
Key Words: fibroblast growth factor • cardiomyocyte coupling • connexin43 • phosphorylation
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Introduction |
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Gap junctions are aggregates of intercellular channels that permit exchange of ions and metabolites between cardiac myocytes.1 2 Cardiac GJ channels mediate intercellular communication and ensure propagation of electrical signals and coordinated contractile activity. Abnormalities in their distribution and/or function are linked to arrhythmias, as those, for instance, following an ischemic episode.3 4 In addition, GJs, by allowing passage of signaling molecules between cells, participate in the control of growth, differentiation, and morphogenesis.1 5 They are composed of a family of related proteins referred to as connexins.1 2 Cx43 is a major connexin of mammalian cardiac myocytes; other connexins, such as Cx40 and Cx45, are also expressed in the heart and myocytes.6 7 Regulation of GJ-mediated communication can occur at many levels—acutely by "gating," but also on a long-term basis by changing overall connexin expression levels and/or switching to different connexin isoforms.1 2 Phosphorylation of Cx43, predominantly on serine but also on tyrosine residues, appears to be an important mechanism regulating function at the level of assembly as well as conductance.8 9 Although ionic coupling via GJs is essential for the heart to beat in a synchronized manner, metabolic coupling is important for tissue homeostasis and is implicated in the regulation of growth and differentiation.1 2 5 Cx43 was recently shown to be essential for normal cardiac development.10 It is therefore important to identify factors that may regulate GJ function and expression in vivo, in development and in disease.
Supportive evidence for the link between growth and GJ-mediated intercellular communication has been provided by several studies demonstrating that polypeptide growth factors can affect intercellular coupling as well as expression/properties of connexins.1 2 5 11 For the most part, an inverse relationship appears to exist between growth and connexin expression/intercellular coupling5 11 ; eg, overexpression of Cx43 in glioma C6 cells results in decreased proliferation.12 Also, activation of receptors for EGF and platelet-derived growth factor (receptor protein kinases) has been shown to decrease intercellular communication in nontransformed cells.5 13 On the other hand, FGF-2 was shown by us14 and others15 to enhance coupling between cardiac fibroblasts and capillary endothelial cells, respectively, by inducing increased Cx43 accumulation.
FGF-2, a major member of a heparin-binding family of growth factors (FGF-1/9),16 is of particular interest in the heart, where it is believed to be involved in the regulation of hyperplastic and hypertrophic growth, cardiogenesis, and angiogenesis.17 Endogenous FGF-2 is increased in the heart after injury, especially at the borders between healthy myocytes and scar tissue18 19 ; this is the same area believed to be involved in the formation of slow heterogeneous electrical conduction.4 As a first step toward understanding the relationship between FGF-2 and GJs, we have examined short-term effects of FGF-2 on cardiomyocyte Cx43 expression and phosphorylation as well as GJ-mediated intercellular communication in vitro. In the present study, we report that although FGF-2 did not affect Cx43 expression (estimated by protein and mRNA accumulation) or change gross Cx43 distribution at sites of intercellular contact (assessed by immunolocalization with a polyclonal anti-Cx43 serum), it (1) decreased intermyocyte metabolic coupling, assessed by scrape dye loading as well as dye transfer, (2) decreased staining with a monoclonal anti-Cx43 antibody preparation at sites of intercellular contact, and (3) increased serine Cx43 phosphorylation within 30 minutes of administration. Our data are consistent with the notion that FGF-2 induces a tyrosine phosphorylation–requiring signal transduction cascade, leading to Cx43 phosphorylation, epitope masking, and decreased metabolic coupling of cardiac myocytes. Local increases in FGF-2 as seen after myocardial infarction could therefore be considered to contribute to changes in communication between cardiomyocytes.
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Cell Culture
Media, sera, and antibiotics were purchased from GIBCO/BRL. Cultureware was purchased from Corning (Baxter Corp). Myocyte cultures were obtained from neonatal rat hearts, as described previously.20 Briefly, the hearts from 36 Sprague-Dawley rat pups (1 to 2 days old) were minced in Ca2+-free F-10 medium and dissociated into single cells by gently stirring in a 0.1% trypsin solution (Sigma Chemical Co) maintained at 35°C in a water-jacketed spinner flask. Myocyte suspensions were passed through a discontinuous Percoll (Sigma) gradient and were plated at a density of 600 000 cells per 35-mm culture dish in F-10 medium containing 10% FCS, 10% horse serum, and 1x penicillin/streptomycin. Cells were switched to DMEM/F-12 medium containing 0.5% FBS, 20 nmol/L selenium, 10 µg/mL insulin, 10 µg/mL transferrin, 2 mg/mL BSA, and 20 µg/mL ascorbic acid 1 day after plating and were maintained in this medium for 6 to 7 days (replacing the medium every 48 hours) before FGF-2 treatment. Cells were maintained in a humidified incubator (37°C, 5% CO2).
Human recombinant FGF-2, purchased from Upstate Biotechnology Inc, was added to the cells at 10 ng/mL. Genistein was purchased from Calbiochem. Medium from cells to be treated with genistein was aspirated, supplemented with genistein at a final concentration of 20 µmol/L, and returned to cells. Myocytes were incubated thus for 30 minutes before FGF-2 treatment. Pilot experiments using 10, 20, and 40 µmol/L genistein indicated that although at 10 µmol/L results were variable and at 40 µmol/L toxic effects on the myocytes (morphological evaluation) were noted, a 20 µmol/L genistein concentration produced maximal and consistent results, including decreased FGF-induced tyrosine phosphorylation of myocardial proteins (authors' unpublished data, 1996).
RNA Isolation and Northern Blotting
RNA was isolated from control or FGF-2–treated myocytes using the acid phenol-guanidine-isothiocyanate method of Chomczynski and Sacchi.21 Total RNA (10 µg per lane) was run on a 1% agarose gel containing 0.22 mol/L formaldehyde, transferred to a nylon membrane (Nytran-Plus, Schleicher & Schuell), and probed with a 1.4-kb EcoRI cDNA fragment specific for Cx43 (a gift from Dr Eric Beyer, Washington University School of Medicine, St. Louis, Mo). Scanning densitometry of autoradiograms of blots reprobed with a 700-kb GAPDH probe (GenBank/EMBL: M17851) was used to normalize loading. Probes were labeled with [32P]dCTP (DuPont) by the random oligonucleotide primer procedure (GIBCO/BRL kit).
Primary Antibodies
A previously characterized22 23 rabbit antiserum raised against a peptide spanning residues 346 to 363 of rat Cx43 was used for immunoprecipitation, Western blotting, and immunofluorescence. The monoclonal anti-Cx43 preparation used for immunofluorescence (No. K22755M, clone No. CON 11-2, lot No. 791) was raised against residues 252 to 270 of rat Cx43 and was purchased from Biodesign International. This product became discontinued and is now owned by Transduction Laboratories.
Synthetic peptides containing residues 252 to 270 (GPLSPSKDCGSPKYAYFNG) or 261 to 270 (GSPKYAYFNG) of rat Cx43 were obtained commercially from ImmunoDynamics Inc. A peptide containing residues 252 to 260 (GPLSPSKDC) was purchased from Genemed Biotechnologies, Inc. These peptides were used at 0.01 mg/mL final concentration in antibody-blocking experiments.
Protein Extraction, Immunoprecipitation, and Western Blotting
Total protein extracts were obtained from control or FGF-2–treated cell cultures after lysis with 1% SDS, 50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, 2 µg/mL leupeptin, 2 µg/mL pepstatin, and 1 mmol/L PMSF. Lysates were boiled for 5 minutes, sonicated (three pulses of 10 seconds each at full power), and centrifuged for 10 minutes in a microfuge. Protein content of supernatants was measured using the BCA assay (Sigma). Samples containing 5 µg total protein were analyzed on 10% SDS-polyacrylamide gels.24
For immunoprecipitations of phosphotyrosine-containing proteins followed by Western blotting for Cx43, 150 µg of total protein obtained as above was diluted 10-fold with IP (1% Triton X-100, 0.5% Nonidet P-40, 10 mmol/L Tris [pH 7.4], 150 mmol/L NaCl, 1 mmol/L EDTA, and 1 mmol/L EGTA with the phosphatase and protease inhibitors listed above) and incubated with 25 µL of monoclonal anti-phosphotyrosine IgG conjugated to agarose beads (4G10, Upstate Biotechnology Inc) in IP for 1 hour. Beads were then washed five times with IP, eluted by boiling into double-strength SDS-PAGE sample buffer, and analyzed on a 10% gel. Proteins were transferred to Immobilon-P membrane (Millipore) by electroblotting, and membranes were blocked overnight in 10% skim milk powder/PBS. Blots were then incubated with the rabbit anti-Cx43 serum at 1:10 000 dilution in Tris-buffered saline containing 0.1% Tween-20 (Bio-Rad) for 1 hour at room temperature with constant agitation. Antigen-antibody complexes were visualized by an ECL reaction catalyzed by horseradish peroxidase–linked secondary antibodies (ECL kit, Boehringer Mannheim) and exposure to x-ray film.
Scrape Loading
Scrape loading was carried out as described by El-fouly et al,25 with minor modifications. Confluent monolayers of control or FGF-2–treated myocytes were washed with warm (37°C) CMF-PBS and were then covered with a preheated (37°C) solution of 0.05% 6-CF and 0.05% tetramethylrhodamine dextran (Sigma) dissolved in CMF-PBS. For each dish of cells, four parallel scrape lines were made through the monolayer with a sharp scalpel. Two minutes after the scraping was performed, the dye was removed, the plate was washed with PBS, and the cells were fixed with ice-cold 1% paraformaldehyde in PBS (pH 7.4). Dye migration from primary loaded cells along the scrape line to adjacent cells beyond the scrape line was then assessed using epifluorescence microscopy.
Microinjection and Dye Transfer
Myocyte microinjections were done essentially as previously described26 27 with the help of a Yarishige micromanipulator (Nikon), a Medical Systems picoinjector, and a Nikon Diaphot microscope equipped with epifluorescence and phase-contrast capabilities. Myocytes were kept at 37°C with a stage heater (Medical Systems), and the pH was maintained at 7.4 by adding 15 mmol/L HEPES to the medium. A 0.05% solution of 6-CF dissolved in 100 mmol/L KCl and 5 mmol/L KH2PO4, pH 7.2, was back-loaded into a glass micropipette pulled by a Sutter Instrument Co pipette puller set at heat=740, pull=90, voltage=120, time=250, pressure=600. Myocytes were injected for 30 milliseconds with a pressure of 20 psi and then viewed with epifluorescence and low-light phase-contrast microscopy for 30 seconds. Cells that had become loaded with dye during this period were scored and placed, by comparing fluorescence and phase-contrast views, in three categories27 : first order, primary cells (ie, cells directly connected to the injected cell); second order, cells located immediately next to the primary cells; and third order, cells located next to the second-order group and distal from the first-order group.
Immunofluorescence
Myocytes were grown on collagen-coated glass coverslips in 35-mm dishes and fixed as described previously.28 Briefly, coverslips were rinsed three times with ice-cold CMF-PBS, fixed with 1% fresh paraformaldehyde/CMF-PBS for 15 minutes at 4°C, rinsed with CMF-PBS, permeabilized with 0.1% Triton X-100 in CMF-PBS for 15 minutes at 4°C, and then rinsed thoroughly with CMF-PBS. Primary rabbit or mouse anti-Cx43 antibodies were diluted in 1% BSA/PBS (at 1:2000 or 1:50, respectively) and carefully applied to coverslips. For some experiments, antibodies, diluted as above, were preincubated for 1 hour at ambient temperature in the presence of 0.01 mg/mL synthetic peptides before being applied on the coverslips. After incubating with the primary antibody overnight at 4°C, coverslips were rinsed with CMF-PBS and then incubated with secondary antibodies (purified anti-mouse immunoglobulin [IgG] conjugated to Texas red or purified anti-rabbit IgG conjugated to biotin [Amersham Corp] for the monoclonal and polyclonal antibodies, respectively) diluted 1:25 in 1% BSA/PBS for 1 hour at room temperature. Subsequently, monoclonal IgG-treated coverslips were rinsed and mounted, whereas rabbit IgG-treated coverslips required a further 1-hour incubation at room temperature with streptavidin-fluorescein (Amersham Corp) diluted 1:25 in 1% BSA/PBS. Coverslips were rinsed five times with CMF-PBS, mounted on slides, and viewed with a Nikon Diaphot epifluorescence microscope equipped with appropriate filters, as described previously.29
Immunoprecipitation of 32P-Labeled Myocytes
Myocytes were labeled for 3 hours by incubation in phosphate-depleted DMEM supplemented with [32P]orthophosphoric acid (NEN/DuPont) in water (100 µCi/mL), as described previously.7 All treatments with FGF-2 were coordinated so that they ended at the end of the labeling period. At the end of the treatment, cells were rinsed three times with ice-cold CMF-PBS containing (mmol/L) PMSF 1, sodium orthovanadate 1, and sodium fluoride 10 as well as 2 µg/mL leupeptin and 2 µg/mL pepstatin and scraped into 1% SDS/50 mmol/L Tris (pH 7.4) containing the inhibitors listed above. Samples were boiled for 5 minutes and centrifuged 10 minutes in a bench-top microfuge, and the protein concentration of the supernatants was determined using the BCA assay. Samples containing 100 µg of total protein were diluted 10 times with 1x IP and then incubated with 2 µL of rabbit anti-Cx43 serum for 1 hour at 4°C with gentle shaking. To collect the antibody-antigen complexes, 100 µL of a 1:3 dilution of protein A–Sepharose beads (Pharmacia) in 1x IP buffer was added, and the whole suspension was incubated for another hour at 4°C. Immunoprecipitated proteins, obtained after centrifugation for 30 seconds at room temperature followed by four washes in IP, were analyzed on 10% SDS-PAGE gels. The gel was subsequently dried and processed for autoradiography using a cassette with intensifying screen.
Phosphoamino Acid Analysis
This procedure was performed as described previously.30 31 Briefly, 32P-labeled Cx43 (immunoprecipitated from 150 µg of total lysate) was recovered from a gel slice after SDS-PAGE and autoradiography and was then hydrolyzed with constant boiling HCl for 1 hour at 110°C. The hydrolyzed samples were lyophilized and mixed with a solution of all unlabeled phosphoserine, phosphotyrosine, and phosphothreonine (Sigma) dissolved in pH 1.9 buffer (2.5% [vol/vol] formic acid [88%] and 7.8% [vol/vol] glacial acetic acid). Samples were electrophoresed on cellulose thin-layer chromatography plates with the pH 1.9 buffer in the first dimension for 45 minutes at 1000 V and in pH 3.5 buffer (0.5% [vol/vol] pyridine and 5% [vol/vol] glacial acetic acid) in the second dimension for 15 minutes at 1000 V. Standards were visualized by ninhydrin staining, and the plates were placed in a PhosphorImager (Molecular Dynamics) cassette for 24 hours. The image from the PhosphorImager was exported as a TIFF file, which was filtered (3x3 median filter to reduce background), cropped, and labeled using the program NIH Image.
Determination of Cx43 Protein Synthesis
Myocytes were labeled for 2 hours in methionine-depleted DMEM supplemented with 100 µCi/mL EXPRE35S35S protein labeling mix (NEN) as described previously.7 FGF-2 was added for the last 30 minutes of the labeling period. Total protein extracts were prepared as described above, and protein content was determined with the BCA assay. Samples containing 100 µg of total protein were immunoprecipitated with 2 µL of rabbit anti-Cx43 serum, followed by collection of antibody-antigen complexes with protein A–Sepharose. Precipitates were washed four times with IP buffer and then analyzed by SDS/PAGE, followed by gel staining with Coomassie blue, destaining, incubation in Amplify fluorographic reagent (Amersham) for 30 minutes, gel drying, and autoradiography for 48 hours. Density of bands was quantified by densitometry.
Statistical Analysis
ANOVA, followed by Fisher's protected least significant difference post hoc test, was used to evaluate the significance of the effect of FGF-2 on Cx43 mRNA levels and protein levels at various time points up to 24 hours after FGF-2 administration (P<.05). ANOVA was also used to evaluate statistical significance of changes in Cx43 phosphorylation (32Pi-labeled cardiomyocytes) at several time points of incubation with FGF-2 (P<.01). An unpaired t test was used to determine statistical significance of differences (1) between cardiomyocyte [35S]Cx43 synthesis in the absence or presence of FGF-2 and (2) between the number of cardiomyocytes loaded with the fluorescent dye in the absence or presence of FGF-2; in the latter case, this test was done at three "distances" from the originally injected cell (P<.01, P<.001). Results are presented as mean±SE of at least three separate experiments. Calculations were performed on a Macintosh computer running StatView 4.0 (Abacus Concepts).
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All experiments were conducted 6 to 7 days after cell plating. At this time point, cardiac myocytes maintained under low (0.5%) serum conditions no longer divide,32 have well-developed myofibrils, and were observed as a synchronously and vigorously contracting monolayer, containing <10% non–muscle cell contamination, assessed by immunfluorescence with striated muscle–specific antibodies (authors' unpublished data, 1996). In addition, pilot experiments (immunofluorescence and Western blotting) indicated that these cultures consistently expressed higher levels of Cx43 and were better coupled as assessed by scrape-loading assays compared with myocytes maintained in high (10% to 20%) serum or myocytes tested within 3 to 4 days of plating (authors' unpublished data, 1995).These cells were treated with 10 ng/mL of FGF-2 for up to 30 minutes and in some cases up to 24 hours. This FGF-2 concentration was chosen because it fully activates the FGF-2 signal transduction cascade leading to the stimulation of cardiomyocyte DNA synthesis33 and it induces maximal phosphorylation of cardiomyocyte protein, as detected by immunostaining with anti-phosphotyrosine antibodies.34
We examined the effect of FGF-2 on cardiac myocyte metabolic coupling by scrape loading and by dye transfer. It was necessary to keep cells in CMF solution for a brief period (2 minutes) during the scrape-loading manipulations to prevent GJ closure resulting from Ca2+ overload of the dissected myocytes. As seen in Fig 1A
and 1C, untreated myocytes remained well coupled metabolically under these conditions, as testified by the presence of the dye 6-CF in myocytes located at several cells' distance from the scrape line. Similar results were obtained when the dye LY was used. We also used a GJ-impermeant dye, tetramethylrhodamine-dextran, to control for non–GJ-mediated dye loading, which may be caused by generalized cell injury after scrape loading. Only primary loaded cells of the scrape line were observed to contain tetramethylrhodamine-dextran, indicating that non–GJ-mediated dye loading in our cultures was insignificant (authors' unpublished data, 1995). Effects of FGF-2 on GJ-mediated dye coupling is shown in Fig 1B and 1D. Unlike control cultures, dye loading was not evident in cells away from the scrape line. FGF-2 treatment appeared to decrease the distance migrated by 6-CF, resulting in more intense localization near the primary loaded cells (Fig 1, compare panels A and C with panels B and D). This decrease in intercellular communication by FGF-2 was seen consistently (repeated three times) and could be inhibited by the tyrosine phosphorylation inhibitor genistein at 20 µmol/L (Fig 1F); genistein treatment itself did not decrease intermyocyte coupling (Fig 1E).
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Intermyocyte dye coupling was also examined by microinjection of 6-CF as well as LY.26 27 We introduced 6-CF or LY in cardiomyocytes and then scored the number of cells taking up the dye in the immediate vicinity (first order) as well as more distally (second and third orders) from the primary loaded cell within a set time.27 Myocytes were maintained in the presence of physiological concentrations of Ca2+ and Mg2+ for this experiment. Combined results from microinjection of 6-CF are presented in Fig 2A. In the presence of FGF-2, the dye remained noticeably closer to the microinjected cell compared with control cells. Significantly fewer dye-containing second- and third-order cells were seen in the presence of FGF-2 compared with control cells. In total, dye was transferred to 6.0±0.3 neighbor cells in control cultures compared with 3.3±0.2 cells in FGF-2–treated cultures, indicating a significant (P<.0001, unpaired t test) reduction of dye coupling induced by FGF-2. Similar results were obtained when LY was used. Fig 2B shows a characteristic set of results with LY dye injection of control and FGF-2–treated myocytes.
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We subsequently examined the effects of FGF-2 on Cx43 mRNA or protein accumulation by Northern and Western blotting, respectively. In the first case and in order to correct for loading variations, relative Cx43 RNA levels were calculated by taking the ratio of the intensity of Cx43 bands over that of their corresponding GAPDH bands as assessed by densitometry of autoradiograms of blots reprobed with the GAPDH cDNA. Results are shown in Fig 3A1
and 3A2. No changes in relative Cx43 mRNA accumulation were seen after FGF-2 addition. To assess relative Cx43 protein levels, equal amounts of total protein lysates (10 µg per lane) from untreated and FGF-2–treated cells were analyzed by gel electrophoresis, followed by transfer to Immobilon membrane and immunoblotting for Cx43. Immunoreactive bands (visualized by chemiluminescence) were quantified by densitometry of the x-ray film. In addition, after immunoblotting, transfer membranes were stained with amido black and scanned by densitometry to determine accuracy of initial total protein determination and loading. Less than 5% variation in total transferred protein was routinely seen between the various lanes. Results from quantifying the anti-Cx43 immunoblots by densitometry are shown in Fig 3B1 and 3B2. Cx43 overall protein levels did not change significantly after FGF-2 addition. Finally, levels of newly synthesized 35S-labeled Cx43 were assessed by immunoprecipitation, SDS-PAGE, and autoradiography. Results are shown in Fig 3C1 and 3C2. FGF-2 treatment did not cause short-term changes in newly synthesized Cx43 levels. As expected, newly synthesized Cx43 migrated with a mobility corresponding to 41-kD and 43- to 45-kD bands, presumably corresponding to unphosphorylated and phosphorylated forms of Cx43, respectively.
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To assess whether FGF-2 induced changes in Cx43 subcellular distribution, we examined immunofluorescence localization with two different anti-Cx43 antibody preparations (Fig 4). Using a rabbit antiserum raised against Cx43 residues 346 to 363, anti-Cx43 immunofluorescence staining intensity and pattern appeared similar in cultures of control or FGF-2–treated myocytes (Fig 4a and 4b, respectively). Staining was punctate and localized mainly between myocytes. Fluorescent "dots" of variable size were discerned between the cells, and some staining was also observed around the nucleus. Staining with this antibody is abolished after preabsorption with the synthetic peptide used as antigen.22 23 The monoclonal anti-Cx43 antibody also produced a punctate staining pattern between cardiomyocytes in control cultures that was similar in overall shape and intensity to the one produced by the rabbit antiserum (Fig 4c). Staining with the monoclonal antibody produced "dots" of variable sizes at sites of intercellular contact; weaker perinuclear staining was also seen. After 30 minutes of FGF-2 treatment, however, staining of cardiomyocytes with the monoclonal anti-Cx43 preparation at sites of cell-to-cell contact was clearly reduced compared with untreated cells (Fig 4b). Immunofluorescence staining elicited by the monoclonal anti-Cx43, raised against Cx43 residues 252 to 270, was completely abolished by preabsorption with synthetic peptides containing residues 252 to 270 or 261 to 270 of Cx43 (Fig 5) but not with synthetic peptides containing residues 252 to 260 or 346 to 362 of Cx43, indicating that this antibody preparation recognizes predominantly epitopes contained or affected by residues 261 to 270 of Cx43. As expected, peptides 252 to 260, 262 to 270, and 250 to 270 did not quench staining by the rabbit anti-Cx43 serum, which was completely eliminated by peptides 346 to 363 (authors' unpublished data, 1996).
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We then examined whether FGF-2 induced changes in phosphorylation levels of Cx43. Cardiac myocytes were metabolically labeled with [32P]orthophosphoric acid and then treated with FGF-2 for up to 30 minutes; this was followed by immunoprecipitation with rabbit anti-Cx43 antibodies and analysis by autoradiography. A typical outcome is shown in Fig 6A. FGF-2 treatment resulted in time-dependent increases in Cx43 phosphorylation. Combined results from three separate experiments show a more than twofold increase in label associated with Cx43 after 30 minutes of treatment (Fig 6B). To examine whether phosphorylation of Cx43 resulted in qualitative changes in electrophoretic mobility of Cx43, we analyzed control and FGF-2–treated myocyte lysates on the same gel with immunoprecipitated [32P]Cx43 from treated and untreated myocyte cultures, including molecular weight markers (ovalbumin, 45 kD) in intervening gel lanes. The gel was transferred to an Immobilon membrane, which was processed first for detection of Cx43 by Western blotting and ECL (30-second exposure of the x-ray film) and then for autoradiographic detection of [32P]proteins (48-hour exposure). The membrane was finally stained with amido black to delineate exact migration of ovalbumin. Comparison and alignment of the Western blot image with that produced by autoradiography and amido black staining allowed us to accurately determine migration of Cx43 bands relative to ovalbumin. Results are shown in Fig 6C. Most of the immunoreactive Cx43 in the Western blot appears to migrate slightly below 45 kD (at 
43 kD) either before or after treatment with FGF-2. A faint immunoreactive band at 45 kD can also be discerned in both lanes. Immunoprecipitated [32P]Cx43 migrated at 45 kD, irrespective of FGF-2 treatment.
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Treatment of myocytes with FGF-2 results in increased tyrosine phosphorylation of several proteins (References 17 and 34; authors' unpublished data, 1995). To obtain an indication as to whether Cx43 was phosphorylated on tyrosine residues after FGF-2 treatment, myocyte extracts were immunoprecipitated with monoclonal anti-phosphotyrosine antibodies covalently linked to agarose beads. Immunoprecipitated proteins were then analyzed for Cx43 by Western blotting with rabbit anti-Cx43 antibodies. Typical results (reproduced four times) are shown in Fig 7A. There were no anti-Cx43 immunoreactive bands in immunoprecipitated protein from nontreated control cultures. After FGF-2 treatment, however, Cx43 immunoreactive bands were clearly immunoprecipitated by antiphosphotyrosine. These bands disappeared after preabsorption of the antibody with the synthetic peptide containing residues 346 to 363 of Cx43, confirming their identity as Cx43 (authors' unpublished data, 1996). When cells were treated with 20 µmol/L genistein, anti-phosphotyrosine antibodies did not immunoprecipitate Cx43 in either control or FGF-2–treated myocytes (Fig 7A). To test for specificity of the anti-phosphotyrosine antibodies, immunoprecipitation was performed in the presence of an excess of phosphotyrosine, phosphoserine, or phosphothreonine. As shown in Fig 7B, only excess phosphotyrosine competed effectively with the antibodies, confirming their specificity for phosphotyrosine residues. One interpretation of these results is that FGF-2 treatment induced tyrosine phosphorylation of Cx43, which was then recognized by anti-phosphotyrosine antibodies. An alternate interpretation is that after FGF-2 treatment Cx43 may have associated and thus coprecipitated with phosphotyrosine-containing protein(s). To discriminate between the two possibilities, we examined the phosphoamino acid composition of [32P]Cx43 before and after FGF-2 treatment. As shown in Fig 8, Cx43 appeared to be phosphorylated exclusively on serine in both control and FGF-2–treated myocytes. The overall amount of label was higher in FGF-2–treated samples, as expected from the results shown in Fig 6.
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FGF-2 is linked to stimulation of growth and is present in the heart at all developmental stages.35 This factor is released by cardiomyocytes during contraction and in response to catecholamine stimulation36 37 and is upregulated locally after myocyte damage in several models of injury.18 19 35 Since cardiomyocytes express functional FGF receptors at all developmental stages,17 38 they are presumably capable of responding to changing FGF-2 levels. Such potential responses include stimulation of DNA synthesis20 33 and/or hypertrophy.36 37 39 Because of the link between growth and GJ-mediated communication,11 we examined how FGF-2 may affect intercellular metabolic coupling in cardiac myocytes. We also examined the effects of FGF-2 on Cx43 accumulation, localization, and phosphorylation. We focused on Cx43, since it is a major myocyte GJ protein, and therefore, factors regulating its abundance and/or properties are likely to affect communication.
Since we were interested in relatively acute changes in communication, most studies were performed within 30 minutes of FGF-2 administration. Using a mostly qualitative (scrape-loading) and a more quantitative (dye-transfer) approach, we were able to demonstrate that unlike our findings with cardiac fibroblasts, intercellular metabolic coupling as reflected by the passage of fluorescent dyes between myocytes was significantly reduced by FGF-2 treatment of cardiac myocytes. This reduction in permeability was clearly seen within 30 minutes from FGF-2 administration and was still evident in cells maintained in the presence of FGF-2 for 24 hours (data not shown). Since rat cardiomyocyte GJs are composed of Cx43 as well as Cx45,7 the question arises as to whether the effect of FGF-2 on communication reflected an effect on Cx43- and/or Cx45-composed GJ channels or possibly on all GJ channels. We suggest that since rat Cx43 channels are highly permeable to 6-CF,40 scrape-loading as well as dye-transfer assays used here reflect predominantly Cx43-mediated coupling. Although we do not know the contributions of rat Cx45 channels in 6-CF dye transfer observed in cardiomyocytes, it is presumed to be minimal, since chicken Cx45 channels are reported to be totally impermeable to 6-CF.41
Again, in contrast to the findings concerning cardiac fibroblasts, FGF-2 had no effect on Cx43 mRNA or protein accumulation in cardiac myocytes treated from 30 minutes to 24 hours. Therefore, the effects of FGF-2 on Cx43 expression and GJ-mediated communication appear to be cell specific, although the mechanism underlying this phenomenon is unknown at present. Cardiac fibroblasts as well as myocytes respond to FGF-2 by increased DNA synthesis,33 and preliminary evidence from cross-linking of 125I–FGF-2 to plasma membranes from fibroblasts or myocytes indicated that these cell types share similar FGF receptor composition (authors' unpublished data, 1994). It is possible that differences in the intracellular milieu of the two cell types affect FGF-2–triggered cascades and protein targets.
Reduction of communication by a growth factor or a growth-promoting stimulus, as seen here for cardiac myocytes, is not unexpected; an inverse relationship appears to exist in many instances between growth stimulation and intercellular coupling.1 2 11 A well-studied example of decreased communication in response to a growth factor is that of EGF and its receptor-mediated effects on Cx43 expression and communication of rat liver epithelial cells.42
Induction of decreased coupling without affecting overall Cx43 protein accumulation in cardiomyocytes suggested two possible scenarios as to mechanism: First, FGF-2 may have affected Cx43 distribution and/or assembly and thus effectively reduced the number of functional Cx43-composed membrane channels. Regulation of coupling at the level of Cx43 assembly has been reported previously.43 In the same context, FGF-2 may affect intercellular adhesion mediated by cadherins and thus again interfere with functional channel formation.43 Second, FGF-2 might cause functional modification(s) on Cx43 and/or induce protein-Cx43 interaction(s), resulting in channel "closure" or "blocking."
To begin examining these possibilities, we determined localization of Cx43 in cardiomyocyte cultures before and after FGF-2 addition, using two different preparations of anti-Cx43 antibodies reacting with different cytoplasmic domains of Cx43. Like other connexins, Cx43 has four membrane-spanning regions, one intracellular and two extracellular loops, and cytoplasmic amino and carboxy terminals.44 Whereas the rabbit antibody recognizes residues very near the C-terminal end of Cx43, the monoclonal antibody recognizes residues at a distance from the C-terminal and near the fourth membrane-spanning domain. It should be noted that as assessed by Western blotting, both of these antibodies recognized bands of 43 to 45 kD in cardiac GJ-enriched membrane preparations and in total cardiomyocyte culture lysates or bands of 41 to 43 kD in brain extracts (authors' unpublished data, 1996), consistent with recognition of the same protein. Immunostaining with the rabbit preparation produced the standard punctate pattern of Cx43 distribution between myocytes. This pattern of immunostaining of Cx43 did not change after FGF-2 administration. It is conceivable that it may not be possible to detect staining intensity changes between two states if an antibody preparation is used at too high a concentration. This did not appear to be the case in our system, since pattern/intensity of staining remained independent of FGF-2 addition even at higher antibody dilutions (1:2000 and 1:3000, authors' unpublished data, 1995). Staining with the rabbit anti-Cx43 indicated that FGF-2 did not cause any major redistribution of Cx43 between plasma membrane and cytoplasm, nor did it cause obvious changes in abundance of Cx43-composed plaques between cells. Furthermore, we did not observe any loss of adhesion between cells or between cells and substrate, as assessed by morphological examination of cultures (phase-contrast microscopy) and of detached cells. However, we cannot exclude the possibility that FGF-2 may have caused finer changes in assembly, plaque formation, or intercellular adhesion that would not be discerned by this approach.
Staining of control cultures with the monoclonal anti-Cx43 preparation presented a pattern similar to that of the rabbit antibody. Unlike staining with the rabbit anti-Cx43 serum, however, immunostaining intensity elicited by the monoclonal anti-Cx43 diminished dramatically after FGF-2 addition. "Dots" could only faintly be discerned between cells after treatment. In view of the findings from the rabbit antibody, loss of staining intensity with the monoclonal antibody at sites of cell-cell contact could not have been caused by loss nor gross redistribution of Cx43. Rather, FGF-2 treatment is likely to have caused a masking of epitopes recognized by the monoclonal but not the rabbit antibody preparation. This is quite important, since it indicated that FGF-2 treatment could "visibly" affect Cx43 in situ. Furthermore, since FGF-2 decreased Cx43 channel permeability, an effect of FGF-2 on Cx43 properties in situ is very likely to be relevant to the loss of coupling. It would be important to examine the nature of Cx43 change(s) caused by FGF-2. Masking of epitopes can come about by (1) a conformational change of the protein induced by a change in the local environment (eg, as in pH or Ca2+ concentration)1 2 ; (2) alteration of the reactive epitopes through modifications such as phosphorylation; (3) interaction with another molecule(s), resulting in concealment of epitopes; or (4) any combination of the above. At this point, we suggest that all of these scenarios are plausible. FGF-2 is known to induce transient Ca2+ increases in cells, including myocytes,45 and these changes may contribute to the observed effect on Cx43 as well as to the loss of coupling. In addition, as we will discuss below, we showed that FGF-2 induced phosphorylation of Cx43 on serine, and we provided some evidence for Cx43 interaction with an as-yet-unidentified cellular protein(s).
Cardiomyocytes have been shown to express one category of FGF tyrosine kinase receptors, the FGFR1 group, products of the flg gene.17 38 Activation of the receptor by the ligand leads to phosphorylation of several proteins on tyrosine residues.17 34 Tyrosine phosphorylation, and thus presumably FGF receptor activation, appeared essential to the loss of intercellular communication, since the effect of FGF-2 was abolished after pretreatment with genistein, a tyrosine phosphorylation inhibitor. Similar findings have been reported in the EGF receptor–mediated loss of Cx43 intercellular communication42 ; like FGFR1, the EGF receptor belongs to the tyrosine kinase group of receptors.46
Loss of Cx43-mediated intercellular communication induced by growth factors and oncogenes has been linked to Cx43 phosphorylation on serine42 or tyrosine47 residues, respectively. Thus, we examined whether FGF-2 affected the phosphorylation levels of Cx43. Immunoprecipitation of 32P-labeled myocytes with the rabbit anti-Cx43 serum and subsequent quantification of labeled Cx43 demonstrated a consistent, time-dependent, more than twofold increase in Cx43 phosphorylation after FGF-2 treatment. It should be noted that cardiac Cx43 already exists in a largely phosphorylated state, and this is reflected, in addition to phosphoamino acid analysis and labeling studies, by its electrophoretic mobility at 43 to 45 kD.48 Immunoprecipitated [32P]Cx43 in our system migrated at 45 kD, irrespective of FGF-2 treatment. Therefore, FGF-2–induced phosphorylation did not produce additional Cx43 bands, but it did increase abundance of the 45-kD species. Migration at 45 kD presumably reflects additional phosphorylation of the already phosphorylated Cx43 (migrating at 43 kD), according to previous reports.43
Phosphorylation of Cx43 after FGF-2 stimulation of cardiomyocytes identifies this protein as one of the targets of FGF-2–triggered signal transduction and provides an important and functionally relevant end point to dissect this process. It is highly probable that phosphorylation of Cx43 caused or contributed to the loss of coupling between cardiomyocytes, as has been inferred for T51B rat liver epithelial cells.42 Disruption of phosphorylatable Cx43 residues was recently shown to result in abnormalities in conduction in vitro and possibly in vivo.49 It is also likely that phosphorylation of Cx43 may, directly or indirectly, be responsible for epitope masking and loss of reactivity with the monoclonal antibody seen in our system. In the latter case, it is of interest that the sequence between residues 261 to 270, containing the epitope(s) recognized by the monoclonal anti-Cx43 preparation (epitope[s] that becomes masked after FGF-2 addition), also contains potential phosphorylatable residues such as Tyr265 and Ser262.50 This region of the molecule is important for coupling, since its modification (by phosphorylation of Tyr265) has been shown to affect junctional properties in Xenopus oocytes.51 Therefore, it is possible that FGF-2 induces Ser262 phosphorylation of cardiac Cx43, which in a manner analogous to its neighbor Tyr265, contributes to decreased intermyocyte metabolic coupling.
Recently, MAPK, which is activated by FGF-2 in cardiac myocytes,38 52 was shown to be capable of inducing phosphorylation of Cx43 on Ser255, Ser279, and Ser282 in the test tube.53 Residues 261 to 270 of Cx43 are in the immediate vicinity of these amino acids and are likely to be affected by phosphorylation of the latter by MAPK.
We were unable to detect tyrosine phosphorylation on Cx43 by phosphoamino acid analysis of the whole protein, irrespective of FGF-2 treatment. Although we cannot at this point exclude the possibility that the sensitivity of this approach was not sufficient to detect tyrosine phosphorylation, it is certain that serine residues were the primary target for FGF-2–induced cardiomyocyte Cx43 phosphorylation. EGF-induced liver epithelial cell Cx43 phosphorylation also occurs on serine residues and leads to loss of communication.42 A similar mechanism may be operating in cardiac myocytes stimulated by FGF-2.
Cardiomyocyte Cx43 was immunoprecipitated by anti-phosphotyrosine antibodies conjugated to agarose beads after, but not before, FGF-2 stimulation. Specificity of the antibodies for phosphotyrosine residues was ascertained by eliminating the response in the presence of nonlabeled free phosphotyrosine but not phosphoserine or phosphothreonine. In preliminary studies, we have found that anti-phosphotyrosine antibodies associated specifically with 10% of total [32P]Cx43, 30 minutes after FGF-2 addition. If this reflected a fraction of Cx43 phosphorylated on tyrosine, one would have expected to be able to detect it by phosphoamino acid analysis; however, this was not the case. Therefore, we suggest that anti-phosphotyrosine antibodies immunoprecipitated a tyrosine-phosphorylated protein(s) or protein complex that associated with Cx43. Kinases such as PKC or MAPK, which have been proposed to phosphorylate Cx43 on serine residues42 53 54 and which are stimulated by FGF-2 in cardiomyocytes,38 52 are possible candidates for such an interaction. In agreement with this notion, Kwak et al55 have shown that activation of PKC by 12-O-tetradecanoylphorbol 13-acetate leads to decreased dye coupling between neonatal cardiomyocytes. Furthermore, preliminary evidence from our laboratory indicates that PKC inhibitors, such as chelerythrine, block FGF-2–induced Cx43 phosphorylation as well as intermyocyte dye coupling (authors' unpublished data, 1996) of cardiac myocytes.
FGF-2 decreased fluorescent dye coupling between cardiomyocytes, which at the very least points to decreased gap junctional pore permeability and metabolic coupling. Decreased metabolic coupling of cardiac myocytes may be related to the induction of a growth response and/or effects on differentiation by FGF-2, as has been inferred for nonelectrically active tissues, which are nevertheless well coupled via GJs.1 2 11 The effect of FGF-2 on cardiac myocyte conductance remains to be studied. A recent study has shown that the relative selectivity and permeability to ions and dyes were not dependent on channel conductance properties.41 In cardiac myocytes, loss of dye coupling following PKC activation by 12-O-tetradecanoylphorbol 13-acetate was accompanied by increases rather than decreases in overall electrical coupling, as Kwak et al55 have shown. Since FGF-2 also activates PKC pathways in myocytes (Reference 52; authors' unpublished data, 1996), one may speculate that it is likely to alter electrical conductance in a fashion similar to 12-O-tetradecanoylphorbol 13-acetate.
In conclusion, we have shown that FGF-2 induces (1) Cx43 phosphorylation on serine residues, accompanied by (2) loss of intermyocyte dye coupling, and likely including (3) masking of Cx43 epitopes located in residues 261 to 270 and (4) a tyrosine phosphorylation–dependent association of Cx43 with other protein(s). It is proposed, but remains to be demonstrated, that all these phenomena are interdependent and provide a basic mechanism for the reduction in intercellular metabolic coupling induced by FGF-2. It would be important to determine whether, similar to our observations for differentiated neonatal myocytes in culture, FGF-2 affects communication in adult cardiomyocytes in vitro and in vivo. Assuming that this is so, events such as myocardial injury, which cause increases in local FGF-2,18 19 35 would be expected to affect coupling of noninjured myocytes in the vicinity of the lesion. It is intriguing that arrhythmias observed after myocardial infarction are proposed to be caused by abnormal conduction from myocytes at borders of scar areas3 ; these myocytes are also associated with increased pericellular FGF-2,18 19 suggesting that this factor should be considered in situations of altered conduction in vivo.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Heart and Stroke Foundation of Canada (HSFC) (Dr Kardami). B.W. Doble and D.G. Bosc are recipients of studentships from HSFC and the Manitoba Health Research Council, respectively. The salary for Dr Kardami was awarded by the Medical Research Council of Canada (group member). Dr Litchfield is a Research Scientist of the National Cancer Institute of Canada and was supported by funds raised by the Canadian Cancer Society. We thank Dr Elliot L. Hertzberg (Departments of Neuroscience and Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY) for providing us with the rabbit anti-Cx43 antibody and Dr Eric Beyer (Washington University School of Medicine, St. Louis, Mo) for the rat Cx43 cDNA.
Received March 8, 1996; accepted July 15, 1996.
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