J Mol
Cell
Cardiol
Changes
22, 1245-1258
(1990)
in the Expression of Connexin 43, a Cardiac Gap Junctional Protein, During Mouse Heart Development
Catherine
Fromaget
‘, Abdelhakim El Aoumari’, Emmanuel J. P. Briand’ and Daniel Gras’
DuPont
I,*,
‘Laboratoire ‘Institut
de Biologie de la DiJ henciation Cellulaire, URA CNRS 179, Faculti des Sciences de Luminy, Universitt BAix-Marseille II, 13288 Marseille Cedex 9, France, de B’ to lo g ie Molkulaire et Cellulaire, Laboratoire d’lmmunochimie, CNRS, 15 rue Descartes. 67000 Strasbourg, France (Received 28 August 1989, accepted in revised form 23 Mq
1990)
C. FROMAGET, A. EL AOUMARI, E. DUPONT, J. P. BRIAND AND D. GROS. Changes in the Expression of Connexin 43. a Cardiac Gap Junctional Protein, During Mouse Heart Development. Joumol of Molecular and Cellular Cardiology (1990) 22, 1245-1258. A cDNA probe coding for rat connexin 43 (Beyer et al., 1987), a gap junctional protein. was used to detect specific mRNA and estimate its relative abundance in mouse heart at different developmental stages: 11, 14 and 19 days post-coi’tum (dpc); 1, 2 and 3 weeks post-partum (wpp), and at the adult stage. On Northern blots of total cellular RNA, a single 3.0 kb message was detected at all stages of development, and the differential intensities of labeling indicated developmental changes in mRNA abundance. mRNA levels were further investigated by dot-blotting. Densitometric analyses of dot-blot autoradiograms showed a five-fold increase of the mRNA level between 11 dpc and 1 wpp, then a gradual decrease until the adult stage where it reached a value close to that detected at 11 dpc. By comparison, myosin heavy chains and glyceraldehyde-3phosphate dehydrogenase mRNAs were found to peak at 3 wpp and 14 dpc, respectively, The presence and the relative abundance of connexin 43 were investigated at the same developmental stages as previously by immunoblotting of whole-ventricle fractions using antipeptide antibodies specific for this junctional protein. Quantitative data obtained from densitometric analyses ofimmunoblots showed that from 14 dpc to 1 wpp intensity of labeling of connexin 43 was roughly multiplied by a factor of 10. It peaked at 3 wpp before dropping to about 20% at the adult stage. The data obtained with both the cDNA probe and the antibodies were significant as shown by variance analyses. They suggest that expression ofcardiac connexin 43 is developmentally-regulated: at the early stages of heart development the expression levels of the protein would seem to be mainly regulated by mRNA abundance; beyond 2 weeks after birth, the levels of connexin 43 would seem rather to depend upon its stability and/or the efficiency of the translation.
Introduction
Gap junctions (or communicating junctions) (Pitts and Finbow, 1986; Revel et al., 1986; Warner, 1988; Caspar et al., 1988) are plasma membrane structures composed of closely aggregated connexons (Revel and Karnovsky, 1967; Makowski et al., 1977). Each connexon
comprises six subunits delimiting a central transmembrane and hydrophilic channel (Unwin and Zampighi, 1980; Baker et al., 1983). In the narrow junctional extracellular space,connexons of one membrane are tightly associated with connexons of the other apposed membrane in such a way that trans-
Abbreuiationr used: BSA, bovine serum albumine; CX 26, connexin 26; CX 32, connexin 32; CX 43, connexin 43; Con A, concanavalin A; dpc, day post-coi’tum; dpm, disintegrations per minute; EDTA, ethylene diamine tetraaretic acid; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MHC, myosin heavy chains; optical density; PAGE, polyacrylamide electrophoresis: PBS, phosphateOQ gel buffered saline; PMSF, phenylmethysulfonyl fluoride; SDS, sodium dodecyl sulphate; Tris, trihydroxymethyl-amino methane; TRITC, tetraethyl rhodamine isothiocyanate; SSPE, solution of sodium, phosphate and EDT.4 I Maniatis et al., 1982): wpp, week post-partum. These studies were supported 84.05.15.T.016.0), INSERM (grant *Present address: Department of Building, East Lansing (Mi, 48824), 0022-2828/90/l
11245 + 14 $03.00/O
by the 88.50.09) Pediatrics USA
“Fondation pour la Recherche Medicale”, and the CNRS. and Human Development, Michigan State
ANVAR University.
c: 1990 Academic
grant
X--
Life Sciences Press I.imited
1246
C. Fromaget
membrane channels are face-to-face and establish cell-to-cell channels (Sosinsky et al., 1988). Connexon subunits belong to a transmembrane protein family: the connexins (Beyer et a.?.,1987). Connexin 32 (CX32, Mr 26/28 kD) and connexin 26 (CX 26, Mr 21 kD) have been identified in liver (Henderson et al., 1979; Paul, 1986; Kumar and Gilula, 1986; Nicholson et al., 1987; Nicholson and Zhang, 1988; Traub et al., 1983 and 1989; Zhang and Nicolson, 1989); connexin 43 (CX 43, Mr 43 kD) in heart (Beyer et al., 1987 and 1989; DuPont et al., 1988 and 1989; Yancey et al., 1989; El Aoumari et al., 1989 and 1990), connexin 30 and 38 in Xenopus embryo (Gimlich et al., 1988; Ebihara et al., 1989). The presenceof gap junctions is correlated with the capacity for intercellular transfer of ions and small moleculesand for the transmission and regulation of hormonal stimulation (Larsen, 1977; Lawrence et al., 1978; Bennett and Goodenough, 1978; Loewenstein, 1981). Swensonet al. (1989) have recently shown that large, voltage-insensitive conductances develop when connexin 32 and 43 mRNA injected oocytes are paired both with themselves and with each other. In myocardium, gap junctions play an essential role in the transmissionof action potentials from cell-tocell. Transmission of influx in cardiac tissues, through gap junctions, is a basic phenomenon of heart physiology, and consequently myocardial cell coupling has been extensively investigated (Dreifuss et al., 1966; Dtleze, 1987; De Mello, 1987; Imanaga, 1987; Noma and Tsuboi, 1987; Weingart and Maurer, 1987). The ultrastructural aspects and the distribution of gap junctions in adult heart have also been the subject of numerous studies, which have been reviewed by Page and Manjunath (1986). Gap junctions are present in all the cardiac tissues:ventricular, atria1 and conductive tissuesand also in the sino-atria1 node (Masson-Pevet et al., 1978). In embryonic heart, experimental results dealing both with gap junctions and with influx propagation are fragmentary. In rat heart, the first spontaneous rhythmic action potentials occur at the 3-somite stage (9.5 days post-coitum) and are transmitted radially at a uniform rate (0.55 mm/s), suggesting to Hirota et al. (1985) that gap junctions are
et al.
uniformly formed throughout the heart. In chicken heart, the first action potentials are detected at the 7-somite stage, with a radial conduction velocity of 1.5 mm/s. (Hirota et al., 1981, 1983and 1985). From a structural point of view, the formation and growth of gap junctions in the developing heart have been investigated with electron microscopy by Hirakow and Goto (1976) in rat, Mazet (1977) in amphibian, Gros et al. (1978 and 1979) in mouse, and Shibata et al. (1980) in rabbit. Gap junctions appear between cardiac cells, in vivo and in vitro (Gros et al., 1982), as short linear arrays of particles and as development proceeds, they grow by accretion of peripheral particles to form plaques which can reach 0.5 pm2 in adult mouse ventricle (Gros et al., 1978).
The characterization ofcDNAs (Beyer et al., 1987) and antibodies (DuPont et al., 1988 and 1989;Beyer et al., 1989; El Aoumari et al., 1989 and 1990) specific for connexin 43 now offers the opportunity to investigate the molecular events which parallel the assembly of gap junctions within the plasma membrane. In the present study the relative abundance of connexin 43 mRNA was estimated by Northern blot and dot-blotting throughout mouseheart development, using a cDNA probe coding for rat connexin 43 (Beyer et al., 1987). Expression of mouse connexin 43 was also investigated at the samedevelopmental stages by immunoblotting, using antipeptide antibodiesshown to be specific for rat connexin 43 by immunoblotting and immunogold-labeling in electron microscopy (El Aoumari et al., 1989 and 1990). Materials
and Methods
Biological
material
Swiss mice were mated overnight and the fertilized femaleswere selectedthe next morning on the basisof the presenceof a vaginal plug (Rugh, 1968). Ventricles were dissected in cold PBS from 11, 14 and 19 dpc embryonic hearts, from 1, 2 and 3 wpp newborn hearts and from 2-month old adult mouse hearts. For immunoblotting, whole-ventricle fractions of different stageswere prepared as follows. Adult heart ventricles and pooled newborn or embryonic heart ventricles were frozen in Freon 22 and pulverized with a
Connexln
43 in Developing
pestle under liquid nitrogen before being freeze-dried. Samples were stored at -80°C until use. For Northern and dot-blotting, adult heart ventricles and pooled newborn or embryonic heart ventricles were frozen as previously and stored at - 80°C until use. Total cellular RNA of these samples was extracted by the method of Chirgwin et al. (1979) and quantified by spectrophotometry (one A,,, unit = 40 pg RNA). For each RNA preparation the ratio OD,,,/OD,s, (R) was determined to check the homogeneity of RNA samples obtained from different ventricle samples ( 1,96 < R < 2,1). The integrity of each RNA preparation was checked using denaturing gels. Cardiac gap junctions were isolated from adult rat heart in the presence of 1 mM solid PMSF and 0.1 M iodoacetamide as described by DuPont et al. ( 1988 and 1989). Qualitative
analysis of mRNAs
by Northern blot
The cDNA clone Gl (2.5 kb) coding for the rat cardiac CX43 was kindly provided by Dr E. Beyer (Beyer et al., 1987). Two other probes were used in control experiments; a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (1.2 kb) (Hanauer and Mandel, 1984) and a cDNA probe (0.55 kb) hybridizing indifferently with rat myosin 01 and fl heavy chains (MHC) (Dr A. M. Lompre, INSERM, U275, Palaiseau, France). The radiolabeled cDNA probes were synthesized by nick-translation (Maniatis et a/., 1982), using (CI-32P)dCTP and a labeling kit (Amersham), according to manufacturer’s instructions. RNA samples (5 pg of total cellular RNA per well) were fractionated by electrophoresis on 10; agarose/8% formaldehyde gel, then capillary blotted onto nitrocellulose membrane (Hybond C Extra, Amersham) (Mania tis et al., 1982). RNA was cross-linked to the membrane by baking for 2 h at 80°C. High stringency blots were prehybridized in (5 x SSPE; 5Ou/o formamide; 5 x Denhart’s; O.5n/o SDS; 250pg/ml salmon sperm DNA) for 4 h at 42°C then Hybridized overnight at 42°C in the same buffer with the labeled probe (3. lo8 dpm/pg). The blots were then washed in (2 x SSPE; O.lO/, SDS) at room tempera-
Heart
1247
ture three times for 5 min each and twice 35 min at 65°C in (0.5 x SSPE; 0.1 “,. SDS) before autoradiography. Exposure to Hyperfilm (Amersham) was carried out at -80°C with intensifying screens. Dehybridizations were performed for l-2 h at 65°C in (5 mM Tris-HCl, pH 8; 2 mM EDTA; 0.1 x Denhart’s). mRNA sizes and 28s and 18s rRNAs were determined by reference to standard RNA Ladder (0.24 kb9.5 kb) (Gibco BRL). Quantitative
analysis of mR.NAs ty dot-blotting
For each developmental stage investigated, total cellular RNA was sampled as follows. For the adult stage, each sample (3) was extracted from the ventricular tissue of a single heart. For the 1,2 and 3 wpp stages, RNA was prepared from ventricular tissue batches, each of which were constituted with the ventricles of 10 to 15 hearts, then distributed into three samples. For the embryonic stages, RNA was prepared from batches constituted with the ventricles of about 50, 100 and 200 hearts of 19, 14 and 11 dpc embryos, respectively; then RNA extracted from each batch was distributed into three samples. 6, 7, 8, 9 and 10 pg of RNA were analyzed in parallel experiments per sample investigated. RNA was denatured with formaldehyde under the conditions described by White and Bancroft (1982) before being dotted onto nitrocellulose membranes (Hybond C Extra, Amersham) using a template-manifold apparatus (Minifold I SRC 96, Schleicher and Schiiellj to ensure a uniform dot size. The radiolabelled cDN,4 probes as well as the conditions for hybridization, dehybridization and autoradiography were the same as those previously described for the qualitative analyses of RNA by Northern blots. Quantitative estimation of the amount of specific mRNA species was performed by measuring autoradiographic dot intensities at 405 nm with a Titertek Multiskan photometer. Time exposures of the films were 7, 10, 14 and 21 h. Densitometric analyses of dots showed that the intensities measured for 14-h exposures fell into the linear response region of films whatever the cDNA probe used or the developmental stage investigated. Consequently, only the measurements performed for 14-h time exposures were retained for further
1248
C. Fromuget
analyses. Five, four and three experiments were performed with CX43, MHC and GAPDH cDNA probes, respectively. For each stage and each probe the dot intensities measured for the various amounts of RNA were pooled and averaged. For each cDNA probe, an analysis of variance was performed to assessthe influence of the developmental stage on the abundance of mRNA. This analysis, carried out with the VAR3 program described by Rouanet and Lepin ( 1977), enables one to compare the observed OD values to the null hypothesis for which there is no effect of the considered factor. The data are expressedas percentages of mean ODs plotted against the developmental stages; the 100% points being arbitrarily defined as the highest values of mean ODs.
et UC.
PAGE under reducing conditions as described by DuPont et al. (1988 and 1989). Molecular weights were estimated by reference to standard proteins (phosphorylase a, Mr 94000; bovine serum albumin, Mr 87000; ovalbumin, Mr 43000; carbonic anhydrase, Mr 29 000; soybean trypsin inhibitor, Mr 2 1000; lysozyme, Mr 14000). Gels were stained with CoomassieBrilliant Blue R-250. Samples fractionated by electrophoresis were transferred (Towbin et al., 1979) onto a nitrocellulose membrane (0.22 pm; Schleicher and Schuell) at constant voltage (25 V) for 12-15 h with 0.02% SDS in the electrode buffer. Immunoreplicas were first blocked with 4% dehydrated milk reconstituted in 40 mM Tris (pH 7.5), Tween 0.1% (BLOTTO solution, Johnson et al., 1984) then incubated overnight at 4°C with affinity-purified antiPreparation and purification of antipeptide peptide IgGs (2 ,ug/ml in BLOTTO). Treatantibodies ment of replicas with a secondary antibody The peptide SAEQNRMGQY (Ser- 1O- (biotinylated-goat antirabbit F(ab’)2, JackTyr) was synthesized in solid phase accord- son Immunoresearch Lab. Inc.) then with Uackson ing to Merrifield (1963). The sequence peroxidase-labeled streptavidin Immunoresearch Lab. Inc.) before detection SAEQNRMGQ corresponds to residues3 14 322 of rat connexin 43 (Beyer et al. 1987). This of peroxidase activity with chloronaphtol was sequence is located in the cytoplasm (El asdescribed in detail by DuPont et al. (1988). Aoumari et al., 1989 and 1990), 60 amino- Pre-immune serum affinity-purified fractions acids away from the carboxy-terminus of were used, undiluted, in control experiments. the protein. A tyrosyl residue was added to the carboxy-terminus of the peptide Semi-quantitative anaylsis of connexin 43 SAEQNRMQ to facilitate coupling to a car- Sampling of freeze-dried ventricles was similar rier protein. Coupling of the synthetic peptide to that of RNA. Total protein titration of to BSA by means of bis-diazobenzidine, whole-ventricle fractions of different stages immunization of rabbits by injection of BSA- was carried out according to Sheffield et al. peptide complexes into the popliteal lymph (1987). Briefly, freeze-dried fractions (e 1 mg) nodesand purification of antipeptide IgGs by were solubilized for 30 min at room temperaaffinity chromatography from immune sera ture in sample buffer (62.5 mM Tris-HCI, pH have been described in detail by DuPont et 6.8; 20% SDS; 5% P-mercaptoethanol; 10 mM al. (1988) in a previous paper dealing with EDTA; 5% glycerol; 1 mM PMSF) (DuPont et the characterization of antibodies (anti- al., 1988), sonicated and centrifuged (5 min, SALGKLLDKVQAY antibodies) directed to 13000 g) to pellet down insoluble material. a amino-terminus domain of connexin 43. Known amounts of supernatants were diluted Pre-immune sera were subjected to the to I:20 with bidistilled water and 2 ~1 of same purification steps as immune sera samples was applied to nitrocellulose. After (DuPont et al., 1988). Fractions collected from drying the membrane was treated with methelutions with HCl-glycine were usedin control anol and amino black as described by Shefexperiments and will be referred to as: “pre- field et al. (1987). The spotswere cut out and immune serum affinity-purified fractions”. transferred into a 96-well microtiter plate. Two hundred microliters of 0.1 N NaOH were Electrophoresis and immunoblotting added to each well and the dye was allowed to Isolated gap junctions and freeze-dried whole- elute for 10 min. Optical density (OD) was ventricle fractions were analysed by SDS- measured at 650 nm in a Biotech EIA reader.
Connedn
43 in Developing
Samples were run in triplicate. Standard curves were established using BSA samples treated under the same experimental conditions as the heart samples. Known amounts of supernatants, containing the solubilized samples,were immunoblotted aspreviously described. Intensity ofperoxidase reaction product on the nitrocellulose membranes was estimated using a Vernon PHI 5 densitometer scan in the reflectance mode. Preliminary experiments carried out with adult heart samplesshowedthat intensity of peroxidase reaction product was linear for total protein amounts ranging from 10 to 100 pg. The amount of protein loaded per well never exceeded 100 pg. For each developmental stage, the experiments were performed in triplicate. As previously, an analysis of variance was performed to assess the stage effect. The data are expressedas percentage of labeling intensity plotted against the developmental stages, the 100% points being arbitrarily defined as the highest values of labeling intensity.
Heart
1249
(a) 12345670 *,
28sb
_
,.
t
18s.b cx43
MHC (b)
Immunojluorescence
Ventricles from embryonic and newborn mouse heart were frozen in liquid nitrogen and stored at -80°C. Frozen sectioning (4-5 pm) was performed at -20°C using a Reichert-Jung cryostat. Sections were reacted successivelywith FITC-labeled Con A diluted in PBS ( 100 pg/ml), with affinity-purified antipeptide antibodies diluted in PBS (6 fig/ ml) containing 0.1o/0 gelatin, then with TRITC-labeled sheepanti-rabbit IgGs (Jackson Immuno-research Lab. Inc.). Control experiments were performed by omission of the incubation step with primary antibodies or by using pre-immune sera affinity-purified fractions instead of primary antibodies. Preparations were examined with a fluorescent Zeissphotomicroscope III.
Results
Under high stringency conditions of hybridization and washing, the rat CX 43 cDNA probe detects a single messageof 3.0 kb in RNA extracted from adult rat ventricle (Fig. 1(a) lane 8) aspreviously reported by Beyer el al. ( 1987). Under the sameconditions a single
FIGURE 1. Qualitative analyses by Northern blots ot cardiac RNAs extracted at different developmental stages. Lanes 1 to 8 correspond to RNA extracted from I 1 1 14 and 19dpc embryonic mouse heart (lanes I, 2 and 3, respectively), from 1, 2 and 3 wpp new-born mouse heart (lanes 4, 5 and 6, respectively) and from adult mouse and rat hearts (lanes 7 and 8, respectively). (a). RNAs (5 fig per well) were fractionated by elertrophoresis, blotted onto nitrocellulose and hybridized at high stringency with G I clone cDNA specific for CX 43 (Beyer el al., 1987). A single 3.0 kb signal was dt-tected whatever the developmental stage investigated. Arrowheads on the right indicate the positions of 28 S and 18 S rRNA subunits. The arrow indicates the top of thr Northern blot. (b) and (c). The blot shown in (a) was rchybridized with a rat cDNA MHC probe (b) then with a human GAPDH probe (c), successively. Only the regions corrcsponding to MHC and GAPDH signals are shown in (b) and (c). The mRNA sizes are 7 kb and 1.3 kb for MHC and GAPDH, respectively. Note that for each probe, differential intensities of labeling indicate developmental changes in mRNA abundance.
1250
C. Fromaget
messageof the same size is seen in mouse ventricle RNA, whatever the stageof development investigated (Fig. 1(a), lanes 1 to 7). Furthermore, differential intensities of labeling indicate developmental changes in the abundance of mRNA (compare lane 1 to lane 4 of Figure 1, for example). Northern blots were stripped and rehybridized successively with rat MHC [Fig. l(b)] and human GAPDH [Fig. l(c)] cDNA probes. Transcripts of 7.0 kb and 1.3 kb were detected with MHC and GAPDH probes, respectively. In both cases,the intensity of labeling indicates that the abundance of mRNA varies during myocardial development. In order to be certain that degradation was not occuring in RNA samples,the RNA was run on denaturing formaldehyde gels, blotted and probed as described. By this assay, CX43, MHC and GAPDH mRNAs appeared undegraded (data not shown). Relative amounts of CX43 mRNA during mouseheart differentiation were further investigated by dot-blotting using the same conditions of hybridization and washing as for Northern blots. Figure 2 showsan autoradio-
et al.
graph of dot-blots of serial amounts of RNA extracted from mouse and rat ventricles and hybridized with the radiolabeled rat CX43 probe. After exposures to autoradiographic films, the dot-blots were stripped and rehybridized with rat MHC and human GAPDH probes, successively(not shown). Quantitative data obtained from dot-blot analyses are shown in Figure 3. At the earliest time assayed (11 dpc) Cx 43 mRNA level is approximately 20% of their maximum value [Fig. 3(a)]. mRNA level risescontinuously from 11 dpc to peak by 1 wpp. By Pwpp CX43 mRNA level declines progressively until the adult stage to reach a value comparable to that detected at 11 dpc. The relative amounts of MHC and GAPDH mRNAs have a totally different time course. The level of GAPDH mRNA is maximum at 14 dpc, then decreasesuntil adult-
lO(
(a)
5( u
cx43 -lJ\
8
(
8
IO
Cell
Cardiol
Changes
22, 1245-1258
(1990)
in the Expression of Connexin 43, a Cardiac Gap Junctional Protein, During Mouse Heart Development
Catherine
Fromaget
‘, Abdelhakim El Aoumari’, Emmanuel J. P. Briand’ and Daniel Gras’
DuPont
I,*,
‘Laboratoire ‘Institut
de Biologie de la DiJ henciation Cellulaire, URA CNRS 179, Faculti des Sciences de Luminy, Universitt BAix-Marseille II, 13288 Marseille Cedex 9, France, de B’ to lo g ie Molkulaire et Cellulaire, Laboratoire d’lmmunochimie, CNRS, 15 rue Descartes. 67000 Strasbourg, France (Received 28 August 1989, accepted in revised form 23 Mq
1990)
C. FROMAGET, A. EL AOUMARI, E. DUPONT, J. P. BRIAND AND D. GROS. Changes in the Expression of Connexin 43. a Cardiac Gap Junctional Protein, During Mouse Heart Development. Joumol of Molecular and Cellular Cardiology (1990) 22, 1245-1258. A cDNA probe coding for rat connexin 43 (Beyer et al., 1987), a gap junctional protein. was used to detect specific mRNA and estimate its relative abundance in mouse heart at different developmental stages: 11, 14 and 19 days post-coi’tum (dpc); 1, 2 and 3 weeks post-partum (wpp), and at the adult stage. On Northern blots of total cellular RNA, a single 3.0 kb message was detected at all stages of development, and the differential intensities of labeling indicated developmental changes in mRNA abundance. mRNA levels were further investigated by dot-blotting. Densitometric analyses of dot-blot autoradiograms showed a five-fold increase of the mRNA level between 11 dpc and 1 wpp, then a gradual decrease until the adult stage where it reached a value close to that detected at 11 dpc. By comparison, myosin heavy chains and glyceraldehyde-3phosphate dehydrogenase mRNAs were found to peak at 3 wpp and 14 dpc, respectively, The presence and the relative abundance of connexin 43 were investigated at the same developmental stages as previously by immunoblotting of whole-ventricle fractions using antipeptide antibodies specific for this junctional protein. Quantitative data obtained from densitometric analyses ofimmunoblots showed that from 14 dpc to 1 wpp intensity of labeling of connexin 43 was roughly multiplied by a factor of 10. It peaked at 3 wpp before dropping to about 20% at the adult stage. The data obtained with both the cDNA probe and the antibodies were significant as shown by variance analyses. They suggest that expression ofcardiac connexin 43 is developmentally-regulated: at the early stages of heart development the expression levels of the protein would seem to be mainly regulated by mRNA abundance; beyond 2 weeks after birth, the levels of connexin 43 would seem rather to depend upon its stability and/or the efficiency of the translation.
Introduction
Gap junctions (or communicating junctions) (Pitts and Finbow, 1986; Revel et al., 1986; Warner, 1988; Caspar et al., 1988) are plasma membrane structures composed of closely aggregated connexons (Revel and Karnovsky, 1967; Makowski et al., 1977). Each connexon
comprises six subunits delimiting a central transmembrane and hydrophilic channel (Unwin and Zampighi, 1980; Baker et al., 1983). In the narrow junctional extracellular space,connexons of one membrane are tightly associated with connexons of the other apposed membrane in such a way that trans-
Abbreuiationr used: BSA, bovine serum albumine; CX 26, connexin 26; CX 32, connexin 32; CX 43, connexin 43; Con A, concanavalin A; dpc, day post-coi’tum; dpm, disintegrations per minute; EDTA, ethylene diamine tetraaretic acid; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MHC, myosin heavy chains; optical density; PAGE, polyacrylamide electrophoresis: PBS, phosphateOQ gel buffered saline; PMSF, phenylmethysulfonyl fluoride; SDS, sodium dodecyl sulphate; Tris, trihydroxymethyl-amino methane; TRITC, tetraethyl rhodamine isothiocyanate; SSPE, solution of sodium, phosphate and EDT.4 I Maniatis et al., 1982): wpp, week post-partum. These studies were supported 84.05.15.T.016.0), INSERM (grant *Present address: Department of Building, East Lansing (Mi, 48824), 0022-2828/90/l
11245 + 14 $03.00/O
by the 88.50.09) Pediatrics USA
“Fondation pour la Recherche Medicale”, and the CNRS. and Human Development, Michigan State
ANVAR University.
c: 1990 Academic
grant
X--
Life Sciences Press I.imited
1246
C. Fromaget
membrane channels are face-to-face and establish cell-to-cell channels (Sosinsky et al., 1988). Connexon subunits belong to a transmembrane protein family: the connexins (Beyer et a.?.,1987). Connexin 32 (CX32, Mr 26/28 kD) and connexin 26 (CX 26, Mr 21 kD) have been identified in liver (Henderson et al., 1979; Paul, 1986; Kumar and Gilula, 1986; Nicholson et al., 1987; Nicholson and Zhang, 1988; Traub et al., 1983 and 1989; Zhang and Nicolson, 1989); connexin 43 (CX 43, Mr 43 kD) in heart (Beyer et al., 1987 and 1989; DuPont et al., 1988 and 1989; Yancey et al., 1989; El Aoumari et al., 1989 and 1990), connexin 30 and 38 in Xenopus embryo (Gimlich et al., 1988; Ebihara et al., 1989). The presenceof gap junctions is correlated with the capacity for intercellular transfer of ions and small moleculesand for the transmission and regulation of hormonal stimulation (Larsen, 1977; Lawrence et al., 1978; Bennett and Goodenough, 1978; Loewenstein, 1981). Swensonet al. (1989) have recently shown that large, voltage-insensitive conductances develop when connexin 32 and 43 mRNA injected oocytes are paired both with themselves and with each other. In myocardium, gap junctions play an essential role in the transmissionof action potentials from cell-tocell. Transmission of influx in cardiac tissues, through gap junctions, is a basic phenomenon of heart physiology, and consequently myocardial cell coupling has been extensively investigated (Dreifuss et al., 1966; Dtleze, 1987; De Mello, 1987; Imanaga, 1987; Noma and Tsuboi, 1987; Weingart and Maurer, 1987). The ultrastructural aspects and the distribution of gap junctions in adult heart have also been the subject of numerous studies, which have been reviewed by Page and Manjunath (1986). Gap junctions are present in all the cardiac tissues:ventricular, atria1 and conductive tissuesand also in the sino-atria1 node (Masson-Pevet et al., 1978). In embryonic heart, experimental results dealing both with gap junctions and with influx propagation are fragmentary. In rat heart, the first spontaneous rhythmic action potentials occur at the 3-somite stage (9.5 days post-coitum) and are transmitted radially at a uniform rate (0.55 mm/s), suggesting to Hirota et al. (1985) that gap junctions are
et al.
uniformly formed throughout the heart. In chicken heart, the first action potentials are detected at the 7-somite stage, with a radial conduction velocity of 1.5 mm/s. (Hirota et al., 1981, 1983and 1985). From a structural point of view, the formation and growth of gap junctions in the developing heart have been investigated with electron microscopy by Hirakow and Goto (1976) in rat, Mazet (1977) in amphibian, Gros et al. (1978 and 1979) in mouse, and Shibata et al. (1980) in rabbit. Gap junctions appear between cardiac cells, in vivo and in vitro (Gros et al., 1982), as short linear arrays of particles and as development proceeds, they grow by accretion of peripheral particles to form plaques which can reach 0.5 pm2 in adult mouse ventricle (Gros et al., 1978).
The characterization ofcDNAs (Beyer et al., 1987) and antibodies (DuPont et al., 1988 and 1989;Beyer et al., 1989; El Aoumari et al., 1989 and 1990) specific for connexin 43 now offers the opportunity to investigate the molecular events which parallel the assembly of gap junctions within the plasma membrane. In the present study the relative abundance of connexin 43 mRNA was estimated by Northern blot and dot-blotting throughout mouseheart development, using a cDNA probe coding for rat connexin 43 (Beyer et al., 1987). Expression of mouse connexin 43 was also investigated at the samedevelopmental stages by immunoblotting, using antipeptide antibodiesshown to be specific for rat connexin 43 by immunoblotting and immunogold-labeling in electron microscopy (El Aoumari et al., 1989 and 1990). Materials
and Methods
Biological
material
Swiss mice were mated overnight and the fertilized femaleswere selectedthe next morning on the basisof the presenceof a vaginal plug (Rugh, 1968). Ventricles were dissected in cold PBS from 11, 14 and 19 dpc embryonic hearts, from 1, 2 and 3 wpp newborn hearts and from 2-month old adult mouse hearts. For immunoblotting, whole-ventricle fractions of different stageswere prepared as follows. Adult heart ventricles and pooled newborn or embryonic heart ventricles were frozen in Freon 22 and pulverized with a
Connexln
43 in Developing
pestle under liquid nitrogen before being freeze-dried. Samples were stored at -80°C until use. For Northern and dot-blotting, adult heart ventricles and pooled newborn or embryonic heart ventricles were frozen as previously and stored at - 80°C until use. Total cellular RNA of these samples was extracted by the method of Chirgwin et al. (1979) and quantified by spectrophotometry (one A,,, unit = 40 pg RNA). For each RNA preparation the ratio OD,,,/OD,s, (R) was determined to check the homogeneity of RNA samples obtained from different ventricle samples ( 1,96 < R < 2,1). The integrity of each RNA preparation was checked using denaturing gels. Cardiac gap junctions were isolated from adult rat heart in the presence of 1 mM solid PMSF and 0.1 M iodoacetamide as described by DuPont et al. ( 1988 and 1989). Qualitative
analysis of mRNAs
by Northern blot
The cDNA clone Gl (2.5 kb) coding for the rat cardiac CX43 was kindly provided by Dr E. Beyer (Beyer et al., 1987). Two other probes were used in control experiments; a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (1.2 kb) (Hanauer and Mandel, 1984) and a cDNA probe (0.55 kb) hybridizing indifferently with rat myosin 01 and fl heavy chains (MHC) (Dr A. M. Lompre, INSERM, U275, Palaiseau, France). The radiolabeled cDNA probes were synthesized by nick-translation (Maniatis et a/., 1982), using (CI-32P)dCTP and a labeling kit (Amersham), according to manufacturer’s instructions. RNA samples (5 pg of total cellular RNA per well) were fractionated by electrophoresis on 10; agarose/8% formaldehyde gel, then capillary blotted onto nitrocellulose membrane (Hybond C Extra, Amersham) (Mania tis et al., 1982). RNA was cross-linked to the membrane by baking for 2 h at 80°C. High stringency blots were prehybridized in (5 x SSPE; 5Ou/o formamide; 5 x Denhart’s; O.5n/o SDS; 250pg/ml salmon sperm DNA) for 4 h at 42°C then Hybridized overnight at 42°C in the same buffer with the labeled probe (3. lo8 dpm/pg). The blots were then washed in (2 x SSPE; O.lO/, SDS) at room tempera-
Heart
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ture three times for 5 min each and twice 35 min at 65°C in (0.5 x SSPE; 0.1 “,. SDS) before autoradiography. Exposure to Hyperfilm (Amersham) was carried out at -80°C with intensifying screens. Dehybridizations were performed for l-2 h at 65°C in (5 mM Tris-HCl, pH 8; 2 mM EDTA; 0.1 x Denhart’s). mRNA sizes and 28s and 18s rRNAs were determined by reference to standard RNA Ladder (0.24 kb9.5 kb) (Gibco BRL). Quantitative
analysis of mR.NAs ty dot-blotting
For each developmental stage investigated, total cellular RNA was sampled as follows. For the adult stage, each sample (3) was extracted from the ventricular tissue of a single heart. For the 1,2 and 3 wpp stages, RNA was prepared from ventricular tissue batches, each of which were constituted with the ventricles of 10 to 15 hearts, then distributed into three samples. For the embryonic stages, RNA was prepared from batches constituted with the ventricles of about 50, 100 and 200 hearts of 19, 14 and 11 dpc embryos, respectively; then RNA extracted from each batch was distributed into three samples. 6, 7, 8, 9 and 10 pg of RNA were analyzed in parallel experiments per sample investigated. RNA was denatured with formaldehyde under the conditions described by White and Bancroft (1982) before being dotted onto nitrocellulose membranes (Hybond C Extra, Amersham) using a template-manifold apparatus (Minifold I SRC 96, Schleicher and Schiiellj to ensure a uniform dot size. The radiolabelled cDN,4 probes as well as the conditions for hybridization, dehybridization and autoradiography were the same as those previously described for the qualitative analyses of RNA by Northern blots. Quantitative estimation of the amount of specific mRNA species was performed by measuring autoradiographic dot intensities at 405 nm with a Titertek Multiskan photometer. Time exposures of the films were 7, 10, 14 and 21 h. Densitometric analyses of dots showed that the intensities measured for 14-h exposures fell into the linear response region of films whatever the cDNA probe used or the developmental stage investigated. Consequently, only the measurements performed for 14-h time exposures were retained for further
1248
C. Fromuget
analyses. Five, four and three experiments were performed with CX43, MHC and GAPDH cDNA probes, respectively. For each stage and each probe the dot intensities measured for the various amounts of RNA were pooled and averaged. For each cDNA probe, an analysis of variance was performed to assessthe influence of the developmental stage on the abundance of mRNA. This analysis, carried out with the VAR3 program described by Rouanet and Lepin ( 1977), enables one to compare the observed OD values to the null hypothesis for which there is no effect of the considered factor. The data are expressedas percentages of mean ODs plotted against the developmental stages; the 100% points being arbitrarily defined as the highest values of mean ODs.
et UC.
PAGE under reducing conditions as described by DuPont et al. (1988 and 1989). Molecular weights were estimated by reference to standard proteins (phosphorylase a, Mr 94000; bovine serum albumin, Mr 87000; ovalbumin, Mr 43000; carbonic anhydrase, Mr 29 000; soybean trypsin inhibitor, Mr 2 1000; lysozyme, Mr 14000). Gels were stained with CoomassieBrilliant Blue R-250. Samples fractionated by electrophoresis were transferred (Towbin et al., 1979) onto a nitrocellulose membrane (0.22 pm; Schleicher and Schuell) at constant voltage (25 V) for 12-15 h with 0.02% SDS in the electrode buffer. Immunoreplicas were first blocked with 4% dehydrated milk reconstituted in 40 mM Tris (pH 7.5), Tween 0.1% (BLOTTO solution, Johnson et al., 1984) then incubated overnight at 4°C with affinity-purified antiPreparation and purification of antipeptide peptide IgGs (2 ,ug/ml in BLOTTO). Treatantibodies ment of replicas with a secondary antibody The peptide SAEQNRMGQY (Ser- 1O- (biotinylated-goat antirabbit F(ab’)2, JackTyr) was synthesized in solid phase accord- son Immunoresearch Lab. Inc.) then with Uackson ing to Merrifield (1963). The sequence peroxidase-labeled streptavidin Immunoresearch Lab. Inc.) before detection SAEQNRMGQ corresponds to residues3 14 322 of rat connexin 43 (Beyer et al. 1987). This of peroxidase activity with chloronaphtol was sequence is located in the cytoplasm (El asdescribed in detail by DuPont et al. (1988). Aoumari et al., 1989 and 1990), 60 amino- Pre-immune serum affinity-purified fractions acids away from the carboxy-terminus of were used, undiluted, in control experiments. the protein. A tyrosyl residue was added to the carboxy-terminus of the peptide Semi-quantitative anaylsis of connexin 43 SAEQNRMQ to facilitate coupling to a car- Sampling of freeze-dried ventricles was similar rier protein. Coupling of the synthetic peptide to that of RNA. Total protein titration of to BSA by means of bis-diazobenzidine, whole-ventricle fractions of different stages immunization of rabbits by injection of BSA- was carried out according to Sheffield et al. peptide complexes into the popliteal lymph (1987). Briefly, freeze-dried fractions (e 1 mg) nodesand purification of antipeptide IgGs by were solubilized for 30 min at room temperaaffinity chromatography from immune sera ture in sample buffer (62.5 mM Tris-HCI, pH have been described in detail by DuPont et 6.8; 20% SDS; 5% P-mercaptoethanol; 10 mM al. (1988) in a previous paper dealing with EDTA; 5% glycerol; 1 mM PMSF) (DuPont et the characterization of antibodies (anti- al., 1988), sonicated and centrifuged (5 min, SALGKLLDKVQAY antibodies) directed to 13000 g) to pellet down insoluble material. a amino-terminus domain of connexin 43. Known amounts of supernatants were diluted Pre-immune sera were subjected to the to I:20 with bidistilled water and 2 ~1 of same purification steps as immune sera samples was applied to nitrocellulose. After (DuPont et al., 1988). Fractions collected from drying the membrane was treated with methelutions with HCl-glycine were usedin control anol and amino black as described by Shefexperiments and will be referred to as: “pre- field et al. (1987). The spotswere cut out and immune serum affinity-purified fractions”. transferred into a 96-well microtiter plate. Two hundred microliters of 0.1 N NaOH were Electrophoresis and immunoblotting added to each well and the dye was allowed to Isolated gap junctions and freeze-dried whole- elute for 10 min. Optical density (OD) was ventricle fractions were analysed by SDS- measured at 650 nm in a Biotech EIA reader.
Connedn
43 in Developing
Samples were run in triplicate. Standard curves were established using BSA samples treated under the same experimental conditions as the heart samples. Known amounts of supernatants, containing the solubilized samples,were immunoblotted aspreviously described. Intensity ofperoxidase reaction product on the nitrocellulose membranes was estimated using a Vernon PHI 5 densitometer scan in the reflectance mode. Preliminary experiments carried out with adult heart samplesshowedthat intensity of peroxidase reaction product was linear for total protein amounts ranging from 10 to 100 pg. The amount of protein loaded per well never exceeded 100 pg. For each developmental stage, the experiments were performed in triplicate. As previously, an analysis of variance was performed to assess the stage effect. The data are expressedas percentage of labeling intensity plotted against the developmental stages, the 100% points being arbitrarily defined as the highest values of labeling intensity.
Heart
1249
(a) 12345670 *,
28sb
_
,.
t
18s.b cx43
MHC (b)
Immunojluorescence
Ventricles from embryonic and newborn mouse heart were frozen in liquid nitrogen and stored at -80°C. Frozen sectioning (4-5 pm) was performed at -20°C using a Reichert-Jung cryostat. Sections were reacted successivelywith FITC-labeled Con A diluted in PBS ( 100 pg/ml), with affinity-purified antipeptide antibodies diluted in PBS (6 fig/ ml) containing 0.1o/0 gelatin, then with TRITC-labeled sheepanti-rabbit IgGs (Jackson Immuno-research Lab. Inc.). Control experiments were performed by omission of the incubation step with primary antibodies or by using pre-immune sera affinity-purified fractions instead of primary antibodies. Preparations were examined with a fluorescent Zeissphotomicroscope III.
Results
Under high stringency conditions of hybridization and washing, the rat CX 43 cDNA probe detects a single messageof 3.0 kb in RNA extracted from adult rat ventricle (Fig. 1(a) lane 8) aspreviously reported by Beyer el al. ( 1987). Under the sameconditions a single
FIGURE 1. Qualitative analyses by Northern blots ot cardiac RNAs extracted at different developmental stages. Lanes 1 to 8 correspond to RNA extracted from I 1 1 14 and 19dpc embryonic mouse heart (lanes I, 2 and 3, respectively), from 1, 2 and 3 wpp new-born mouse heart (lanes 4, 5 and 6, respectively) and from adult mouse and rat hearts (lanes 7 and 8, respectively). (a). RNAs (5 fig per well) were fractionated by elertrophoresis, blotted onto nitrocellulose and hybridized at high stringency with G I clone cDNA specific for CX 43 (Beyer el al., 1987). A single 3.0 kb signal was dt-tected whatever the developmental stage investigated. Arrowheads on the right indicate the positions of 28 S and 18 S rRNA subunits. The arrow indicates the top of thr Northern blot. (b) and (c). The blot shown in (a) was rchybridized with a rat cDNA MHC probe (b) then with a human GAPDH probe (c), successively. Only the regions corrcsponding to MHC and GAPDH signals are shown in (b) and (c). The mRNA sizes are 7 kb and 1.3 kb for MHC and GAPDH, respectively. Note that for each probe, differential intensities of labeling indicate developmental changes in mRNA abundance.
1250
C. Fromaget
messageof the same size is seen in mouse ventricle RNA, whatever the stageof development investigated (Fig. 1(a), lanes 1 to 7). Furthermore, differential intensities of labeling indicate developmental changes in the abundance of mRNA (compare lane 1 to lane 4 of Figure 1, for example). Northern blots were stripped and rehybridized successively with rat MHC [Fig. l(b)] and human GAPDH [Fig. l(c)] cDNA probes. Transcripts of 7.0 kb and 1.3 kb were detected with MHC and GAPDH probes, respectively. In both cases,the intensity of labeling indicates that the abundance of mRNA varies during myocardial development. In order to be certain that degradation was not occuring in RNA samples,the RNA was run on denaturing formaldehyde gels, blotted and probed as described. By this assay, CX43, MHC and GAPDH mRNAs appeared undegraded (data not shown). Relative amounts of CX43 mRNA during mouseheart differentiation were further investigated by dot-blotting using the same conditions of hybridization and washing as for Northern blots. Figure 2 showsan autoradio-
et al.
graph of dot-blots of serial amounts of RNA extracted from mouse and rat ventricles and hybridized with the radiolabeled rat CX43 probe. After exposures to autoradiographic films, the dot-blots were stripped and rehybridized with rat MHC and human GAPDH probes, successively(not shown). Quantitative data obtained from dot-blot analyses are shown in Figure 3. At the earliest time assayed (11 dpc) Cx 43 mRNA level is approximately 20% of their maximum value [Fig. 3(a)]. mRNA level risescontinuously from 11 dpc to peak by 1 wpp. By Pwpp CX43 mRNA level declines progressively until the adult stage to reach a value comparable to that detected at 11 dpc. The relative amounts of MHC and GAPDH mRNAs have a totally different time course. The level of GAPDH mRNA is maximum at 14 dpc, then decreasesuntil adult-
lO(
(a)
5( u
cx43 -lJ\
8
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8
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