© 1999 American Heart Association, Inc.
Original Contribution |
Downregulation of Connexin 45 Gene Products During Mouse Heart Development
From the Laboratoire de Génétique et Physiologie du Développement (S.A., M.T-R., T.J-G., I.M., E.T., J-P.C., D.B.G.), Institut de Biologie du Développement de Marseille, CNRS/INSERM/AP Marseille/Université de la Méditerranée, Campus de Luminy, Marseille, France; Department of Anatomy and Embryology (A.F.M.M., W.H.L.), Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; and Laboratoire d'Immunochimie des Peptides et des Virus (J-P.B.), Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France. The current affiliation for E.T. is the Centre for Genome Research, University of Edinburgh, United Kingdom.
Correspondence to Daniel Gros, LGPD-IBDM, Campus de Luminy, Case 907, 13288 Marseille Cédex 09, France. E-mail gros{at}lgpd.univ-mrs.fr
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Abstract—The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase–polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.
Key Words: connexin 45 • heart • development
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The gap junctions are clusters of transmembrane channels that mediate direct communication between the cytoplasmic compartments of adjacent cells. These channels, permeable to ions and small molecules (<900 Da), including second messengers, are the structural components responsible for intercellular electrical and metabolic coupling. The proteins that form these channels are encoded by a multigene family, the connexin (Cx) family. Each Cx forms channels that have unique properties of conductance and permeability. The structure and the oligomerization of Cxs into gap junction channels, and the properties and the functions of these channels, have recently been reviewed.1 2 3 4 5 6 7 8
In excitable tissues, such as the myocardial tissues, the gap junction channels are the sites of electrical coupling between myocytes and are responsible for the coordinated propagation of electrical activity that triggers the sequential contraction of the cardiac chambers.9 10 Understanding this conduction phenomenon requires the identification of Cx genes expressed in the myocytes and a detailed investigation of the spatial distribution of Cxs in the heart. It has thus been demonstrated that 3 Cxs are associated with the myocytes in the mammalian heart, Cx43, Cx40, and Cx45, and that each Cx is expressed with its own unique characteristics (for review, see Reference 1010 ).
Cx43 is the major gap junction protein in the adult mammalian heart. It
is abundantly expressed in the different myocardial tissues
(ventricles, atria, septa, His bundle, etc), but its putative presence
in some specific regions such as the sinoatrial and
atrioventricular nodes is still a subject of
controversy.11 12 In mouse, Cx43 protein was detected as
early as 9.5 days postcoitum (dpc) in the primitive ventricle and then
at 12.5 dpc in the atria.13 From this stage its abundance
steadily increases up to the adult stage.14
Analysis of the heart of mice homozygous or heterozygous for a
Cx43 null mutation has demonstrated that this Cx is involved in the
propagation of the electrical activity, mainly in the ventricles, but
that, in addition, and unexpectedly, Cx43 is essential for normal
myocardial development.15 16 17 18 The second Cx expressed in
the myocytes is Cx40. A characteristic that emerges from the numerous
investigations on Cx40 in adult mammalian heart is its expression in
the atrial and conduction tissues associated with its nonexpression in
the working ventricular tissue.10 12 19 This
distribution pattern of Cx40 in adult heart is the consequence of
strong spatiotemporal downregulation that occurs during fetal
life.13 20 The abundance of Cx40 in the
ventricular conduction system has been interpreted as
evidence of the specific implication of this Cx in the fast-conduction
phenomenon. Electrophysiological analysis
of the heart of mice homozygous for a Cx40 null mutation has confirmed
this interpretation and, in addition, demonstrated that the absence of
Cx40 leads to cardiac rhythm defects and conduction
blocks.21 22 Of the 3 Cxs associated with
cardiomyocytes, Cx45 is probably the least known. Human
Cx45 channels, for example, have 2 major characteristics, as follows:
(1) their unitary conductance (
j) is low (15
to 30 pS)23 24 25 compared with the unitary conductance of
other gap junctional channels, and (2) their macroscopic conductance
(gj) is strongly voltage
sensitive,10 24 in contrast to the weak voltage dependence
of Cx43 and Cx40 channels.10 24 This latter property
implies that a slight difference in membrane potential (equivalent to a
transjunctional voltage) between myocytes, such as may occur during the
normal cardiac cycle, can potentially close Cx45 channels. Cx45 gene
transcript was detected in low abundance in adult and embryonic
mammalian hearts.20 26 27 28 29 Cx45 protein, which is
phosphorylated,30 31 32 was reported to have
a widespread distribution in dog and human heart and to be a component
of the gap junctions of all types of myocytes (working, conductive, and
nodal myocytes).27 28 33 34 35 36 This Cx was also shown to be
expressed in quantities that are far from negligible in cultured
neonatal rat heart myocytes31 32 33 34 35 36 37 and also in rabbit
atrial and ventricular
myocardium.38 In marked contrast with these
results, recent investigations have demonstrated that Cx45 was very
weakly expressed in adult and embryonic mouse and rat heart and had a
distribution pattern restricted to the conduction
system.29 39
In this study, we report the expression of Cx45 gene products in mouse heart from early stages of cardiac morphogenesis (8.5 dpc) to the adult stage and demonstrate that Cx45, distributed in all cardiac compartments at early stages, is subject to spatiotemporal regulation that restricts both its expression level and distribution in adult heart.
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Biological Material
The human SKHep1 and HeLa cells and the rat UMR 106 cells (wild types) were cultured as described previously.24 40 41 Ovaries and hearts were dissected from adult mice. Hearts and forelimb buds were collected from mouse embryos at different developmental stages. The developmental stage of these embryos was checked by determining the number of somites and analyzing the morphology of the heart.42
For the reverse transcriptase (RT)–polymerase chain reaction (PCR) experiments, the hearts from mouse embryos and newborn and adult mice were collected under sterile conditions, and great care was taken to ensure that only the heart was removed.13 RNA was extracted from hearts using the guanidinium thiocyanate-phenol-chloroform method.43
pBluescripts containing either the complete coding region of the mouse Cx45 (pF9-7 and pIVT)26 or the 3' end of the rat sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2 (SERCA 2; pRH39)44 45 were propagated in Escherichia coli XL1 Blue and purified according to standard protocols.
Identification of Transcripts by RT-PCR
RT-PCR Experiments
RNA samples (2 µg) extracted from mouse hearts at different
developmental stages (from 8.5 dpc to the adult stage) were reverse
transcribed using the oligo-dT primers included in the First Strand
cDNA Synthesis Kit (Pharmacia Biotech). The PCR experiments were
carried out as described13 using the murine sequences of
GAPDH, Cx45, and desmin genes26 46 47
(Table
). For the 8.5-dpc samples,
products amplified with primers specific for Cx45 were subjected to
a second step of PCR using internal primers (Table; nested PCR).
All of the RNA samples were tested for DNA contamination with
experiments carried out in the absence of RT. Blank reactions were also
performed without RNA.
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Analysis of Amplification Products
To determine that the DNA fragments amplified with the primers
specific for Cx45 were those predicted, the fragments were digested
with endonucleases. The analysis of the mouse Cx45 sequence
indicates that the PCR-amplified 915-bp fragment (Table
) has to
contain unique restriction sites for BglII (+65) and
BamHI (+407).26 The 915-bp fragment was
digested with these enzymes, and the digestion products were
analyzed with 1% agarose gel stained with ethidium bromide.
Alternatively, the 915-bp fragment was also amplified by nested PCR as
described above for the 8.5-dpc samples.
In Situ Hybridization
Whole-Mount In Situ Hybridization
The whole-mount in situ hybridizations carried out with mouse
embryos ranging from 8.0 to 12.5 dpc were performed as previously
described in detail.13 Control experiments were carried
out with anti-digoxigenin antibodies on nonhybridized embryos and
embryos treated with the sense probe.
pF9-7 was modified by excision of the 1.6-kb fragment included between the 2 XbaI restriction sites localized in the polylinker and the Cx45 coding region (+667). The single-stranded antisense and sense RNA probes were synthesized from the modified plasmid in the presence of UTP-digoxigenin (Boehringer Mannheim) according to the manufacturer's instructions. The 0.95-kb Cx45 antisense probe was synthesized with T7 RNA-polymerase from the XbaI-linearized plasmid; the sense probe was synthesized with T3 RNA-polymerase from the XhoI-linearized plasmid. The probes matched 521 bp of the C-terminal coding region of the mouse Cx45 gene plus 429 bp of the 3'-untranslated region.26
In Situ Hybridization on Sections
In situ hybridizations were carried out on serial sections of
14-, 16-, and 18-dpc embryos as previously described.45
The hybridizations were performed with 30 pg of
35S-labeled antisense RNA (16 to 18 hours;
54°C) in 6 to 10 µL of hybridization mixture per section (50%
formamide, 10% dextransulfate, 2x Denhardt's solution, 0.1% Triton
X-100, and 200 ng/µL of herring sperm DNA, and [in mmol/L]
NaCl 300, sodium citrate 30, and DTT 10; pH 7.4). The specific activity
of the cRNA was 1.5x109 cpm/µg. After
hybridization, the sections were washed and treated with RNase (30
minutes; 37°C) to reduce nonspecific binding.
The antisense RNA probes used to detect the Cx45 and SERCA2a
transcripts were synthesized in the presence of both
[35S]UTP and [35S]CTP
with T3 RNA-polymerase from the KpnI-linearized pIVT
or the XhoI-linearized pRH39,
respectively.17 45 These 1.6- to 1.7-kb probes were
degraded to fragments of 
100 bp by hydrolysis in 80 mmol/L
NaHCO3 and 120 mmol/L
Na2CO3 at 60°C for 10 to
20 minutes.
Preparation of Antibodies Directed to Residues 285 to 298 of
Cx45
The peptides, (Y)SAPPGYNIAVKPDQ (corresponding to amino acids
285 to 298 of mouse Cx45),26 (Y)SAPPGY, and NIAVKPDQ, were
synthesized in solid phase. The first peptide, (Y)SAPPGYNIAVKPDQ, which
is conserved in mouse, chick, dog, and human
Cx45,26 33 35 48 was conjugated to BSA using
bis-diazobenzidine as a cross-linker. The protocols used for the
immunization of rabbits with the BSA-peptide complex, the affinity
purification of antibodies from immune sera, and the ELISAs have been
described previously.13 49 The antibodies directed to
residues 285 to 298 of the mouse Cx45 will be referred to as antibodies
285/298. The preimmune sera were subjected to the same purification
steps as the immune sera. The collected fractions, used in control
experiments, will be referred to as preimmune fractions.
Immunoblotting Analysis
The samples were homogenized in a solubilization
buffer (150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1%
SDS, and 50 mmol/L Tris-HCl, pH 7.4) supplemented with
inhibitors (2.5 mmol/L pretreated orthovanadate,
2 mmol/L phenylarsine oxide, and 125 µmol/L
phenylmethylsulfonyl fluoride) and processed as
described.13 Alternatively, the samples were frozen in
liquid nitrogen; freeze dried; solubilized for 30 minutes in a solution
containing 62.5 mmol/L Tris-HCl (pH 6.8), 20% SDS, 5%
mercaptoethanol, 10 mmol/L EDTA, and 5% glycerol, supplemented
with the inhibitors described above; and then
centrifuged for 30 minutes at 13 000g at 4°C. The
supernatants containing the solubilized material according to both
protocols were recovered, and the total amount of protein was
quantified using a bicinchoninic acid assay (Sigma Aldrich
Chimie).
Solubilized proteins were separated by SDS-PAGE and then electrotransferred onto Nitroscreen membranes (Amersham) according to standard procedures. The transferred proteins were probed for the presence of Cx43 or Cx45 using rabbit anti-Cx43 antibodies (0.2 to 0.5 µg/mL)50 or antibodies 285/298 (2.5 to 5 µg/mL) (see above), as described.13 The membranes were then incubated with biotin-conjugated F(ab')2 fragments from goat anti-rabbit IgGs (Immunotech) (dilution 1:1500; 1 hour at room temperature) and then with peroxidase-conjugated streptavidin (Immunotech) (dilution 1:2500; 1 hour at room temperature). Peroxidase was detected with a chemiluminescence detection reagent kit (Boehringer Mannheim) according to the manufacturer's instructions. The chemiluminescence reaction was visualized on Hyperfilm-MP (Amersham). After exposure, some membranes were washed for 90 seconds with Tris-buffered saline containing 0.2% Tween 20 before being re-exposed. The specificity of immunodetections was checked by (1) the omission of the primary antibodies (antibodies 285/298), (2) the replacement of the primary antibodies by a preimmune fraction, and (3) the replacement of the primary antibodies by a solution of primary antibodies preincubated overnight at 4°C with the immunogenic peptide (100 µg/mL).
Immunofluorescence Analyses
Antibodies
The anti-desmin mouse monoclonal antibody (clone DE-U-10, Sigma
Aldrich Chimie), diluted to 1:50, was used as marker of
myocytes.51 The anti-Cx43 mouse monoclonal antibody (Zymed
Laboratories, Inc), the rabbit polyclonal anti-Cx40
antibodies,13 and the rabbit polyclonal antibodies 285/298
were used at 2, 3, and 3 to 10 µg/mL, respectively.13
The antibodies, including the secondary antibodies (see below), were
diluted in a saturation solution containing 1% (wt/vol) BSA and 0.05%
(vol/vol) saponin.
Single-Labeling Experiments
SKHep1, HeLa, and UMR 106 cells were cultured on gelatin-coated
coverslips, fixed, and permeabilized according to a
previously described procedure.13 The cells were incubated
overnight at 4°C with antibodies 285/298, washed with PBS, and
incubated for 1 hour at room temperature with TRITC-labeled goat
anti-rabbit IgGs (Sigma Aldrich Chimie; dilution, 1:50) and then
processed as described.13
The different organs that were collected were embedded in Tissue-Tek OCT compound (Miles Laboratories), after fixation (15 minutes in 2% formaldehyde solution in PBS) or without fixation, and then frozen in isopentane precooled at -30°C. Frozen sections, mounted on gelatin-coated slides, were incubated overnight at 4°C with antibodies 285/298 and then processed as described above for the cells. No difference was detected between fixed and unfixed samples.
Specimens were examined with a Zeiss III light microscope equipped for epifluorescence. The specificity of the immunolabelings was checked by carrying out control experiments similar to those described above for the immunoblotting analyses.
Double-Labeling Experiments
Samples (young embryos; hearts dissected from embryos, newborns,
and adults; and interventricular septa dissected from adult
hearts) were embedded in Tissue-Tek OCT compound as above, after
fixation or without fixation, and then frozen, and serial sectioned.
The sections were incubated overnight at 4°C with the rabbit
polyclonal antibodies 285/298 and then for 1 hour at room temperature
with the anti-desmin or anti-Cx43 mouse monoclonal antibody. The
sections were washed with PBS and then incubated for 1 hour with
TRITC-labeled goat anti-rabbit IgGs (see above) or when mentioned with
indocarbocyanine (Cy3)–conjugated donkey anti-rabbit
F(ab')2 fragments (Jackson ImmunoResearch
Laboratories; dilution, 1:400), mixed with FITC-labeled sheep
anti-mouse IgGs (Sigma Aldrich Chimie; dilution, 1:200). The
sections were processed and analyzed as described above.
Frozen sections from fixed (see above) or unfixed mouse ovary were incubated overnight at 4°C with the rabbit antibodies 285/298 and then for 1 hour at room temperature with the anti-Cx43 mouse monoclonal antibody. The primary antibodies were revealed using a mixture of TRITC-labeled goat anti-rabbit IgGs and FITC-labeled sheep anti-mouse IgGs (see above). The sections were analyzed by confocal laser scanning (CLS) microscopy using a Zeiss LSM 410 system mounted on a Zeiss Axiovert microscope (model 135M). An argon laser and a HeNe laser, both equipped with attenuating filters, were used as excitation sources at 488 and 543 nm, respectively. The confocal system was equipped with filters and detectors designed for either the simultaneous or the separate recording of FITC and TRITC. The emitted images were digitized, and pictures from screen images were printed with an 8800 Sony video printer.
Immunoelectron Microscopy
Samples of mouse ovary were fixed as described52
and embedded in LR Gold resin (TAAB Laboratories Equipment,
Ltd). Ultrathin sections were incubated successively with antibodies
285/298 (5 µg/mL) and 10-nm gold-labeled goat anti-rabbit IgGs
(Aurion) diluted to 1:30.53 Sections were examined with a
Zeiss EM 912 electron microscope. Control experiments were similar to
those carried out for immunoblotting
analyses.
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Cx45 Gene Transcript Is Present in Heart From Early Developmental Stages (8.5 dpc) to the Adult Stage
In mouse, the heart starts to beat at 8.5 dpc.42 54 It is at this stage an S-shaped tube traversed by slow peristaltic contractions. This primitive cardiac activity, which has been well characterized in rat heart,55 implies that the myocytes express 1 or more Cxs assembled into gap junction channels. To complete previous investigations on Cx43 and Cx40,13 we have searched for Cx45 gene transcript in mouse heart from the first contraction stage to the adult stage.
RNA extracted from embryonic and fetal hearts (8.5, 9, 10, 11, 12, 14,
16, and 19 dpc), from young mouse heart (1 and 2 weeks postpartum), and
from adult heart was reversed transcribed as indicated in Materials and
Methods. The cDNAs generated by RT were amplified by PCR or nested PCR
(8.5-dpc samples) with primer pairs unique for Cx45 (Table), and
the resulting amplicons were analyzed by electrophoresis. At
all stages investigated, the amplicons were of the predicted size, 363
or 915 bp, for nested PCR or PCR experiments, respectively (Figure 1A). The identity of 915-bp
amplicons was confirmed by enzymatic digestion with BglII
and BamHI (Figure 1B) and by nested PCR (not shown).
RNA extracted from 8.5-dpc samples was also shown to contain desmin
gene transcript (Figure 1A, lane 1), which confirmed the
presence of differentiated myocytes in these
samples.57 Analyses of RT-PCR control
experiments revealed no signal. The contamination of the
analyzed samples with noncardiac cells had low probability, but
it cannot be completely ruled out. These results indicate that Cx45
gene transcript was present in mouse heart as early as the
beginning of cardiac activity and throughout development up until the
adult stage. The next step was aimed at investigating by in situ
hybridization the spatial distribution of this transcript and
identifying the cardiac cells that expressed it.
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Changes of Expression Pattern of Cx45 Gene Transcript During Heart
Development, as Seen by In Situ Hybridization
The 8- to 8.5-dpc embryos hybridized with antisense Cx45 probe
presented a strong and specific labeling of the ectoderm
(Figure 2A and 2B), which substantially weakened at further developmental
stages. At 8 dpc, the whole primitive heart appeared to be labeled with
a stronger expression of the transcript in the inflow tract (Figure 2B). At 8.5 dpc, the transcript was detected in all of the
cardiac compartments (Figure 2C), including again the inflow
tract, and this expression pattern persisted until 10 dpc (Figure 2D, 9 dpc; Figure 2E, 2G, and 2H, 10 dpc). At this stage
the most remarkable labelings were those of the
atrioventricular canal and the outflow tract (Figures 2G, 2H, 3A, and 3D). The 11-dpc
stage was characterized by a weakening of the labeling in the left
ventricle, as was clearly visible in ventral views of the heart (Figure 2I), whereas the expression of the transcript was maintained in
the other cardiac regions (Figure 2J). In the ventricles of 12-
to 12.5-dpc hearts, the transcript was expressed, weakly and diffusely
(Figure 2K and 2L), whereas more distinct expression was always
seen in the outflow tract (Figure 2K) and the anterior part of
the right ventricle (Figure 2L). Weak labeling was sometimes
observed in the atria at 12 dpc, but it was no longer detected by 12.5
dpc (Figure 2L). In previous experiments,13 the
detection of Cx40 transcript by whole-mount in situ hybridization in
9- to 11-dpc embryonic hearts had required 2- to 4-hour incubation
times (4°C) with the chromogenic substrate. For the
detection of Cx45 transcript, no labeling was seen for incubations
shorter than 8 to 10 hours (4°C), which testifies to the low
abundance of this transcript in heart.
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Whole-mount hybridized embryos were serial sectioned, and the sections
were examined to identify the cardiac cells expressing Cx45 transcript.
In the heart, myocytes were seen to express the transcript, and this is
illustrated in Figure 3 for the 10-dpc stage. In the thin atrial
walls, all of the myocytes were equally labeled (Figure 3A and 3B); in contrast, in the ventricular walls, an expression
gradient was observed, extending from the outermost-layer myocytes
forming a compact layer, which were distinctly labeled, to the
trabecular myocytes, which were not labeled or were very
weakly labeled (Figure 3A and 3C). The labeling of the
atrioventricular canal observed in whole-mount embryos
was confirmed by examination of sections (Figure 3D).
At developmental stages later than 12.5 dpc, the hybridization
experiments were carried out on sections to avoid the problem of the
penetration of the probe in the tissues. These experiments are
illustrated in Figure 4 for the
14-, 16-, and 18-dpc stages. The expression of SERCA2 transcript was
used to identify the myocardium45 (Figure 4B, 4D, and 4F). At the 3 developmental stages investigated,
very weak and diffuse labeling was detected in the ventricles, with a
slightly more distinct staining in the outflow tract
myocardium (Figure 4A and 4C). Besides this
labeling, the coronary vessels were also faintly positive in
18-dpc samples (Figure 4E), recalling the expression pattern of
Cx40 transcript in the ventricular
walls.20 56
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Immunodetection of Cx45 Protein in Mouse Heart Myocytes Depends on
the Developmental Stages Investigated and the Cardiac Tissues
Analyzed
The studies described above, dealing with the expression pattern
of Cx45 gene transcript, were completed with investigations of the
distribution of Cx45 protein at different developmental stages. The
first step of these investigations was the characterization of
antibodies specific for Cx45. This characterization is reported in the
paragraph below.
In a first step, it was demonstrated, using ELISA assays, that the
affinity-purified antibodies 285/298 specifically recognized both the
immunogenic peptide YSAPPGYNIAVKPDQ and its carboxyl-terminal domain
NIAVKPDQ (not shown). In contrast, no affinity was detected between
antibodies 285/298 and the amino-terminal domain (YSAPPGY) of the
immunogenic peptide. In a second step, biological samples known to
express Cx45 gene products abundantly or in low amounts, such as
mouse ovary, SKHep1, HeLa, and UMR 106 cell
lines,24 26 40 41 57 were used to characterize by
immunoblotting antibodies 285/298. A protein with a
Mr of 48 000 was detected in the
immunoreplicas of all of these samples (Figure 5A, lane b, ovary; lane c, HeLa cells;
and lane d, SKHep1 cells) (UMR 106 cells are not shown), and the
intensity of signals reflected the amount of Cx45 assumed to be
expressed in these same samples. No labeling was seen in the
immunoreplicas of the control experiments (not shown). Furthermore,
Cx43 isoforms present in the ovaries were shown to migrate faster
than the above-mentioned 48-kDa protein (compare lanes a and b in
Figure 5A). These results were in agreement with those
previously published,24 30 40 57 although the
electrophoretic mobility of the detected protein (48 000) was slightly
slower than that reported (45 000). This could be due to either the
uncertainty of the Mr measurements or the
composition of the polyacrylamide gels used.
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The specificity of antibodies 285/298 was further investigated by
immunofluorescence. In some HeLa cell preparations,
infrequent and dispersed tiny spots were observed between adjacent
cells (not shown), as reported29 ; in others, no labeling
was observed. In contrast, in SKHep1 cells (Figure 6C and 6D) and in UMR 106 cells (not
shown), conspicuous immunoreactive sites were observed at the cell
periphery, as expected for junctional labeling, and also, but to a
lesser extent, inside the cells. In the ovaries, immunoreactivity was
seen to be mainly associated with the granulosa cells (Figure 6A and 6B) and partially colocalized with Cx43 (Figure 7), as previously reported for anti-Cx45
antibodies.57 No immunoreactive site was seen in the above
samples subjected to the control experiments (not shown). In adult
mouse heart, where, as in the ovaries, Cx43 is strongly expressed, the
major part of the working myocardium was immunonegative to
antibodies 285/298 (Figure 10C through 10G). The specificity of
antibodies 285/298 was finally investigated by immunoelectron
microscopy. In ovary sections treated for immunoelectron microscopy,
numerous gap junctions, between the granulosa cells, were seen
decorated with gold particles (Figure 8),
whereas the intracellular organelles and the cytosol were free of
labeling. No immunoreactivity was detected in the sections prepared
according to the control procedures (not shown).
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The results described above demonstrate that antibodies 285/298 are specific for Cx45, and they will hereafter be referred to as anti-Cx45 antibodies. They were used in the experiments described below to investigate the expression and the distribution of Cx45 in mouse heart during its morphogenesis.
In the 8.5-dpc cardiac tube, the myocardium was identified
by its immunoreactivity with anti-desmin antibodies.58 It
surrounded the endocardium, which was desmin negative (Figure 9A and 9B). At this stage the detection
of Cx45 was homogeneous throughout the
myocardium, and the periphery of the myocytes appeared to
be more or less regularly labeled with anti-Cx45 antibodies (Figure 9C and 9D). At 9.5 dpc, the primitive atrium is separated from
the common ventricular chamber by the
atrioventricular canal (Figure 9E). A few
immunoreactive sites were observed in the thin atrial wall, always
associated with the outermost myocytes (Figure 9E through 9G).
Myocytes expressing Cx45 were also seen in the
atrioventricular canal as shown in Figure 9H and 9I. In the ventricular wall, Cx45 was clearly detected, but
its distribution was polymorphic. In some samples, most of the
myocytes forming the compact ventricular layer were labeled
(Figure 9J through 9L); in others, only a few myocytes were seen
to express Cx45. This latter pattern of expression was found again, in
even more marked form, in the 10.5-dpc heart ventricular
wall, where Cx45 was detected only in some rare myocytes (not shown).
At this same stage, a strong labeling caused by anti-Cx45 antibodies
was observed between the differentiating keratinocytes
of the forelimb buds (Figure 10A and 10B), indicating that the loss of immunoreactivity described above was
specific and endogenous to the myocardium.
Beyond 11 dpc, and until the 7-day postpartum (dpp) stage, no
significant immunofluorescence signal was detected
in the atrial and ventricular free walls. This also held
true at the adult stage for the major part of the working
myocardium (Figure 10C through 10G), as
reported.29 However, in a small number of sections, Cx45
was detectable at high magnification, in trace amounts, in some small
loci localized in the ventricles (Figure 10H and 10I). In
addition, at this same stage, Cx45 was seen in low amounts in the
anterior region of the interventricular septum (Figure 10J through 10L), associated with the common His bundle, as
previously described.29 39 No attempt was made to
investigate further the expression of Cx45 in the conduction system
(but see References 29 and 3929 39 ). This would have been beyond the scope
of the present work. The specificity of the labelings described
above was checked at all stages investigated with appropriate control
experiments (see Materials and Methods).
In preliminary experiments, several attempts were made by
immunoblotting to check for the presence or the absence
of Cx45 in the homogenates of hearts collected at the same
developmental stages as those used for the
immunofluorescence experiments. Because of the
poorly reproducible results obtained for certain developmental stages,
we finally focused the Western blot experiments on 3 stages: 9.5 dpc, 7
dpp, and the adult stage. The expression of Cx45 in early embryonic
heart was confirmed: in 9.5-dpc heart homogenates, Cx45 was
detected as a 48-kDa protein, in agreement with the above results, but
it was found to be consistently associated with a 52-kDa
polypeptide, whatever the amount of material analyzed (Figure 5B, lanes e and f). No protein was revealed in immunoreplicas of
control experiments (not shown). The 52-kDa polypeptide, which was
shown not to be a phosphorylated form of Cx45 (S.
Alcoléa et al, unpublished results, 1998), might be
derived from the 48-kDa protein by an as-yet-unidentified
post-translational modification. Two proteins with
Mrs similar to those reported above were
also detected in immunoprecipitates resulting from the treatment of
cultured neonatal rat myocyte homogenates with anti-Cx45
antibodies (see, eg, Figures 6A and 8A in Reference 3737 ). We observed no
significant fluorescence signal in neonatal mouse heart
sections treated with anti-Cx45 antibodies, but a 48-kDa protein was
specifically detected in 7-dpp heart homogenates,
associated or not with the 52-kDa polypeptide (Figure 5B, lanes
g and h). The detection of this polypeptide was indeed demonstrated to
be dependent on the protein load (Figure 5B, lanes g and h),
which suggests a quantitative decrease of this molecular species during
heart development. Finally, no significant signal was revealed in the
immunoreplicas of adult heart homogenates probed with
anti-Cx45 antibodies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cx45 in Developing Mouse Heart
A combination of techniques was used to investigate the expression and the spatiotemporal regulation of Cx45 gene products in mouse heart during development. RT-PCR experiments have conclusively demonstrated, by the completion of previous partial results obtained by Northern blotting,20 that the Cx45 gene transcript was present in mouse heart from the early stages of cardiac morphogenesis (8.5 dpc, stage of the first contractions) to the adult stage. The presence of this transcript in embryonic and fetal hearts was confirmed by in situ hybridization experiments. In addition, these experiments have demonstrated that (1) the transcript was equally expressed in all cardiac compartments, including the inflow and outflow tracts and the atrioventricular canal, at early stages of morphogenesis (8.5 to 10.5 dpc), and (2) the abundance of transcript, which is constitutively low compared with that of Cx40 and Cx43 (see also Reference 2020 ), was downregulated from 11 to 12 dpc onward. At subsequent developmental stages, it was detected in the ventricles only, in a diffuse way, but with a more distinct expression level in the outflow tract.
Examination of sections from whole-mount in situ hybridized embryos has
shown that the Cx45 transcript was associated with myocytes. In
addition, the expression of Cx45 protein in the myocytes of young
embryonic hearts (8.5 to 9.5 dpc) was demonstrated by its
immunodetection by fluorescence microscopy. Surprisingly,
beyond 10.5 dpc (with the exception of the adult heart, which will be
discussed later), Cx45 protein was no longer detected by conventional
immunofluorescence microscopy in the embryonic,
fetal, or neonatal working myocardium, even though the
transcript was present in heart at these developmental stages (as
demonstrated by RT-PCR and in situ hybridization experiments), and the
protein was detected in Western blots of 7-dpp heart
homogenates. The discrepancy between these results is
perhaps only apparent, and the following hypothesis might provide the
explanation. In early stages of cardiac morphogenesis (8.5 to 9.5 dpc),
Cx45 is sufficiently concentrated in the plasma membrane of myocytes to
be immunodetected by fluorescence microscopy. At later
developmental stages, subsequent to the downregulation of the
transcript, the synthesis of Cx45 decreases and its concentration in
the membrane of ventricular myocytes is below the detection
limit of the fluorescence microscope. One might postulate that
the low number of Cx45 molecules present in the membrane could be
aggregated into immunofluorescence-detectable gap
junctions, but this is apparently not the case, because the channels
they form probably remain dispersed in the membrane. The expression of
Cx45 in HeLa cells is a similar example that supports the above
hypothesis. Although no gap junction plaques have been observed in
wild-type HeLa cell plasma membranes,41 high-resolution
electrophysiological
measurements,23 Western blotting (Figure 5A), and
immunogold labeling of freeze-fractured membrane41 have
demonstrated that Cx45 channels were present, albeit in very low
amounts, in wild-type HeLa cells. Nevertheless, the immunodetection of
these channels by fluorescence microscopy remains elusive.
Immunoreactivity, when detected (References 29 and 3029 30 and this study),
is only seen as infrequent and tiny spots between adjacent cells,
probably because most of the Cx45 channels are not aggregated into gap
junction plaques but dispersed in the plasma membrane, as has been
recently demonstrated.41 The elegant technique of
freeze-fracture immunogold labeling, associated with the double-patch
clamp technique on freshly dissociated embryonic myocyte pairs, should
go some way toward confirming the above hypothesis. This hypothesis,
which accounts for our results on the working myocardium,
does not exclude the possibility that Cx45 might be sufficiently
expressed to be immunodetected in some specialized regions of the
heart. It is indeed the case in the conduction system of adult and
fetal mouse heart, as recently demonstrated.29 39
Strangely enough, the intensity of signal we have detected on sections
by in situ hybridization in the fetal conduction system was as low as
that seen in the working myocardium. This may suggest a
differential regulation of Cx45 expression according to the cardiac
tissue in which this Cx is synthesized.
Cx45 was undetectable in the major part of adult mouse heart. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Although this remains to be demonstrated, one might speculate that these immunopositive foci contain terminal elements of the conduction system (ie, Purkinje fibers). In a general way, expression of Cx45 in adult heart is extremely low, as indicated by the failure to detect it by immunoblotting in adult heart homogenates (Reference 2929 and this study). This would appear to reflect the general downregulation of Cx45 gene products, which starts quite early during cardiac morphogenesis. The results we have described are, in addition, in complete disagreement with those reported for dog and human heart, in which Cx45 was shown to be expressed, fairly strongly, in all cardiac tissues.27 28 33 34 35 36 Species difference, or the characteristics of the antibodies used, might account for this discrepancy.
The myocytes have been demonstrated to synthesize Cx45, but other
cardiac cells are probably also capable of expressing it. Clues are
given in Figures 2E and 4E in which the paired dorsal
aortae and coronary vessels, respectively, are shown to express
Cx45 gene transcript. In addition, faint and specific immunolabeling
was detected very infrequently in coronary vessels of adult
mouse heart (not shown). These observations indicate that vessels,
including coronary vessels, can express Cx45. This is
remarkably well illustrated by the strong expression of Cx45 in the
smooth muscle layers of pig heart coronary vessels (D. Gros et
al, unpublished results, 1998).
Cx45, Cx of the Primitive Heart
At the stage of the first contractions (8.5 dpc), the Cx40 and
Cx43 gene transcripts have been demonstrated to be present in
heart, as is confirmed by the results of RT-PCR
experiments,13 but at this same stage Cx40 and Cx43
proteins have not been detected. They are detected successively
in the embry-onic ventricle at 9.5 dpc for Cx40 and then in
the atrium at 10.5 dpc for Cx4313 (Figure 11). How then is the electrical
activity propagated from the pacemaker, located in the inflow tract, to
the primitive ventricle at the stage of the first contractions?
Clearly, Cx45 channels play a role as early as this stage in the
conduction phenomenon. The Cx45 gene transcript was shown to be
expressed in the heart before the first contractions, at 8 dpc; this
transcript and the Cx45 protein were both detected in the 8.5-dpc
heart. The coordination of the myocytes during the slow peristaltic
contractions that occur at this stage would then be controlled by the
low unitary conductance of Cx45 channels. Cx45 could thus be termed the
"primitive cardiac Cx," because it is, so far, the only Cx
expressed in the primitive tubular heart.
|
The expression of Cx40 and Cx43 in the early embryonic heart only
concerns the ventricular and atrial tissues (Figure 11). At no developmental stages were these Cxs shown to be
associated with the myocytes of the inflow and outflow tracts or the
atrioventricular canal.12 13 Remarkably,
the Cx45 gene was demonstrated to be expressed in those compartments
that are characterized by slow impulse conduction.59 The
downregulation of Cx45 that occurs in embryonic heart is
counterbalanced by the spatiotemporal upregulation of both Cx40 and
Cx43. This is illustrated in Figure 11, which clearly shows the
complementary expression, sometimes partially redundant, of the 3 Cxs
associated with the myocytes in the embryonic heart. Cx43 was shown to
be involved in the development of the right ventricle and the impulse
conduction in the ventricular walls.16 17 18 19
Cx40 was demonstrated to be essential for the rapid conduction of
impulse in the His-Purkinje system.21 22 The
analysis of heart from Cx45 null mice should confirm, or
invalidate, the primordial role that Cx45 should play in the
development and the physiology of this organ given the unique
characteristics of its expression.
|
Acknowledgments |
|---|
This investigation was supported by grants from the Association Française contre les Myopathies and the European Economic Community (grant BMH4-CT 96-1427) (to D.B.G.). We thank Dr K. Willecke for the F9-7 and IVT clones, Dr A-M. Lompré for the SERCA2 clone, and Dr R. Civitelli for the UMR 106 cells. C. Moretti, P. Weber, and G. Turini were of invaluable help in the analyses by CLS microscopy and the photographic work.
Received October 1, 1998; accepted April 5, 1999.
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J. E. I. Gittens, A. A. Mhawi, D. Lidington, Y. Ouellette, and G. M. Kidder Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis Am J Physiol Cell Physiol, April 1, 2003; 284(4): C880 - C887. [Abstract] [Full Text] [PDF]
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 X. Li and J. M. Simard Increase in Cx45 Gap Junction Channels in Cerebral Smooth Muscle Cells from SHR Hypertension, December 1, 2002; 40(6): 940 - 946. [Abstract] [Full Text] [PDF]
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I. Kehat, A. Gepstein, A. Spira, J. Itskovitz-Eldor, and L. Gepstein High-Resolution Electrophysiological Assessment of Human Embryonic Stem Cell-Derived Cardiomyocytes: A Novel In Vitro Model for the Study of Conduction Circ. Res., October 18, 2002; 91(8): 659 - 661. [Abstract] [Full Text] [PDF]
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 N. M. Rummery, H. Hickey, G. McGurk, and C. E. Hill Connexin37 Is the Major Connexin Expressed in the Media of Caudal Artery Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1427 - 1432. [Abstract] [Full Text] [PDF]
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C. M. Johnson, E. M. Kanter, K. G. Green, J. G. Laing, T. Betsuyaku, E. C. Beyer, T. H. Steinberg, J. E. Saffitz, and K. A. Yamada Redistribution of connexin45 in gap junctions of connexin43-deficient hearts Cardiovasc Res, March 1, 2002; 53(4): 921 - 935. [Abstract] [Full Text] [PDF]
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 S. Fujigaki, K. Saito, M. Takemura, N. Maekawa, Y. Yamada, H. Wada, and M. Seishima L-Tryptophan-L-Kynurenine Pathway Metabolism Accelerated by Toxoplasmagondii Infection Is Abolished in Gamma Interferon-Gene-Deficient Mice: Cross-Regulation between Inducible Nitric Oxide Synthase and Indoleamine-2,3-Dioxygenase Infect. Immun., February 1, 2002; 70(2): 779 - 786. [Abstract] [Full Text] [PDF]
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X. Li and J. M. Simard Connexin45 gap junction channels in rat cerebral vascular smooth muscle cells Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H1890 - H1898. [Abstract] [Full Text] [PDF]
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T. A.B. van Veen, H. V.M. van Rijen, and T. Opthof Cardiac gap junction channels: modulation of expression and channel properties Cardiovasc Res, August 1, 2001; 51(2): 217 - 229. [Abstract] [Full Text] [PDF]
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D. Franco and J. M. Icardo Molecular characterization of the ventricular conduction system in the developing mouse heart: topographical correlation in normal and congenitally malformed hearts Cardiovasc Res, February 1, 2001; 49(2): 417 - 429. [Abstract] [Full Text] [PDF]
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S. Kirchhoff, J.-S. Kim, A. Hagendorff, E. Thonnissen, O. Kruger, W. H. Lamers, and K. Willecke Abnormal Cardiac Conduction and Morphogenesis in Connexin40 and Connexin43 Double-Deficient Mice Circ. Res., September 1, 2000; 87(5): 399 - 405. [Abstract] [Full Text] [PDF]
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T. A.B. van Veen, H. V.M. van Rijen, and H. J. Jongsma Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation Cardiovasc Res, June 1, 2000; 46(3): 496 - 510. [Abstract] [Full Text] [PDF]
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V. Valiunas, R. Weingart, and P. R. Brink Formation of Heterotypic Gap Junction Channels by Connexins 40 and 43 Circ. Res., February 4, 2000; 86 (2): e42 - e49. [Abstract] [Full Text] [PDF]
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O Kruger, A Plum, J. Kim, E Winterhager, S Maxeiner, G Hallas, S Kirchhoff, O Traub, W. Lamers, and K Willecke Defective vascular development in connexin 45-deficient mice Development, January 10, 2000; 127(19): 4179 - 4193. [Abstract] [PDF]
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M Kumai, K Nishii, K Nakamura, N Takeda, M Suzuki, and Y Shibata Loss of connexin45 causes a cushion defect in early cardiogenesis Development, January 8, 2000; 127(16): 3501 - 3512. [Abstract] [PDF]
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D. Vaidya, H. S. Tamaddon, C. W. Lo, S. M. Taffet, M. Delmar, G. E. Morley, and J. Jalife Null Mutation of Connexin43 Causes Slow Propagation of Ventricular Activation in the Late Stages of Mouse Embryonic Development Circ. Res., June 8, 2001; 88(11): 1196 - 1202. [Abstract] [Full Text] [PDF]
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