Colin R. Green

Immunolabelling patterns of gap junction connexins in the developing and mature rat heart

SummaryThe distribution of gap junctions in prenatal, postnatal, and adult rat hearts was studied by laser scanning confocal microscopy, using antiserum raised to a peptide (HJ) matching part of the sequence of connexin43 (a cardiac gap junction protein). Using digital reconstruction of optically-sectioned tissue volumes, a highly sensitive detection of immunolabelled gap junctions was achieved. The distribution of positive anti-HJ immunolabelling was regionalised in the prenatal heart from its first detection at 10 days post-coitus. High levels of immunopositive staining occurred in the trabeculae of the embryonic ventricles. Other zones of the early myocardium including early central conduction tissues had no detectable signal. The prenatal outflow tract, interventricular septum and a narrow zone of myocardium subjacent to the epicardial free wall also had low levels of immunopositive signal. During postnatal growth and in the adult rat heart, a marked distinction emerged between the central conducting tissues of the atria and ventricles. Whilst small immunostained gap junctions became detectable within the atrioventricular node on the atrial side of the junction, between the interatrial and interventricular septa, no immunolabelling was found within the ventricular branching bundle. This difference between the atrioventricular node and branching bundle is consistent with potential functional distinctions between these two structures, and is not consistent with the recent proposal that the His bundle and its branches act as an extended atrioventricular node in smaller mammals such as the rat. Ventricular Purkinje fibres, distal to the branching bundle, showed high levels of anti-HJ immunostaining. Organisation of gap junctions into intercalated disks within the ventricle proceeded late into the adolescent stages of heart growth. The distribution of a second connexin protein, MP70, not previously characterised in the heart, was studied using monoclonal antibodies. MP70 was transiently immunolabelled in the heart during the postnatal period, but only within valves. Previously, this protein has been reported only in the eye lens. MP70-containing gap junctions may represent a specialisation in avascular tissues, since blood vessels are not present in either the eye lens or the cusps of heart valves.

Evidence for a distinct gap-junctional phenotype in ventricular conduction tissues of the developing and mature avian heart.

The gap-junctional proteins connexin43 and connexin42 have been shown to be expressed in the developing and mature avian heart, but their respective spatiotemporal distributions are unknown. In the present study, we have immunolocalized connexin42 in the conduction tissues of the adult avian heart (nonbranching bundle, bundle branches, and Purkinje fibers) and vascular endothelial cells. Connexin43 immunolabeling was confined to vascular smooth muscle. A novel microwave-based method was used to label connexin42 and connexin43 in the same tissue section. Neither connexin42 nor connexin43 was immunolocalized in working myocardium, atrioventricular node, and atrioventricular ring tissue of the bird heart. Although connexin42 first appeared in periarterial conduction myocytes and vascular endothelium at 9-10 embryonic days, the central conduction tissues, including the nonbranching bundle and proximal branches, remained immunonegative for connexin42 up until hatching (approximately 20 embryonic days). During the early postnatal period (1-14 days), connexin42 immunolabeling progressively spread up the bundle branches toward the nonbranching bundle. Connexin42 appeared uniformly distributed along the left bundle branch by 14 postnatal days. The distribution and spread of connexin42 immunoreactivity suggest that the emergence of specialized junctional contacts along ventricular fascicles occurs relatively late in heart development and coincides with the emergence of the chick from incubation within the egg.

The incidence and size of gap junctions between the bone cells in rat calvaria

Polyclonal antisera to synthetic peptides matching sequences on the cytoplasmic regions of connexin-43, a gap junction protein first identified in rat heart, have been used to immunolabel gap junctions in the calvarial bone, maintained intact as in vivo, of 1- to 2-week-old rats. The specimens were examined in reflection and fluorescence modes by scanning laser confocal microscopy, and the numbers of gap junctions and their sizes estimated. The mean number of connexin-43 immunolabelled junctions per osteoblast (n=65) was 15.3 (SD ± 4.5). The mean length of 227 junctions, selected for the sharpness of the image of the fluorescent spot, was 0.67 μm (SD ± 0.18; range 0.37–1.29 μm) and their mean area 0.26 μm2 (SD ± 0.145; range 0.075–0.93 μm2); these probably fell within the upper half of the total size range. Gap junctions were detected between preosteoblasts, osteoblasts, osteocytes and chondrocytes, and between these juxtaposed cell types. In addition, connexin-43 immuno-labelled junctions were found between some osteoclasts and overlying mononuclear cells at active sites of resorption.

Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease

In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131–142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal the presence of large junctions arranged as a peripheral ring around the disk, with smaller junctions in an interior zone: an arrangement that may facilitate efficient intercellular transfer of current. By applying our immunolabelling techniques to tissue from hearts removed from transplant patients with advanced ischaemic heart disease, we have demonstrated that gap junction distribution between myocytes at the border zone of healed infarcts is markedly disordered. This abnormality may contribute to the genesis of reentrant arrhythmias in ischaemic heart disease.

Analysis of the Rat Liver Gap Junction Protein: Clarification of Anomalies in its Molecular Size

The major gap junction polypeptide in most tissues has an apparent molecular mass of 27 kDa with a 47 kDa dimer present in junction-enriched fractions. However, a 54 kDa protein recognized by gap junction-specific antibodies has been reported and a complementary DNA (cDNA) sequence for both human and rat liver gap junctions codes for a 32 kDa protein. In this paper we show that these are all forms of the same gap junction protein that can be observed on SDS–polyacrylamide gels simply by varying the concentration of acrylamide in the gels. A 64 kDa dimer is also obtainable. Antibodies to the gap junction protein or to a synthetic peptide constructed to match the rat liver gap junction amino-terminal sequence recognize all of these forms. Under some conditions a 54 kDa dimer is ‘preferred’, explaining the presence of this species in whole tissue homogenate Western blots. These results clarify several controversies and indicate that the protein forming the gap junction channel probably undergoes no major post-translational modification as the cDNA sequence codes for a protein of molecular mass 32 kDa and this protein species and its 64 kDa dimer are demonstrable on SDS–polyacrylamide gels under appropriate conditions.

Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model

Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co‐ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction—the cardiac gap junction—is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low‐resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart.

Connexin43 gap junction protein plays an essential role in morphogenesis of the embryonic chick face.

Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co‐ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx‐1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face.

Functional Block Of Gap Junctional Communication Using Antipeptide Antibodies: Molecular Localisation Of The Putative Binding Sites

We have tested a range of antipeptide antibodies, against various domains of connexins 32 and 43, for their ability to block dye transfer in the 8-16 cell stage mouse embryo. The mouse embryo at this stage is well coupled by gap junctions which are formed exclusively from the connexin 43 protein. We report that only antibodies which immunostain gap junctions in the embryo have the ability to block dye transfer. Epitopes on the cytoplasmic loop are particularly sensitive to antibody binding and will bring about block of dye transfer even when antibodies in the form of Fab fragments are used. Such sensitivity to antibody binding is not exhibited by the epitope on the carboxyl tail; in this case although block occurs when IgG antibodies bind, Fab fragments have no effect. A full account of this work is currently under review.