The longstanding quest for the anatomical basis of the Wolff-Parkinson-White syndrome has left many unanswered questions. The ultrastructural morphology of the myocytes comprising accessory atrioventricular pathways, which are capable of rapid and variable conduction, is central to understanding the development and behaviour of this congenital anomaly, but remains unknown. Examination of three surgically resected pathways was performed to determine their underlying cellular morphology and the pattern of intercellular coupling, by correlative light microscopy, electron microscopy and confocal scanning laser microscopy combined with immunohistochemical localization of the cardiac gap-junctional protein, connexin43. Two left-sided pathways were composed of myocardium of 'normal working ventricular' type. The right-sided pathway was composed almost entirely of highly abnormal myocytes characterized by aberrant myofibril organisation, with a lack of A-band material and abnormal mitochondria, but normal intact intercalated disks no different from those seen in left-sided pathways. The gap junctions of all pathways were composed of connexin43 distributed as in ventricular myocardium, and not as found in atrial or atrioventricular nodal tissues. While myocytes of abnormal structure were present in one of the accessory atrioventricular pathways examined, all pathways had morphologically normal gap junctions, the structures responsible for efficient intercellular coupling, with a pattern of distribution suggestive of working ventricular myocardium.

SUMMARY 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. Freeze‐fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision‐related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasicrystalline arrangement, not a ‘random’ arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap‐junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap‐junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131–142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three‐dimensional precision mapping of gap junctions through thick slices of cardiac tissue. By exploiting the serial optical sectioning ability of the confocal microscope, we have succeeded in (1) elucidating the organization of gap junctions at the intercalated disc, (2) establishing temporal and spatial patterns of gap‐junctional protein expression in embryogenesis that correlate with functional differentiation in subsets of cardiac cells, and (3) demonstrating abnormalities of gap‐junction distribution and quantity that may cont...

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.

Gap junctions allow direct communication between cells without recourse to the extracellular space and have been widely implicated as important mediators of cell-cell signalling. They are constructed from the connexin proteins, which form a large family, and individual connexins show complex spatial and temporal variations in their expression patterns. Understanding how this variation contributes to the control of intercellular signalling, both in the adult and during embryonic development, is an important problem that would be aided by reagents that interfere with gap junctional communication through specific connexins. We have begun to address this issue by raising antibodies to peptides derived from connexin43 and connexin32. Connexin43 peptides were located in the amino terminus, cytoplasmic loop and carboxytail. Connexin32 peptides came from the cytoplasmic loop and the first extracellular loop. Immunoblotting and immunostaining properties of purified IgGs were characterized on mouse heart, liver and the 8- to 16-cell mouse embryo. Effects on transfer through gap junctions were assessed in the fully compacted 8-cell mouse embryo by co-injection with Lucifer Yellow or Cascade Blue. Embryos were maintained in culture to assess the developmental consequences of injection. Peptide competition was used to confirm the specificity of immunostaining and inhibition of dye transfer. All connexin specific antibodies recognized their parent connexin on immunoblots and showed no 43/32 cross-reactivity. The connexin32 extracellular loop antibody recognized both connexin 32 and 43 on immunoblots, as predicted by the amino acid sequence homology in this region, but did not immunostain intact gap junctions. Connexin specific antibodies that immuno-stained showed the predicted connexin specificity. Antibodies to either connexin43 amino acids (AA) 1–16 (amino terminus) or AA 101–112 (cytoplasmic loop) neither immunostained nor prevented functional communication through 8-cell embryo gap junctions. Antibodies to AA 123–136 and AA 131–142 in the cytoplasmic loop immunostained heart and 8-cell embryo gap junctions and blocked transfer through them with high efficiency. Fab' fragments were equally effective. Peptide competition showed that both antibodies contained epitopes within AA 131–136 of connexin43. Antibodies against AA 313–324 in the carboxytail immunostained heart and the 8-cell embryo and, as IgGs, prevented dye transfer. Fab' fragments were ineffective. All connexin43 antibodies that blocked gap junctional communication between cells of the 8-cell mouse embryo induced non-communicating cells subsequently to withdraw from compaction.(ABSTRACT TRUNCATED AT 400 WORDS)