INTERCELLULAR-JUNCTIONS AND THE APPLICATION OF MICROSCOPIC TECHNIQUES- THE CARDIAC GAP JUNCTION AS A CASE MODEL

Intercellular junctions are fundamental to the interactions between ce lls. By means of these junctions, the activities of the individual cel ls that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the au thors on one specific type of junction-the cardiac gap junction-is pre sented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can h elp extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid i mpulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct inte rcellular communication, a function of particular importance for morph ogenetic signalling during development. The work discussed demonstrate s some of the applications of techniques in electron microscopy, immun ocytochemistry and confocal scanning laser microscopy to the understan ding of the structural basis of the function of gap junctions in the n ormal adult heart, the developing heart and the diseased heart. Freeze -fracture electron microscopy of heart tissue prepared by rapid freezi ng techniques, in which excision-related structural damage to the cell s is minimized or avoided, makes it possible to deduce the structure o f the functioning gap junction in vivo. Gap junctions in hearts that a re beating normally in the living animal until the very instant of fre ezing consist of connexons (transmembrane channels) organized in a qua sicrystalline arrangement, not a 'random' arrangement as proposed in t he original hypothesis on the structural correlates of gap junction fu nction. Alterations in connexon arrangement occur in response to ischa emia and hypoxia, though the relationship of these to gap-junctional p ermeability 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 ga p-junctional protein reported in the heart, were raised. The specifici ty of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (tha t raised to residues 131-142) was found to be particularly effective a s a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable th e three-dimensional precision mapping of gap junctions through thick s lices of cardiac tissue. By exploiting the serial optical sectioning a bility of the confocal microscope, we have succeeded in (1) elucidatin g the organization of gap junctions at the intercalated disc, (2) esta blishing temporal and spatial patterns of gap-junctional protein expre ssion in embryo-genesis that correlate with functional differentiation in subsets of cardiac cells, and (3) demonstrating abnormalities of g ap-junction distribution and quantity that may contribute to the genes is of arrhythmias in ischaemic heart disease.