Skip to main content
SpringerLink
Log in

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

  • Robert Feulgen Prize Lecture
  • Published:
Histochemistry Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Baldwin K (1981) Cell-to-cell tracer movement in cardiac muscle. Ruthenium red vs lanthanum. Cell Tissue Res 221:279–294

    Google Scholar 

  • Barr L, Dewey MM, Berger W (1965) Propagation of action potentials and the structure of the nexus in cardiac muscle. J Gen Physiol 48:797–823

    Google Scholar 

  • Bennett MVL, Barrio LC, Bargiello TA, Spray DC, Hertzberg E, Sáez JC (1991) Gap junctions: new tools, new answers, new questions. Neuron 6:305–320

    Google Scholar 

  • Beyer EC, Paul DL, Goodenough DA (1987) Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621–2629

    Google Scholar 

  • Beyer EC, Kistler J, Paul DL, Goodenough DA (1989) Antisera directed against connexin43 peptides react with a 43-kd protein localised to gap junctions in myocardium and other tissues. J Cell Biol 108:595–605

    Google Scholar 

  • Beyer EC, Paul DL, Goodenough DA (1990) Connexin family of gap junction proteins. J Membr Biol 116:187–194

    Google Scholar 

  • Branton D, Bullivant S, Gilula NB, Karnovsky MJ, Moor H, Muhlethaler K, Northcote DH, Packer L, Satir B, Speth V, Staehelin LA, Steere RL, Weinstein RS (1975) Freeze-etching nomenclature. Science 190:54–56

    Google Scholar 

  • Davies MJ (1990) A macro and micro view of coronary vascular insult in ischemic heart disease. Circulation 82 [Suppl II]:46

    Google Scholar 

  • Dermietzel R, Hwang TK, Spray DS (1990) The gap junction family: structure, function and chemistry. Anat Embryol 182:517–528

    Google Scholar 

  • Dillon SM, Allessie MA, Ursell PC, Wit AL (1988) Influences of anisotropic tissue structure on reentrant circuits in the epicardial border zone of subacute canine infarcts. Circ Res 63:182–206

    Google Scholar 

  • Dupont E, El Aoumari A, Roustiau-Severe S, Briand JP, Gros D (1988) Immunological characterization of rat cardiac gap junctions: presence of common antigenic determinants in heart of other vertebrate species and in various organs. J Membr Biol 104:119–128

    Google Scholar 

  • Forbes MS, Sperelakis N (1985) Intercalated discs of mammalian heart: a review of structure and function. Tissue Cell 17:605–648

    Google Scholar 

  • Fuster V, Stein B, Ambrose JA, Badimon L, Badimon JJ, Chesebro JH (1990) Atherosclerotic plaque rupture and thrombosis. Evolving concepts. Circulation 82 (Supp II]:47–59

    Google Scholar 

  • Geiger B, Volk T, Volberg T (1985) Molecular heterogeneity of adherens junctions. J Cell Biol 101:1523–1531

    Google Scholar 

  • Gourdie RG, Green CR, Severs NJ (1988) The development of detergent-free methods for cardiac gap junction isolation. In: Dickinson HG, Goodhew PG (eds) Institute of Physics Conference Series (93), vol 3. Eurem 88, York. IOP, Bristol, pp 139–140

  • Gourdie RG, Harfst E, Severs NJ, Green CR (1990) Cardiac gap junctions in rat ventricle: localization using site-directed antibodies and laser scanning confocal microscopy. Cardioscience 1:75–82

    Google Scholar 

  • Gourdie RG, Green CR, Severs NJ (1991) Gap junction distribution in adult mammalian myocardium revealed by an anti-peptide antibody and laser scanning confocal microscopy. J Cell Sci 99:41–55

    Google Scholar 

  • Gourdie RG, Green CR, Severs NJ, Anderson RH, Thompson RP (1993) Evidence for a distinct gap-junctional phenotype in ventricular conduction tissue of the developing and mature avian heart. Circ Res 72: in press

  • Gourdie RG, Green CR, Severs NJ, Thompson PR (1992) Immunolabelling patterns of gap junction connexins in the developing and mature rat heart. Anat Embryol 185:363–378

    Google Scholar 

  • Green CR (1988) Evidence mounts for the role of gap junctions during development. BioEssays 8:7–10

    Google Scholar 

  • Green CR, Severs NJ (1983) A simplified method for the rapid isolation of cardiac intercalated discs. Tissue Cell 15:17–26

    Google Scholar 

  • Green CR, Severs NJ (1984a) Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks. J Cell Biol 99:453–463

    Google Scholar 

  • Green CR, Severs NJ (1984b) Connexon rearrangement in cardiac gap junctions: evidence for cytoskeletal control? Cell Tissue Res 237:185–186

    Google Scholar 

  • Harfst E, Gourdie RG, Severs NJ, Green CR, Powell T (1988) Gap junctions from rabbit heart and dissociated myocytes — development of detergent-free isolation methods. J Mol Cell Cardiol 20 [Suppl 5]:S77

  • Harfst E, Severs NJ, Green CR (1990) Cardiac myocyte gap junctions: evidence for a major connexon protein with an apparent relative molecular mass of 70 000. J Cell Sci 96:591–604

    Google Scholar 

  • Harlow E, Lane D (1988) Antibodies: a laboratory manual, Cold Spring Harbor, Laboratory, Cold-Spring Harbor, New York

    Google Scholar 

  • Hawkes R, Niday E, Gordon J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119:142–147

    Google Scholar 

  • Hennemann H, Suchyna T, Lechtenberg-Fraté H, Jungbluth S, Dahl E, Schwarz J, Nicholson BJ, Willecke K (1992) Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung. J Cell Biol 117:1299–1310

    Google Scholar 

  • Hertzberg EL (1984) A detergent-independent procedure for the isolation of gap junctions from rat liver. J Biol Chem 259:9936–9943

    Google Scholar 

  • Hoffman BF, Dangman KH (1987) Review. Mechanisms for cardiac arrhythmias. Experientia 43:1049–1056

    Google Scholar 

  • Hoh JH, Lal R, John SA, Revel J-P, Arnsdorf MF (1991) Atomic force microscopy and dissection of gap junctions. Science 253:1405–1408

    Google Scholar 

  • Houghten RA (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interactions at the level of individual amino acids. Proc Natl Acad Sci USA 82:5131–5135

    Google Scholar 

  • Hoyt RH, Cohen ML, Saffitz JE (1989) Distribution and three-dimensional structure of intercellular junctions in canine myocardium. Circ Res 64:563–574

    Google Scholar 

  • Imanaga I (1987) Cell-to-cell coupling studied by diffusional methods in myocardial cells. Experientia 43:1080–1083

    Google Scholar 

  • Jones P, Jackson P, Price GJ, Patel B, Ohanion V, Lear AL, Critchley DR (1989) Identification of a talin binding site in the cytoskeletal protein vinculin. J Cell Biol 109:2917–2927

    Google Scholar 

  • Jongsma HJ, Gros D (1991) The cardiac connection. News Physiol Sci 6:34–40

    Google Scholar 

  • Kanter HL, Saffitz JE, Beyer EC (1992) Cardiac myocytes express multiple gap junction proteins. Circ Res 70:438–444

    Google Scholar 

  • Kleber AG (1987) Review. Conduction of the impulse in the ischemic myocardium — implications for malignant ventricular arrhythmias. Experientia 43:1056–1061

    Google Scholar 

  • Koteliansky VE, Shirinsky VP, Gneushev GN, Chernousov MA (1985) The role of actin binding proteins vinculin, filamin, and fibronectin in intracellular and intercellular linkages in cardiac muscle. Adv Myocardiol 5:215–221

    Google Scholar 

  • Laird DWA, Revel J-P (1990) Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes. J Cell Sci 97:109–117

    Google Scholar 

  • Luke RA, Saffitz JE (1991) Remodeling of ventricular conduction pathways in healed canine infarct border zones. J Clin Invest 87:1594–1602

    Google Scholar 

  • Luke RA, Beyer EC, Hoyt RH, Saffitz JE (1989) Quantitative analysis of intercellular connections by immunohistochemistry of the cardiac gap junction protein connexin43. Circ Res 65:1450–1457

    Google Scholar 

  • Magee AI, Buxton RS (1991) Transmembrane molecular assemblies regulated by the greater cadherin family. Curr Opin Cell Biol 3:854–861

    Google Scholar 

  • Makowski L, Casper DLD, Philips WC, Goodenough DA (1977) Gap junction structures. II. Analysis of the X-ray diffraction data. J Cell Biol 74:629–645

    Google Scholar 

  • Manjunath CK, Page E (1985) Cell biology and protein composition of cardiac gap junctions. Am J Physiol 248:H783-H791

    Google Scholar 

  • Manjunath CK, Goings GE, Page E (1984) Cytoplasmic surface and intramembrane components of rat heart gap-junctional protein. Am J Physiol 246:H865-H875

    Google Scholar 

  • Milks LC, Kumar NM, Houghten R, Unwin N, Gilula NB (1988) Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO J 7:2967–2975

    Google Scholar 

  • Page E (1992) Cardiac gap junctions. In: Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE (eds) The heart and cardiovascular system. Raven Press, New York, pp 1003–1048

    Google Scholar 

  • Parker AE, Wheeler GN, Arnemann J, Pidsley SC, Ataliotis P, Thomas CL, Rees DA, Magee AI, Buxton RS (1991) Desmosomal glycoproteins II and III: cadherin-like junctional molecules generated by alternative splicing. J Biol Chem 266:10438–10445

    Google Scholar 

  • Revel J-P, Karnovsky MJ (1967) Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol 33:C7-C12

    Google Scholar 

  • Risek B, Gilula NB (1991) Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during rat pregnancy. Development 113:165–181

    Google Scholar 

  • Saffitz JE, Hoyt RH, Luke RA, Kanter HL, Beyer EC (1992) Cardiac myocyte interconnections at gap junctions — role in normal and abnormal electrical conduction. Trends Cardiovasc Med 2:56–60

    Google Scholar 

  • Severs NJ (1985) Intercellular junctions and the cardiac intercalated disk. In: Harris P, Poole-Wilson PA (eds) Advances in myocardiology, Plenum Press, New York, pp 223–242

    Google Scholar 

  • Severs NJ (1989a) Gap junction shape and orientation at the cardiac intercalated disk. Circ Res 65:1458–1461

    Google Scholar 

  • Severs NJ (1989b) Constituent cells of the heart and isolated cell models in cardiovascular research. In: Piper HM, Isenberg G (eds) Isolated adult cardiomyocytes, vol 1. CRC Press, Boca Raton, pp 3–41

    Google Scholar 

  • Severs NJ (1990) Review. The cardiac gap junction and intercalated disc. Int J Cardiol 26:137–173

    Google Scholar 

  • Severs NJ, Green CR (1983a) Rapid freezing of unpretreated tissues for freeze-fracture electron microscopy. Biol Cell 47:193–204

    Google Scholar 

  • Severs NJ, Green CR (1983b) Ultrarapid freezing techniques and connexon configuration in cardiac gap junctions. Beitr Elektronenmikroskop Direktabb Oberfl 16:571–578

    Google Scholar 

  • Severs NJ, Robenek H (1992) Constituents of the arterial wall and atherosclerotic plaque: an introduction to atherosclerosis. In: Robenek H, Severs NJ (eds) Cell interactions in atherosclerosis. CRC Press, Boca Raton, pp 1–49

    Google Scholar 

  • Severs NJ, Slade AM, Powell T, Twist VW (1985) Ultrastructure of the sarcolemma and intercalated disc in isolated rat myocytes. Basic Res Cardiol 80 [Suppl 2]:35–40

    Google Scholar 

  • Severs NJ, Shovel KS, Slade AM, Powell T, Twist VW, Green CR (1989) Fate of gap junctions in isolated adult mammalian cardiomyocytes. Circ Res 65:22–42

    Google Scholar 

  • Severs NJ, Slade AM, Powell T, Twist VW, Green CR (1990) Integrity of the dissociated adult cardiac myocyte: gap junction tearing and the mechanism of plasma membrane resealing. J Muscle Res Cell Motil 11:154–166

    Google Scholar 

  • Severs NJ, Peters NS, Gourdie RG, Harfst E, Green CR (1992) Cytochemical labelling of gap junctions in ischaemic heart disease — correlative investigation by laser scanning confocal microscopy and electron microscopy. In: Ríos A, Arias JM, Megías-Megías L, López-Galindo A (eds) Electron microscopy 92, vol 1. Eurem 92, Secretariado de Publicaciones de la Universidad de Granada, Granada, Spain, pp 627–628

    Google Scholar 

  • Severs NJ, Gourdie RG, Harfst E, Peters NS, Green CR (1993) Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model. J Microsc (in press)

  • Shotton DM (1989) Review. Confocal scanning optical microscopy and its applications for biological specimens. J Cell Sci 94:175–206

    Google Scholar 

  • Shovel KS, Severs NJ, Powell T (1988) Remodelling of the intercalated disc in dissociated and cultured adult rabbit ventricular myocytes. In: Dickinson HG, Goodhew PG (eds) Institute of Physics Conference Series (93), vol 3. Eurem 88, IOP, Bristol, pp 131–132

  • Sjöstrand FS, Andersson-Cedergen E (1960) Intercalated disks of heart muscle. In: Bourne GH (ed) The structure and function of heart muscle, vol 1. Academic Press, New York, pp 421–445

    Google Scholar 

  • Smith JH, Green CR, Peters NS, Rothery S, Severs NJ (1991) Altered patterns of gap junction distribution in ischemic heart disease. An immunohistochemical study of human myocardium using laser scanning confocal microscopy. Am J Pathol 139:801–821

    Google Scholar 

  • Sommer JR, Scherer B (1985) Geometry of cell and bundle appositions in cardiac muscle: light microscopy. Am J Physiol 248:H792-H803

    Google Scholar 

  • Spach MS, Miller WT, III, Dolber PC, Kootsey JM, Sommer JR, Mosher CE Jr (1982) The functional role of structural complexities in the propagation of depolarization in the atrium of the dog. Cardiac conduction disturbances due to discontinuities of effective axial resistivity. Circ. Res 50:175–191

    Google Scholar 

  • Spray DC, Burt JM (1990) Structure-activity relations of the cardiac gap junction channel. Am J Physiol 258:C195-C205

    Google Scholar 

  • Stauffer KA, Kumar NM, Gilula NB, Unwin N (1991) Isolation and purification of gap junction channels. J Cell Biol 115:141–150

    Google Scholar 

  • Takeichi M (1990) Cadherins: a molecular family important in selective cell-cell adhesion. Annu Rev Biochem 59:237–252

    Google Scholar 

  • Tamura T, Bauer H, Birr C, Pipkom R (1983) Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src. Sell 34:587–596

    Google Scholar 

  • Uemura K, Pisa Z (1988) Trends in cardiovascular mortality in industrialized countries since 1950. World Health Stat Q 41:155–178

    Google Scholar 

  • Unwin PNT, Ennis PD (1984) Two configurations of a channel-forming membrane protein. Nature 307:609–613

    Google Scholar 

  • Unwin PNT, Zampighi G (1980) Structure of the junction between communicating cells. Nature 283:545–549

    Google Scholar 

  • Ursell PC, Gardner PI, Albala A, Fenoglio JJ, Wit AL (1985) Structural and electrophysiological changes in the epicardial border zone of canine myocardial infarcts during infarct healing. Circ Res 56:436–451

    Google Scholar 

  • Van Kempen MJA, Fromaget C, Gros D, Moorman AFM, Lamers WH (1991) Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res 68:1638–1651

    Google Scholar 

  • White JG, Amos WB, Fordham M (1987) An evaluation of confocal versus conventional imaging of biological structure by fluorescence light microscopy. J Cell Biol 105:41–48

    Google Scholar 

  • Willecke K, Hennemann H, Dahl E, Jungbluth S, Heynkes R (1991) The diversity of connexin genes encoding gap-junctional proteins. Eur J Cell Biol 56:1–7

    Google Scholar 

  • Yancey SB, John SA, Lal R, Austin BJ, Revel J-P (1989) The 43-kD polypeptide of heart gap junctions: immunolocalization, topology and functional domains. J Cell Biol 108:2241–2254

    Google Scholar 

  • Yancey SB, Biswal S, Revel J-P (1992) Spatial and temporal patterns of distribution of the gap junction protein connexin43 during mouse gastrulation and organogenesis. Development 114:203–212

    Google Scholar 

  • Yeager M, Gilula NB (1992) Membrane topology and quaternary structure of cardiac gap junction ion channels. J Mol Biol 223:929–948

    Google Scholar 

  • Zimmer DB, Goldstein MA (1987) DNase I interactions with filaments of skeletal muscles. J Muscle Res Cell Motil 8:30–38

    Google Scholar 

  • Zimmer DB, Green CR, Evans WH, Gilula NB (1987) Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J Biol Chem 262:7751–7763

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy and Developmental Biology, University College London, Gower Street, WC1E 6BT, London, UK

    Colin R. Green

  2. Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, SW3 6LY, London, UK

    Nicholas J. Severs

Authors
  1. Colin R. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicholas J. Severs
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Green, C.R., Severs, N.J. Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease. Histochemistry 99, 105–120 (1993). https://doi.org/10.1007/BF00571871

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00571871

Keywords

Search

Navigation