Fate of gap junctions in isolated adult mammalian cardiomyocytes.

Abstract

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surfflce-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxla had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of"complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its Address for correspondence: Dr. NJ. Severs, Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, England.Received January 7, 1988; accepted November 29, 1988. reflected in distinct differences in their structure, biochemistry, and composition. 5 - 7Refinements in techniques for the dissociation and culture of myocytes from the mature mammalian heart 8-11 have opened new opportunities for exploration of the specific properties of cardiac gap junctions. Isolated pairs of myocytes have already been exploited in electrophysiological and dye transfer studies 12 -14 ; other potential applications include use of dissociated and cultured myocytes for 1) the study of cardiac gap junction degradation and formation, and 2) as a starting material for the isolation of pure cardiac myocyte gap junctions from which contamination with junctions of other cell types in the heart (e.g., endothelial cells, smooth muscle cells, and myofibroblasts) can be e...

Internalization of Large Double-Membrane Intercellular Vesicles by a Clathrin-dependent Endocytic Process

“…Early ultrastructural analyses revealed large vesicular double-membrane GJ structures, AGJs, in the cytoplasm of cells derived from many different tissues (Ginzberg and Gilula, 1979;Larsen et al, 1979;Pfeifer, 1980;Leach and Oliphant, 1984;Mazet et al, 1985;Severs et al, 1989). Here, we demonstrate by expressing Cx43-GFP (a major GJ subunit protein) in HeLa cells, that entire GJ plaques can be internalized into one of the coupled cells to form these cytoplasmic, double-membrane GJ vesicles (Figure 1).…”

Increased Association of ZO-1 With Connexin43 During Remodeling of Cardiac Gap Junctions

“…Previous workers have characterized that this treatment, which results in the break-up of electrical and mechanical intercellular couplings, induces the formation of cytoplasmic gap junctional vesicles known as annular junctions. 21 That this increase in ZO-1 immunolocalization at internalized GJs is accompanied by increases in direct interaction between Cx43 and ZO-1 at the protein-protein level is supported by coimmunoprecipitation analyses. Taken together, our findings are consistent with the growing evidence 29,30 that previously hypothesized functions for MAGUK proteins (eg, ZO-1, dlg, PSD-95 and PSD-93) in active localization of proteins to membrane subdomains and in ion channel aggregation may need reevaluation, or at least, expansion to consider other possible modalities.…”

“…Our data indicate that association between ZO-1 and Cx43 is surprisingly limited to low to moderate levels in intact ventricular myocardium. Furthermore, we find that disruption of intercellular contacts between myocytes, a treatment inducing gap junction endocytosis, 21 results in a dynamic change in the pattern of association between ZO-1 and Cx43. Our data suggests previously unanticipated roles for ZO-1 in the turnover of Cx43 during, or after, gap junctional endocytosis.…”

“…During isolation of adult cardiomyocytes, the entire population of GJs is internalized, undergoing redistribution from the sarcolemma to cytoplasmic vesicles known as annular GJs and cell surface located gap junctional vesicles. 21 We therefore undertook immunoconfocal analyses of freshly isolated ventricular myocytes. In such cells, striking increases in levels of colocalization between immunolabeled Cx43 and ZO-1 were found (Figures 5a through 5c) relative to those observed at GJs in intact myocardium (Figures 1a and b).…”

Internalization and Dephosphorylation of Connexin43 in Hypertrophied Right Ventricles of Rats With Pulmonary Hypertension

“…Annular gap junctions have been recognized as endocytosed fragments of gap junction plaques and early degradation products of gap junction membrane. 11,14,[26][27][28] Intracellular sites and pathways responsible for subsequent gap junction degradation have not been fully understood in cardiac cells, but previous molecular studies have suggested that both the lysosome and proteasome participate in cardiac gap junction degradation. 27,28 Gap junctions have a remarkably dynamic structure, because the turnover of connexin proteins is surprisingly rapid (the half-life of Cx43 in cardiac myocytes rages 1-2 h).…”