PubMed Health⌕ Search

Biomedical subjects

N J Severs

Publications and source records attributed to N J Severs.

At least 91 records · Page 5Linked to original sources
PubMed1993

Robert Feulgen Prize Lecture. 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.

Amino Acid Sequence↗
PubMed1993

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. 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 quasi-crystalline 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Reduced content of connexin43 gap junctions in ventricular myocardium from hypertrophied and ischemic human hearts.

BACKGROUND: Gap junctions are a determinant of myocardial conduction. Disturbances of gap-junctional content may account for abnormalities of impulse propagation, contributing to the arrhythmic tendency and mechanical inefficiency of ischemic and hypertrophied myocardium. The aim of this study was to characterize gap junction organization in normal human ventricular myocardium and to establish whether abnormalities exist in myocardium of chronically ischemic and hypertrophied hearts. METHODS AND RESULTS: Cardiac gap-junctional connexin43 antibodies and confocal microscopy were used in a quantitative immunohistochemical study of surgical myocardial samples to explore the structural basis of electromechanical ventricular dysfunction in chronic ischemic and hypertrophic heart diseases. Normal adult human left ventricular myocardium had a gap-junctional surface area of 0.0051 micron2/micron3 myocyte volume; gap junctions were confined to intercalated disks, of which there was a mean of 11.6 per cell. The right ventricle showed similar gap junction surface area. Left ventricular myocardium from ischemic hearts (distant from any fibrotic scarring), despite normal numbers of intercalated disks per cell, had a reduced gap junction surface area (0.0027 micron2/micron3; P = .02), as did hypertrophied myocardium (0.0031 micron2/micron3; P = .05). The cardiac myocytes in the pathological tissues were larger than normal, and estimated gap-junctional content per cell was reduced in ischemic ventricle (P = .02) compared with normal. CONCLUSIONS: Gap junctions in normal adult human working ventricular myocardium occupy an area of 0.0051 micron2/micron3 myocyte volume. This surface area is reduced in ventricular myocardium from hearts subject to chronic hypertrophy and ischemia, despite a normal number of intercellular abutments, and this alteration may contribute to abnormal impulse propagation in these hearts.

Adult↗
PubMed1993

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.

Age Factors↗
PubMed1993

Structural alterations of airway epithelial tight junctions in cystic fibrosis: comparison of transplant and postmortem tissue.

Previous descriptive freeze-fracture studies have suggested that the airway epithelium in cystic fibrosis (CF) is associated with proliferation and basal extension of tight junctional elements. The significance of these reports in CF has been difficult to evaluate because tight junctions are labile structures, capable of alteration, postmortem. The aim of the present study was to test the hypothesis that the abnormalities in tight junction structure reported previously are unique to CF airways, and to determine the confounding effects of postmortem autolysis. To this end, we analyzed human airway epithelial tight junctions in: (1) histologically normal tissue taken from lungs resected for pulmonary tumor (i.e., resection controls), (2) lungs from patients who died of nonrespiratory causes (i.e., postmortem controls), (3) lungs removed from CF patients undergoing transplantation, and (4) lungs from CF patients postmortem. Tight-junctional depth, strand number, and junctional complexity were analyzed. Basal extensions and isolated foci of tight-junctional elements were found in all groups except for the resection controls. The following two parameters were therefore assessed: (1) the number of times a junction extended below the maximal depth found in the resection controls and (2) the extent of junctional elements that were isolated and distant from the apico-lateral junctional belt. The mean frequencies of occurrence of junctional extension beyond the normal maximum were 4.8% for the CF transplant, 23.6% for the control postmortem, and 48.8% for the CF postmortem group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Validation of immunohistochemical quantification in confocal scanning laser microscopy: a comparative assessment of gap junction size with confocal and ultrastructural techniques.

Confocal scanning laser microscopy (CSLM) is increasingly being used to image antibody-labeled structures visualized with a fluorescent secondary antibody. Such digital images are routinely stored on computer and are well suited to quantitative analysis. Although theoretical aspects of CSLM imaging and resolution are well defined, information is lacking on the relationship observed between measurements of fluorescent antibody-labeled structures and the size of the same structures as determined by electron microscopy (EM). In the present study we examined this relationship for the cardiac gap junction. Data on the size of immunofluorescent-labeled gap junctions were acquired by two methods of analysis from CSLM images and compared statistically with measurements of gap junction size obtained by freeze-fracture EM. The freeze-fracture data were compared before and after exclusion of small junctions, corresponding to those that theoretically would not have been detected in CSLM analysis. The data obtained by the different methods were similar but not identical, reflecting the advantages and limitations of each technique. However, the comparison did indicate that with appropriate sample preparation and orientation, accurate and rapid analysis can be achieved by CSLM, particularly when digital semi-automated techniques are employed.

Animals↗
PubMed1993

The spatial distribution and relative abundance of gap-junctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conduction system.

Electrical coupling between heart muscle cells is mediated by specialised regions of sarcolemmal interaction termed gap junctions. In previous work, we have demonstrated that connexin42, a recently identified gap-junctional protein, is present in the specialised conduction tissues of the avian heart. In the present study, the spatial distribution of the mammalian homologue of this protein, connexin40, was examined using immunofluorescence, confocal scanning laser microscopy and quantitative digital image analysis in order to determine whether a parallel distribution occurs in rat. Connexin40 was detected by immunofluorescence in all main components of the atrioventricular conduction system including the atrioventricular node, atrioventricular bundle, and Purkinje fibres. Quantitation revealed that levels of connexin40 immunofluorescence increased along the axis of atrioventricular conduction, rising over 10-fold between atrioventricular node and atrioventricular bundle and a further 10-fold between atrioventricular bundle and Purkinje fibres. Connexin40 and connexin43, the principal gap-junctional protein of the mammalian heart, were co-localised within atrioventricular nodal tissues and Purkinje fibres. By applying a novel photobleach/double-labelling protocol, it was demonstrated that connexin40 and connexin43 are co-localised in precisely the same Purkinje fibre myocytes. A model, integrating data on the spatial distribution and relative abundance of connexin40 and connexin43 in the heart, proposes how myocyte-type-specific patterns of connexin isform expression account for the electrical continuity of cardiac atrioventricular conduction.

Animals↗
PubMed1993

Gap junction density in human myometrium at term revealed by an anti-peptide antibody and laser scanning confocal microscopy.

The concept that the progesterone-estrogen ratio determines gap junction abundance in myometrium has developed from studies in laboratory animals. In the humans, however, gap junctions form in abundance at term despite a high progesterone-estrogen ratio. We have therefore examined the relationship between contractile activity, progesterone and estrogen levels and gap junction abundance in human myometrium at term. Myometrial tissue obtained from women who had an elective or emergency Cesarean operation at the 38th or 39th week of pregnancy was immunohistochemically examined using a polyclonal antiserum raised against a synthetic peptide matching part of the sequence of connexin43, the principal component of myometrial gap junctions. The concentration of progesterone and estradiol in the maternal blood was measured by means of radio-immunoassay. The number of gap junctions was estimated by counting of punctate staining patterns obtained in myometrial tissue as viewed by laser confocal microscopy and calculated per 1 microns 3 of tissue. The number of gap junctions measured was found to be related to the presence or absence of labor. In the myometrium of women in labor (active phase), the number of gap junctions was highest (45.25 +/- 35). When the myometrium was not contracting (non-labor) and when contractions were weak (pre-labor) the number of gap junctions was low (15.16 +/- 10-5.75 +/- 5). In the pre-and active phase of labor, the concentration of progesterone in maternal blood was significantly higher (P < 0.025 and 0.03, respectively) than in women who were not in labor. The present study shows the potential of using optical section series obtained by laser confocal microscopy for the quantification and mapping of the three-dimensional distribution of gap junctions in human myometrium at term.

Antibodies↗
PubMed1992

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

The 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 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.

Animals↗
PubMed1992

Coated vesicles are implicated in the post-fusion retrieval of the membrane of rat atrial secretory granules.

Using an in situ tannic acid perfusion technique, this study presents evidence that the removal of membrane components from the rat atrial secretory granule membrane after granule exocytosis is mediated by coated vesicles. When tannic acid is used to arrest the post-fusion stages of granule release, coated pit formation occurs on granule membrane, which, although continuous with the sarcolemma, is easily recognised by the membrane omega profile and the continued presence of the granule core. Tannic acid perfusion, before aldehyde fixation, allows a degree of continued cell function, and granule fusions can persist after tannic acid has reached the cell. This results in an increase in the numbers of fusion profiles and the appearance of coated pits on granule membrane at these sites. The proportion of granules with coats increases with perfusion time, suggesting that endocytotic, as well exocytotic events, may be arrested by the action of tannic acid. Coated vesicles are also involved at earlier stages of the release pathway. In other types of secretory system this is considered to represent recycling of membrane proteins as part of the maturation process of the granule. Although arrested granules exhibiting this clathrin coat could have had the coat prior to fusion, as part of the maturation process, our results show that it is more likely to represent a second stage of membrane protein recycling; the postfusion reclamation of proteins from the sarcolemma. This facet of the tannic acid perfusion procedure suggests a general method for quantifying coated pit formation during secretory granule release.

Animals↗
PubMed1992

Freeze-fracture morphology and quantification of human bronchial epithelial tight junctions.

A comprehensive investigation of the morphology of human airway epithelial tight junctions was carried out by freeze-fracture electron microscopy using quantitative methods designed to analyze a range of junctional characteristics. Extrapulmonary bronchi that appeared grossly normal were taken at sites distant from tumor in lungs resected for pulmonary carcinoma. The absence of cellular atypia in the samples was confirmed by histology. Airway levels I (main bronchus; n = 7 subjects) and II (lobar bronchus; n = 5 subjects) were compared with respect to junctional depth, strand number, and junctional complexity. Junctional complexity was assessed by frequency of strand interconnection and numbers of strands per interconnection. Comparisons between airway levels I and II for these parameters showed that there were no significant differences in strand number or junctional complexity between the two airway levels. However, junctional depth was slightly but significantly reduced at level II compared with level I (P less than 0.01). The arrangement of strands varied considerably from one junction to the next, irrespective of the cell types involved. "Parallel" and "network" patterns of junctions were observed; the existence of gradations between these two patterns indicated that they represent opposite extremes of a single junctional form rather than distinct categories of junction. These results have allowed us to establish a data pool for normal human bronchi from which the structure of epithelial cell junctions in bronchial diseases can be compared.

Bronchi↗
PubMed1992

Superior qualities of University of Wisconsin solution for ex vivo preservation of the pig heart.

The components of the University of Wisconsin solution have the potential to enhance and extend heart preservation. We have evaluated University of Wisconsin solution by comparing it with St. Thomas' Hospital cardioplegic solution in the isolated pig heart subjected to 8 hours of ischemia at 4 degrees C (n = 6 in each). The hearts were perfused ex vivo with enriched autologous blood for the control and the postpreservation assessments. Morphologic, metabolic, and functional evaluations were performed. Left and right ventricular function as assessed by the slope values of systolic and diastolic pressure-volume relationships of isovolumically contracting isolated heart was better preserved by University of Wisconsin solution (percent reduction: left ventricular systolic, 52.4% +/- 5.5% versus 17.7% +/- 6.7% [p less than 0.001]; right ventricular systolic, 125.6% +/- 46.4% versus 65.5% +/- 31.4% [p less than 0.05]; right ventricular diastolic, 112.3% +/- 48.7% versus 40.2% +/- 31.3% [p less than 0.02] after St. Thomas' Hospital and University of Wisconsin preservation, respectively). Postischemic recovery of left ventricular rate of rise of pressure and myocardial oxygen consumption were significantly improved after University of Wisconsin preservation (percent reduction, rate of rise of pressure: St. Thomas' Hospital 39.3% +/- 8.1%; University of Wisconsin 18.1% +/- 4.6%; percent reduction, myocardial oxygen consumption St. Thomas' Hospital 55.1% +/- 6.9%, University of Wisconsin 24.8% +/- 6.7%; p less than 0.001). Microvascular functional integrity as assessed by coronary vascular resistance was well maintained throughout the postischemic period and was similar to the preischemic control value in the University of Wisconsin group. By contrast, a significant increase was found at the beginning of postpreservation reperfusion, with a progressive rise thereafter in the St. Thomas' Hospital group (p less than 0.001). Preservation of myocardial adenosine triphosphate was improved and energy charge was unchanged after 8 hours of ischemia and reperfusion in the University of Wisconsin-preserved hearts compared with the St. Thomas' Hospital-preserved hearts (p less than 0.01). Electron microscopic examination revealed substantially better preservation of the contractile apparatus after preservation with University of Wisconsin solution. Myocytes from hearts receiving University of Wisconsin solution, unlike those given St. Thomas' Hospital solution, showed relaxed myofibrils with prominent I-bands. We conclude that University of Wisconsin solution has the potential to improve the preservation of the heart and possibly prolong the ischemic period in clinical cardiac transplantation.

Adenosine↗
PubMed1991

Freeze-fracture cytochemistry: a simplified guide and update on developments.

A wide variety of methods by which cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centered on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen--specifically, turning it over during processing--so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture cytochemistry works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.

Animals↗
PubMed1991

Gap junction distribution in adult mammalian myocardium revealed by an anti-peptide antibody and laser scanning confocal microscopy.

A polyclonal antiserum, raised against a synthetic peptide matching part of the sequence of connexin43 (a rat cardiac gap-junctional protein), was used in combination with laser scanning confocal microscopy to investigate gap junction distribution in cardiac tissues from a range of mammalian species. Comparison of the localised punctate staining patterns obtained in ventricular tissue with the distribution of intercalated disks as viewed by conventional light microscopy and electron microscopy, and with the staining observed by standard light-microscope immunofluorescence using the same anti-serum, demonstrated highly specific labelling of clearly resolved individual gap junctions. Laser scanning confocal microscopy of ventricular myocardium showed the immunostained gap junctions to be confined to well-defined intercalated disks bisecting the long axis of the muscle fibre, whereas in the atrial myocardium, gap junctions were commonly distributed widely over the lateral surfaces of the myocyte body. Rat atrial gap junctions were significantly larger (as measured by the longest axial lengths of fluorescent spots), and showed a narrower spread of sizes, than their counterparts in the ventricle. Ventricular myocardium from six mammalian species including man gave similar immunostaining patterns, indicating conservation both of the epitope(s) detected by the antiserum, and of the general organisation of the cell-to-cell pathways for electrical propagation, in the mammalian heart. Optical section series obtained by laser scanning confocal microscopy permitted the quantification and mapping of the three-dimensional distribution of gap junctions in ventricular intercalated disks with high clarity over substantial specimen depths. A consistent feature of gap junction organisation within disks of ventricular myocardium in all species studied was the presence of a conspicuous ring of large gap junctions around the periphery of the disk. Immunostained gap junctions lying within the interior zone delineated by the peripheral junctions generally occurred at lower numerical densities and were significantly smaller. In all species, less than 3% of all immunolabelled gap junctions measured were greater than 2 microns in maximal length, though a small proportion (0.06%) exceeded 4 microns. The numerical density of immunolabelled gap junctions in the disk was similar between species; however, within species there was a significant decrease in numerical density with increasing disk size. The new features of intercalated disk structure revealed in this study may have an important part to play in the intercellular communication and electrical propagation properties of the mammalian heart.

Analysis of Variance↗
PubMed1991

Altered patterns of gap junction distribution in ischemic heart disease. An immunohistochemical study of human myocardium using laser scanning confocal microscopy.

Arrhythmias are a common and potentially life-threatening complication of myocardial ischemia and infarction in humans. The structural pathways for the rapid intercellular conduction of the electrical impulse that stimulates coordinated contraction in the myocardium are formed by the gap junctions situated at intercalated disks. By raising antibodies to cardiac gap-junctional protein, and using these antibodies in an immunohistochemical procedure in combination with the technique of laser scanning confocal microscopy, we have succeeded in localizing gap junctions, with a clarity not previously possible, through thick volumes of human myocardial tissue. To explore the structural basis for ischemia and infarction-related arrhythmogenesis, antibody labeling and laser scanning confocal microscopy were applied to study the organization, distribution, and other characteristics of gap junctions in the explanted hearts of patients undergoing cardiac transplantation for advanced ischemic heart disease. In areas of myocardium free from histologically detectable structural damage, there was no significant difference in the size of distribution of labeled gap junctions, or in their number per intercalated disk, between left ventricular tissue (in which functional impairment was severe) and right ventricular tissue (in which functional impairment was minimal). However, in myocytes at the border of healed infarcts--zones to which the slow conduction responsible for reentry arrhythmias has been localized--the organization of gap junctions was markedly disordered; instead of being aggregated into discrete intercalated disks, gap-junctional immunostaining was spread extensively over myocyte surfaces. Some infarct zones were bridged by continuous strands of myocytes, coupled to one another by gap junctions, thereby linking healthy myocardium on either side. At their thinnest, these bridges were in some instances no wider than a single attenuated myocyte. The conclusions are 1) a widespread, generalized derangement of gap junction organization does not appear to underlie functional impairment in the ischemic heart, 2) a disorderly arrangement typifies gap junctions in myocytes of the infarct border zone, and this may contribute to alterations in conduction that are capable of precipitating reentry arrhythmias, and 3) delicate chains of myocytes traverse some healed infarcts, apparently forming electrically coupled bridges across what would otherwise constitute blocked zones. The weakest link in this chain can be a single, degenerating myocyte; avoidance of arrhythmia may therefore depend on the continued survival of this single cell.

Antibodies↗
PubMed1991

The pathway of atrial natriuretic peptide release--from cell to plasma.

Tannic acid and dextran have been used to arrest the exocytosis of secretory granules in the atria of the rat heart. By immunogold labeling with silver intensification of ultrathin sections, the arrested exocytosing granules are demonstrated to contain atrial natriuretic peptide (ANP). Extracellular core-like structures found in atria treated with tannic acid are also shown to contain atrial natriuretic peptide. This allows a pathway for the release of atrial natriuretic peptide to be traced from the surface of the myocyte, through the endomysium, into the sub-endothelial space and to the abluminal surface of the capillary endothelium. Uptake of atrial natriuretic peptide into the capillary endothelial cells was also detected. Endothelial transport and release of atrial natriuretic peptide appears to involve the caveolae and smooth vesicles of the non-selective endothelial transport system. No labeling was detected in the endothelial cells of the endocardium or in the mesothelial cells of the epicardium. A disruption of arrested granule cores is seen after dextran treatment. This, and the variation in the labeling of extracellular cores observed, is consistent with the possibility that the cleavage of atrial natriuretic peptide prohormone may be initiated upon the fusion of a granule with the plasma membrane, and continue during the passage of the core material through the extracellular space, with a gradual loss of the active moiety from the disrupted core.

Animals↗
PubMed1991

Acute effects of adriamycin on the macromolecular organization of the cardiac muscle cell plasma membrane.

Adriamycin is a potent chemotherapeutic agent used in the treatment of human neoplastic disease. A major side effect limiting the use of this drug is its toxic effect on the heart, and congestive heart failure becomes an increasingly common complication as the cumulative dose of the drug rises. To learn more about the mechanism of adriamycin cardiotoxicity and, in particular, to investigate its initial effects on the cardiac muscle cell plasma membrane, isolated guinea pig myocytes were exposed to the drug in vitro. Plasma membrane macromolecular structure was examined by freeze-fracture electron microscopy of myocytes exposed to 0.1, 1 and 2 mM adriamycin for periods up to 105 minutes. The principal effect of adriamycin was rapid induction of smooth (protein-poor) domains in the membrane, with displacement and clustering of intramembrane particles (the structures representing integral membrane proteins). These effects, which are time-dependent and dose-dependent, culminate in the formation of saucer-shaped lesions in the membrane, which broadly resemble deformations of the membrane induced by polymyxin B. Both adriamycin and polymyxin are known to have the ability to interact selectively with anionic phospholipids. It is concluded that an important initial effect of adriamycin on cardiac muscle cells is alteration of the macromolecular architecture of the plasma membrane, probably through interaction with anionic phospholipids, and that this may represent the underlying cause of a range of sarcolemmal dysfunctions associated with exposure to the drug.

Animals↗