Emmanuel Dupont

Remodelling of gap junctions and connexin expression in diseased myocardium.

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease.

Gap junctions and connexin expression in human suburothelial interstitial cells.

Objective  To determine whether suburothelial interstitial cells of the human bladder express gap junctions, and if so, to establish their extent and composition, using immunocytochemistry, confocal microscopy and electron microscopy.

Connexin45 expression is preferentially associated with the ventricular conduction system in mouse and rat heart

Cardiac myocytes are electrically coupled by gap junctions, clusters of low-resistance intercellular channels composed of connexins. Variations in the quantities and spatial distribution of different connexin types have been implicated in regional differentiation of electrophysiological properties in the heart. Although independent studies have demonstrated that connexin43 is abundant in working ventricular myocardium and that connexin40 is preferentially expressed in the atrioventricular conduction system of a number of species, information on the spatial distribution of connexin45 in the heart is limited to data obtained using an antibody raised to a single peptide sequence. In the present study, we report on the production and characterization of a new anti-connexin45 antibody and its application to the investigation of connexin45 expression in mouse and rat myocardium. The affinity-purified antiserum, raised in guinea pig to residues 354 to 367 of human connexin45, recognized a single 45-kD band on Western blots of HeLa cells transfected to express connexin45 and gave punctate immunolabeling at the cell borders, demonstrated by freeze-fracture cytochemistry to represent gap junctions. Only low levels of connexin45 mRNA were detected on Northern blots of mouse and rat cardiac tissues, and connexin45 protein levels were below the limit of detection on Western blots. Confocal microscopy of immunolabeled ventricular tissue revealed that the major part of the working myocardium was immunonegative for connexin45. A clearly defined zone containing connexin45-expressing cells was, however, localized to the endocardial surface, overlapping with connexin40-expressing myocytes of the conduction system. As these results contrast with the prevailing view that connexin45 is widely distributed in working ventricular myocytes, we compared the immunolabeling pattern obtained with a commercially supplied anti-connexin45 antiserum raised against the same peptide that was used in previous studies. The commercial connexin45 antiserum gave widespread labeling throughout the ventricular myocardium, but this labeling was inhibited by a six-amino acid peptide matching part of the connexin43 sequence, indicating cross-reaction of the commercial connexin45 antiserum with connexin43 in the tissue. Further evidence for such cross-reactivity came from observations on connexin43-transfected cells, which gave positive immunolabeling with the commercial anti-connexin45 antiserum. Our demonstration of a specific association of connexin45 with connexin40-expressing myocytes in rat and mouse ventricle raises the possibility that connexin45 contributes to the modulation of electrophysiological properties in the ventricular conduction system and highlights the need for reappraisal of the distribution and role of connexin45 in other species.

Downregulation of immunodetectable connexin43 and decreased gap junction size in the pathogenesis of chronic hibernation in the human left ventricle.

BACKGROUND The regional wall motion impairment and predisposition to arrhythmias in human ventricular hibernation may plausibly result from abnormal intercellular propagation of the depolarizing wave front. This study investigated the hypothesis that altered patterns of expression of connexin43, the principal gap junctional protein responsible for passive conduction of the cardiac action potential, contribute to the pathogenesis of hibernation. METHODS AND RESULTS Patients with poor ventricular function and severe coronary artery disease underwent thallium scanning and MRI to predict regions of normally perfused, reversibly ischemic, or hibernating myocardium. Twenty-one patients went on to coronary artery bypass graft surgery, during which biopsies representative of each of the above classes were taken. Hibernation was confirmed by improvement in segmental wall motion at reassessment 6 months after surgery. Connexin43 was studied by quantitative immunoconfocal laser scanning microscopy and PC image software. Analysis of en face projection views of intercalated disks revealed a significant reduction in relative connexin43 content per unit area in reversibly ischemic (76.7+/-34.6%, P<.001) and hibernating (67.4+/-24.3%, P<.001) tissue compared with normal (100+/-30.3%); ANOVA P<.001. The hibernating regions were further characterized by loss of the larger gap junctions normally seen at the disk periphery, reflected by a significant reduction in mean junctional plaque size in the hibernating tissues (69.5+/-20.8%) compared with reversibly ischemic (87.4+/-31.2%, P=.012) and normal (100+/-31.5%, P<.001) segments; ANOVA P<.001. CONCLUSIONS These results indicate progressive reduction and disruption of connexin43 gap junctions in reversible ischemia and hibernation. Abnormal impulse propagation resulting from such changes may contribute to the electromechanical dysfunction associated with hibernation.

Immunocytochemical analysis of connexin expression in the healthy and diseased cardiovascular system.

Gap junctions play essential roles in the normal function of the heart and arteries, mediating the spread of the electrical impulse that stimulates synchronized contraction of the cardiac chambers, and contributing to co‐ordination of activities between cells of the arterial wall. In common with other multicellular systems, cardiovascular tissues express multiple connexin isotypes that confer distinctive channel properties. This review highlights how state‐of‐the‐art immunocytochemical and cellular imaging techniques, as part of a multidisciplinary approach in gap junction research, have advanced our understanding of connexin diversity in cardiovascular cell function in health and disease. In the heart, spatially defined patterns of expression of three connexin isotypes—connexin43, connexin40, and connexin45—underlie the precisely orchestrated patterns of current flow governing the normal cardiac rhythm. Derangement of gap junction organization and/or reduced expression of connexin43 are associated with arrhythmic tendency in the diseased human ventricle, and high levels of connexin40 in the atrium are associated with increased risk of developing atrial fibrillation after coronary by‐pass surgery. In the major arteries, endothelial gap junctions may simultaneously express three connexin isotypes, connexin40, connexin37, and connexin43; underlying medial smooth muscle, by contrast, predominantly expresses connexin43, with connexin45 additionally expressed at restricted sites. In normal arterial smooth muscle, the abundance of connexin43 gap junctions varies according to vascular site, and shows an inverse relationship with desmin expression and positive correlation with the quantity of extracellular matrix. Increased connexin43 expression between smooth muscle cells is closely linked to phenotypic transformation in early human coronary atherosclerosis and in the response of the arterial wall to injury. Current evidence thus suggests that gap junctions in both their guises, as pathways for cell‐to‐cell signaling in the vessel wall and as pathways for impulse conduction in the heart, contribute to the initial pathogenesis and eventual clinical manifestation of human cardiovascular disease. Microsc. Res. Tech. 52:301–322, 2001.

The Gap-Junctional Protein Connexin40 Is Elevated in Patients Susceptible to Postoperative Atrial Fibrillation

Background —Atrial fibrillation (AF), a cardiac arrhythmia arising from atrial re-entrant circuits, is a common complication after cardiac surgery, but the proarrhythmic substrate underlying the development of postoperative AF remains unclear. This study investigated the hypothesis that altered expression of connexins, the component proteins of gap junctions, is a determinant of a predisposition to AF. Methods and Results —The expression of the 3 atrial connexins–connexins 43, 40, and 45 —was analyzed at the mRNA and protein levels by Northern and Western blotting techniques and immunoconfocal microscopy in right atrial appendages from patients with ischemic heart disease who were undergoing coronary artery bypass surgery. Twenty percent of the patients subsequently developed AF, which allowed retrospective division of the samples into 2 groups, non-AF and AF. Connexin43 and connexin45 transcript and protein levels did not differ between the groups. However, connexin40 transcript and protein were expressed at significantly higher levels in the AF group. Connexin40 protein was markedly heterogeneous in distribution. Conclusions —Atrial myocardium susceptible to AF is distinguished from its nonsusceptible counterpart by elevated connexin40 expression. The heterogeneity of connexin distribution could give rise to different resistive properties and conduction velocities in spatially adjacent regions of tissue, which become enhanced and, hence, proarrhythmic the higher the overall level of connexin40.

Individual Gap Junction Plaques Contain Multiple Connexins in Arterial Endothelium

Gap-junctional intercellular communication in endothelial cells is implicated in the coordination of growth, migration, and vasomotor responses. Up to 3 connexin types, connexin40 (Cx40), Cx37, and Cx43 may be expressed in vascular endothelium according to vascular site, species, and physiological conditions. To establish how these connexins are organized at the level of the individual endothelial gap junction, we used affinity-purified connexin-specific antibodies raised in 3 different species to permit double and triple immunolabeling in combination with confocal and electron microscopy. Using HeLa cells transfected with Cx37 and Cx40 for characterization, the anti-Cx37 antibody (raised in rabbit) and the anti-Cx40 antibody (raised in guinea pig) were shown to recognize single bands of 37 and 40 kDa, respectively, on Western blots and to give prominent punctate labeling at the cell borders, specifically in the corresponding transfectant. By applying these antibodies together with mouse monoclonal anti-Cx43 for double and triple immunofluorescence labeling at confocal microscopy, rat aortic and pulmonary arterial endothelia were found to express all 3 connexin types, whereas coronary artery endothelium expressed Cx40 and Cx37 but lacked Cx43. High-resolution en face confocal viewing of the aortic endothelium after double labeling demonstrated frequent colocalization of connexins, with distinct variation in the expression pattern within a given cell, where it made contact with different neighbors. Triple immunogold labeling at the electron-microscopic level revealed that aortic endothelial gap junctions commonly contain all 3 connexin types. This represents the first definitive demonstration of any cell type in vivo expressing 3 different connexins organized within the same gap-junctional plaque.

Gap junction channels and cardiac impulse propagation.

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.

Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos-like syndrome

Background: Desmosomes are cellular junctions important for intercellular adhesion and anchoring the intermediate filament (IF) cytoskeleton to the cell membrane. Desmoplakin (DSP) is the most abundant desmosomal protein with 2 isoforms produced by alternative splicing. Methods: We describe a patient with a recessively inherited arrhythmogenic dilated cardiomyopathy with left and right ventricular involvement, epidermolytic palmoplantar keratoderma, and woolly hair. The patient showed a severe heart phenotype with an early onset and rapid progression to heart failure at 4 years of age. Results: A homozygous nonsense mutation, R1267X, was found in exon 23 of the desmoplakin gene, which results in an isoform specific truncation of the larger DSPI isoform. The loss of most of the DSPI specific rod domain and C-terminal area was confirmed by Western blotting and immunofluorescence. We further showed that the truncated DSPI transcript is unstable, leading to a loss of DSPI. DSPI is reported to be an obligate constituent of desmosomes and the only isoform present in cardiac tissue. To address this, we reviewed the expression of DSP isoforms in the heart. Our data suggest that DSPI is the major cardiac isoform but we also show that specific compartments of the heart have detectable DSPII expression. Conclusions: This is the first description of a phenotype caused by a mutation affecting only one DSP isoform. Our findings emphasise the importance of desmoplakin and desmosomes in epidermal and cardiac function and additionally highlight the possibility that the different isoforms of desmoplakin may have distinct functional properties within the desmosome.

Upregulation of Connexin43 Gap Junctions Between Smooth Muscle Cells After Balloon Catheter Injury in the Rat Carotid Artery

Phenotypic transformation of smooth muscle cells (SMCs) to the synthetic state in vitro and in human coronary atherosclerosis is reported to be associated with upregulation of connexin43 gap junctions. To determine whether cellular interactions mediated by gap junctions participate in the phenotypic transformation of SMCs in arterial injury and disease in general and to establish the spatial and temporal pattern of any such change in relation to neointimal development, we investigated SMC connexin43 gap junction expression during vascular healing in the rat carotid artery after balloon catheter injury. Quantitative immunoconfocal microscopy was applied to localize and to quantify connexin43 gap junctions 1, 3, 9, and 14 days after injury. Parallel studies were conducted by electron microscopy (direct morphological demonstration of SMC gap junctions) and immunoconfocal microscopy (localization of altered actin expression). Synthetic-state SMCs in the neointima (first apparent from 9 days postinjury) revealed abundant expression of gap junctions, with levels of immunodetectable connexin43 threefold greater than those of medial cells. However, the first detectable changes were found in the media, before neointimal formation; at 1 to 3 days postinjury, an increase in SMC gap junction expression was apparent in the innermost (subluminal) zone, the major site from which the cells subsequently found in the neointima are recruited. We conclude that upregulation of connexin43 gap junctions is intimately linked to SMC phenotypic transition and that interactions mediated by gap junctions may be a hitherto unrecognized contributor to the cellular mechanisms underlying the vascular response to injury.