Telomere shortening and telomerase activity in ischaemic cardiomyopathy patients - Potential markers of ventricular arrhythmia.

BACKGROUND: Implantable cardioverter defibrillators (ICDs) reduce mortality in patients with ischaemic cardiomyopathy at high risk of ventricular arrhythmias (VA). However, the current indication for ICD prescription needs improvement. Telomere and telomerase in leucocytes have been shown to associate with biological ageing and pathogenesis of cardiovascular diseases. We hypothesised that leucocyte telomere length, load-of-short telomeres and/or telomerase activity are associated with VA occurrence in ischaemic cardiomyopathy patients. METHODS AND RESULTS: 90 ischaemic cardiomyopathy patients with primary prevention ICDs were recruited. 35 had received appropriate therapy from the ICD for potentially-fatal VA while the remaining 55 patients had not. No significant differences in baseline demographic data relevant to telomere biology were seen between the two groups. There was no significant difference in the age and sex adjusted mean telomere length analysed by qPCR between the groups (p=0.88). In contrast, the load-of-short telomeres assessed by Universal-STELA method and telomerase activity by TRAP assay were both higher in patients who had appropriate ICD therapy and were significantly associated with incidence of ICD therapy (p=0.02, p=0.02). ROC analyses demonstrated that the sensitivity and specificity of these telomere dynamics in predicting potentially-fatal VA was higher than the current gold-standard - left ventricular ejection fraction (AUC 0.82 versus 0.47). CONCLUSION: The load-of-short telomeres and telomerase activity had a significant association with ICD therapy (for VA) in ischaemic cardiomyopathy patients. These biomarkers should be tested in prospective studies to assess their clinical utility in predicting VA after myocardial infarction and guiding primary prevention ICD prescription.

Impedance measurements and connexin expression in human detrusor muscle from stable and unstable bladders.

UNLABELLED: Three of this month's Scientific Discovery papers highlight the importance of collaboration in delivering high quality scientific research. As scientific technology increases in power and cost, and specific areas of interest become more specialized, it is becoming more difficult to cover all aspects of a completeresearch story. Collaborating with other experts in the field or other fields, including industry, allows strong scientific proof to be generated for the hypothesis and aims. Building strong collaborative,inter-disciplinary, multi-institutional, international groups with academic and industrial partners is the way forward for all discovery. We look forward to publishing more of these collaborative papersin future issues of the BJU International. OBJECTIVES: To test the hypothesis that intercellular electrical coupling is altered in human detrusor smooth muscle from patients with unstable bladders. MATERIALS AND METHODS: Human detrusor biopsy samples were obtained from patients with stable and unstable bladders. Intracellular electrical impedance was measured with alternating current (20 Hz-300 kHz) across the ends of detrusor strips in an oil-gap, after correcting for extracellular space resistance. Gap junctions were identified by localization of connexins (Cx), specifically Cx45, Cx43 and Cx40 transcripts, using immunoconfocal microscopy. RESULTS: Total intracellular resistivity was greater in strips from unstable than from stable bladders (median 1246 vs 817 Omega.cm). The increase was attributed to an increase in junctional resistance; cytoplasmic resistance was unchanged. Cx43 was localized to a submucosal layer and to connective tissue; Cx40 label was confined to endothelial cells of blood vessels. Cx45 labelling was localized to detrusor bundles and appeared to be less marked in samples from unstable bladders. Semi-quantitative analysis of Northern blots showed that Cx45 expression in unstable was less than that in stable bladders. CONCLUSIONS: These data suggest that intercellular coupling is reduced in detrusor from unstable bladders. Cx45 was localized to the detrusor layer, with Cx 43 more evident in the suburothelial mucosa. Cx45 labelling was less intense in detrusor samples from unstable bladders. These results are consistent with reduced gap junction coupling in detrusor from unstable bladders.

Multiple connexins localized to individual gap-junctional plaques in human myometrial smooth muscle.

The synchronous contractions of the uterus in labour depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In the human myometrium, gap junctions are scarce in the non-pregnant uterus, but become abundant at term in preparation for labour. We have previously demonstrated that in the human myometrium at term, three different gap-junctional proteins are expressed, connexins 43, 45, and 40. These connexins are known to have distinctive functional capacities in in vitro expression systems but whether, in the human myometrium in vivo, they are co-assembled into the same gap junction or form different types of gap junction has previously been unclear. By applying triple immunogold labelling to sections of Lowicryl-embedded tissue for electron microscopy, together with complementary immunoconfocal microscopy, we demonstrate here that connexins 43, 45, and 40 are commonly present as mixtures within the same gap-junctional plaque. While all gap junctions contain connexin43, the relative signal for each connexin type varies between individual junctions. The presence within single gap-junctional plaques of three different connexins, each with the potential for conferring distinctive channel properties, suggests an inherent versatility for modulation of smooth muscle cell intercellular communication properties during human parturition.

Regional differentiation of desmin, connexin43, and connexin45 expression patterns in rat aortic smooth muscle.

The gap-junctional protein, connexin43, is differentially expressed in vascular smooth muscle cells (SMCs) according to phenotype. Previous studies suggest that desmin-negative SMCs are characterized by high levels of connexin43, whereas desmin-positive SMCs (of a more contractile phenotype) typically have low connexin43 levels. In this study, we examine systematically the inverse relationship between connexin43 and desmin in SMCs of defined regions of the rat aortic media and determine whether additional connexin isotypes are expressed and contribute to this relationship. Immunoconfocal microscopy demonstrated that (1) the inverse relationship between connexin43 and desmin expression holds true for the media of sequential aortic zones, with 1 exception, the ascending aorta, and (2) an additional vascular connexin, connexin45, is expressed by aortic SMCs. Examination of connexin43, connexin45, and desmin expression in sequential aortic zones reveals 3 SMC subpopulations. The first, predominating in the aortic arch and thoracic aorta, is desmin negative and contains high connexin43 levels; the second, predominating in the abdominal aorta and iliac artery, is desmin positive and contains low connexin43 levels; and the third, which is restricted to the ascending aorta, is desmin positive and expresses high connexin43 levels. Connexin45 levels are high in the ascending aorta but low in the other aortic segments. In para-aortic veins, a fourth SMC subpopulation appears, one that is desmin positive and contains connexin45 but not connexin43. These results demonstrate that a diversity of connexin expression patterns characterizes distinctive subpopulations of medial SMCs in situ with a potential to contribute to regional differentiation of vascular function.

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.

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.

Altered connexin expression in human congestive heart failure.

Congestive heart failure is associated with a high risk of life-threatening ventricular re-entrant arrhythmias. Down-regulation of the principal gap-junctional protein of the ventricular myocytes, connexin43, has previously been implicated in arrhythmia in ischaemic heart disease, but it is not known whether connexin43 is similarly reduced in heart failure due to idiopathic dilated cardiomyopathy, whether disease-related connexin43 down-regulation occurs at the level of transcription or translation, or whether the expression of other connexin isotypes is altered in congestive heart failure. We therefore investigated the expression of the four connexins expressed in the heart-connexins 43, 40, 45 and 37-at the mRNA and protein levels in explanted hearts from transplant patients with end-stage heart failure (NYHA class 4) by immunoconfocal analysis, and northern and western blotting. Connexin43 mRNA and protein were markedly downregulated in the left ventricle in end-stage heart failure due both to ischaemic cardiomyopathy and idiopathic dilated cardiomyopathy. Connexin43 content was spatially heterogeneous in the diseased ventricle. Connexin40 mRNA was increased in the ischaemic group, more so in the left ventricle than the right. This correlated with an increased depth of connexin40 protein expression in myocytes at the endocardial surface. Connexin45 mRNA and protein, present only in very low quantities, followed a similar trend to connexin43, while connexin37 (exclusively expressed in endothelium) showed no change. Our findings show that congestive heart failure is associated with significantly reduced levels of the principal gap junction protein, connexin43, in the left ventricle, potentially contributing to enhanced arrhythmogenicity and contractile dysfunction. This down-regulation is due predominantly to a reduced transcript steady-state level. Elevated connexin40 may represent a compensatory response that improves the spread of depolarization in the otherwise compromised ischaemic ventricle.

Comparison of connexin 43, 40 and 45 expression patterns in the developing human and mouse hearts.

The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.

Connexin45 is the first connexin to be expressed in the central conduction system of the mouse heart.

OBJECTIVE: To examine the spatial pattern of labelling for the gap junctional protein, connexin45, in relation to that of the other two cardiac connexins, connexin40 and connexin43, during the development of the central conduction system in mouse heart. ANIMALS AND METHODS: Hearts from Balb-c mice at stages from embryonic day (E) 12.5 to adult were frozen and sectioned. The sections were immunolabelled for connexins 45, 40 and 43 using fully characterized connexin-specific antibodies. Labelled sections were observed using confocal microscopy. Single, double and triple labelling were employed with sequential scanning to record images from multiple-labelled sections for the analysis of the spatial distribution of the three connexin types in relation to each other. RESULTS: High levels of connexin45 label were detected in specific regions within the developing mouse heart. These regions corresponded to the conus myocardium, developing interatrial septum and other developing conduction tissues of the heart. Connexin40 label was initially absent from these tissues but by E15.5 was present in the more distal regions of the conduction system. However, by E17.5, connexin45 and 40 labelling was similar to the pattern observed in the adult heart, with both connexins present in most regions of the conduction system, though they were not completely colocalized. Connexin43 label was not observed in the regions of high connexin45 labelling. CONCLUSIONS: These results show connexin45 to be the earliest detectable connexin in the central conduction system and to be the only connexin present throughout the whole conduction system. A distinct temporal pattern of connexin expression was also shown to occur during the development of the conduction tissues of the mouse heart.

Presence of the Kv1.5 K(+) channel in the sinoatrial node.

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.

Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border.

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.

Chamber-related differences in connexin expression in the human heart.

Electrical coupling in the heart is mediated by gap junctions, aggregates of cell-to-cell channels composed of connexins. The principal cardiac gap-junctional connexin, connexin43 (Cx43), is reduced in diseased human myocardium that is prone to arrhythmia. Three additional connexin isoforms, Cx40, Cx45 and Cx37, of distinctive functional capacities in vitro, are expressed in cardiovascular cells, but our knowledge of their expression patterns in the human heart is fragmentary. In the present study, we therefore applied Northern blotting, Western blotting and immunoconfocal microscopy to analyse and compare the expression of Cx43, Cx40, Cx37 and Cx45 mRNA and protein in the human left ventricle, right ventricle, left atrium and right atrium of the human heart. Cx43 was confirmed to be abundantly expressed at similar levels by myocytes in all four chambers. Cx40 levels varied between chambers in the order right atrium >left atrium >/= right ventricle approximately left ventricle. Cx37 (exclusively expressed in the endothelium) was expressed at similar overall levels in all chambers (as judged from Northern blots). Cx45 was detectable only at very low levels, with a trend toward higher levels in the atria than the ventricles in a pattern similar to Cx40. The results indicate that in humans, the ventricles and atria have distinctive connexin expression profiles, and that the atrial-type connexin profile is more pronounced in the right atrium than the left atrium. While the ventricular connexin expression pattern resembles that of other mammalian species, atrial connexin expression shows greater species variation. These differences contribute to the interpretative framework for examining the potential role of altered connexin expression in ventricular and atrial arrhythmia in the human heart.

Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy.

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.

Connexin45 (alpha 6) expression delineates an extended conduction system in the embryonic and mature rodent heart.

We previously demonstrated that alpha 6 (Cx45), one of the three connexins of the mammalian myocardium, is preferentially expressed in the peripheral portion of the ventricular conduction system in rats and mice. Here we report that alpha 6 is also prominently immunolocalized in the atrioventricular node and His bundle of these species. The distribution of immunolocalized alpha 6 reveals that the node and bundle form part of an extended central conductive network circumscribing the AV and outflow junctional regions of the fetal, and less continuously, the adult heart. Of the three cardiac connexins, alpha 6 is the isoform most continuously expressed by conduction tissues, and may thus account for the recently reported viability of the alpha 5 (Cx40) knockout mouse. It is concluded that alpha 6 expression is a defining feature of the heterogenous tissues comprising the atrioventricular conduction system of the rodent heart.

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.

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.

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.