Steven R. Coppen

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.

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.

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.

Direct Intramyocardial But Not Intracoronary Injection of Bone Marrow Cells Induces Ventricular Arrhythmias in a Rat Chronic Ischemic Heart Failure Model

Background— Therapeutic efficacy of bone marrow (BM) cell injection for treating ischemic chronic heart failure has not been established. In addition, experimental data are lacking on arrhythmia occurrence after BM cell injection. We hypothesized that therapeutic efficacy and arrhythmia occurrence induced by BM cell injection may be affected by the cell delivery route. Methods and Results— Three weeks after left coronary artery ligation, wild-type female rats were injected with 1×107 mononuclear BM cells derived from green fluorescent protein–transgenic male rats through either a direct intramyocardial or a retrograde intracoronary route. Both intramyocardial and intracoronary injection of BM cells demonstrated similar improvement in left ventricular ejection fraction measured by echocardiography and a similar graft size analyzed by real-time polymerase chain reaction for the Y chromosome–specific Sry gene. Noticeably, intramyocardial injection of BM cells induced frequent ventricular premature contractions (108±73 per hour at 7 days after BM cell injection), including multiform, consecutive ventricular premature contractions and ventricular tachycardia for the initial 14 days; intracoronary injection of BM cells and intramyocardial injection of phosphate-buffered saline rarely induced arrhythmias. Immunohistochemistry demonstrated that intramyocardial BM cell injection formed distinct cell clusters containing donor-derived cells and accumulated host-derived inflammatory cells in the infarct border zone, whereas intracoronary BM cell injection provided more homogeneous donor cell dissemination with less inflammation and without disrupting the native myocardial structure. Conclusions— BM cell injection is able to improve cardiac function in ischemic chronic heart failure but has a risk of arrhythmia occurrence when the intramyocardial route is used. Such arrhythmias may be prevented by using the intracoronary route.

Dynamics and mediators of acute graft attrition after myoblast transplantation to the heart

Survival and proliferation of skeletal myoblasts within the cardiac environment are crucial to the therapeutic efficacy of myoblast transplantation to the heart. We have analyzed the early dynamics of myoblasts implanted into the myocardium and investigated the mechanisms underlying graft attrition. At 10 min after implantation of [14C]thymidine‐labeled male myoblasts into female mice hearts, 14C measurement showed that 39.2 ± 3.0% of the grafted cells survived, and this steadily decreased to 16.0 ± 1.7% by 24 h and to 7.4 ± 0.9% by 72 h. PCR of male‐ specific Smcy gene calculated that the total (surviving plus proliferated) number of donor‐derived cells was 18.3 ± 1.6 and 23.3 ± 1.3% at 24 and 72 h, respectively, indicating that proliferation of the surviving cells began after 24 h. Acute inflammation became prominent by 24 h and was reduced by 72 h as indicated by myeloperoxidase activity and histological findings. Multiplex RT‐PCR revealed corresponding changes in IL‐1β, TGF‐β, IL‐6, and TNF‐α expression. Treatment with CuZn‐superoxide dismutase attenuated the initial rapid death and resulted in enhanced cell numbers afterward, giving a twofold increased total number at 72 h compared with the nontreatment. This effect was associated with reduced inflammatory response, suggesting a causative role for superoxide in the initial rapid graft death and subsequent inflammation. These data describe the early dynamics of myoblasts implanted into the myocardium and suggest that initial oxidative stress and following inflammatory response may be important mechanisms contributing to acute graft attrition, both of which could be potential therapeutic targets to improve the efficiency of cell transplantation to the heart.

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.

Gap Junction Localization and Connexin Expression in Cytochemically Identified Endothelial Cells of Arterial Tissue

Vascular endothelial cells interact with one another via gap junctions, but information on the precise connexin make-up of endothelial gap junctions in intact arterial tissue is limited. One factor contributing to this lack of information is that standard immunocytochemical methodologies applied to arterial sections do not readily permit unequivocal localization of connexin immunolabeling to endothelium. Here we introduce a method for multiple labeling with specific endothelial cell markers and one or more connexin-specific antibodies which overcomes this limitation. Applying this method to localize connexins 43, 40, and 37 by confocal microscopy, we show that the three connexin types have quite distinctive labeling patterns in different vessels. Whereas endothelial cells of rat aorta and coronary artery characteristically show extensive, prominent connexin40, and heterogeneous scattered connexin37, the former, unlike the latter, also has abundant connexin43. The relative lack of connexin43 in coronary artery endothelium was confirmed in both rat and human using three alternative antibodies. In the aorta, connexins43 and 40 commonly co-localize to the same junctional plaque. Even within a given type of endothelium, zonal variation in connexin expression was apparent. In rat endocardium, a zone just below the mitral valve region is marked by expression of greater quantities of connexin43 than surrounding areas. These results are consistent with the idea that differential expression of connexins may contribute to modulation of endothelial gap junction function in different segments and subzones of the arterial system.

Role of Interleukin-1β in Acute Inflammation and Graft Death After Cell Transplantation to the Heart

Background—Poor survival of grafted cells is a major factor hindering the therapeutic effect of cell transplantation; however, the causes of cell death remain unclear. We hypothesized that interleukin-1&bgr; (IL-1&bgr;) might play a role in the acute inflammatory response and graft death after cell transplantation and that inhibition of IL-1&bgr; might improve graft survival. Methods and Results—14C-labeled male skeletal muscle precursor cells were implanted into female mouse hearts by direct intramuscular injection. The amount of 14C-label provides an estimate of the surviving cell number, whereas the amount of male-specific Smcy gene measured by polymerase chain reaction indicates the total (surviving+proliferated) number of donor-derived cells. At 10 minutes after implantation, 44.8±2.4% of the grafted cells survived and this steadily decreased to 14.6±1.1% by 24 hours, and to 7.9±0.6% by 72 hours (n=6 in each point). Proliferation of the surviving cells, which began after 24 hours, resulted in an increase in the total cell number from 15.5±0.8% at 24 hours to 24.4±1.6% at 72 hours. Acute inflammation was prominent at 24 hours and was reduced by 72 hours, in parallel with IL-1&bgr; expression. Administration of anti–IL-1&bgr; antibody improved graft survival at both 24 (25.6±1.6%) and 72 hours (14.8±1.1%) and resulted in a 2-fold increase in the total cell number at 72 hours (45.8±2.4%). The effects of IL-1&bgr; inhibition corresponded with a reduced inflammatory response. Conclusion—IL-1&bgr; is involved in acute inflammation and graft death after direct intramyocardial cell transplantation. Targeted inhibition of IL-1&bgr; may be a useful strategy to improve graft survival.