Biomedical subjects

N J Severs

Publications and source records attributed to N J Severs.

At least 37 records · Page 2Linked to original sources

PubMed2000

Species-specific difference in distribution of voltage-gated L-type Ca(2+) channels of cardiac myocytes.

Ca(2+) influx via sarcolemmal voltage-dependent Ca(2+) channels (L-type Ca(2+) channels) is the fundamental step in excitation-contraction (E-C) coupling in cardiac myocytes. Physiological and pharmacological studies reveal species-specific differences in E-C coupling resulting from a difference in the contribution of Ca(2+) influx and intracellular Ca(2+) release to activation of contraction. We investigated the distribution of L-type Ca(2+) channels in isolated cardiac myocytes from rabbit and rat ventricle by correlative immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat myocytes, labeling appeared more intense in T tubules than in the surface sarcolemma. Immunogold electron microscopy extended these findings, showing that the number of gold particles in the surface plasma membrane was significantly higher in rabbit than rat myocytes. In rabbit myocyte plasma membrane, the gold particles were distributed as clusters in both regions that were associated with junctional sarcoplasmic reticulum and those that were not. The findings are consistent with the idea that influx of Ca(2+) via surface sarcolemmal Ca(2+) channels contributes to intracellular Ca(2+) to a greater degree in rabbit than in rat myocytes.

Animals↗

PubMed2000

Expression, localization, and activity of tissue factor pathway inhibitor in normal and atherosclerotic human vessels.

Tissue factor (TF) pathway inhibitor (TFPI) is the major downregulator of the procoagulant activity of the TF-factor VIIa (FVIIa) complex (TF. FVII). The active TF present in the atherosclerotic vessel wall is proposed to be responsible for the major complication of primary atherosclerosis, namely, acute thrombosis after plaque rupture, but our knowledge of the sites of TFPI expression in relation to TF remains fragmentary. The aim of this study was to investigate the expression, localization, and activity of TFPI and its relation to the activity and distribution of TF in the normal and atherosclerotic vessel wall. We applied a novel approach in which serial cross sections of human vascular segments were used to perform a complete set of assays: immunolabeling for TFPI and/or TF, in situ hybridization for the expression of TFPI mRNA, ELISA for the determination of TFPI antigen, and functional assay for the activity of TFPI and TF. In healthy vessels, TFPI protein and mRNA are present in luminal and microvascular endothelial cells (ECs) and in the medial smooth muscle cells (SMCs). In atherosclerotic vessels, TFPI protein and mRNA frequently colocalized with TF in ECs overlying the plaque and in microvessels, as well as in the medial and neointimal SMCs, and in macrophages and T cells in areas surrounding the necrotic core. At the ultrastructural level, immunogold electron microscopy confirmed the localization of TFPI in ECs, macrophages/foam cells, and SMCs. In ECs and SMCs, the gold particles decorated the plasmalemma proper and the caveolae. ELISA on cross sections revealed that atherosclerotic tissues contain more TFPI than do the healthy vessels. TFPI was functionally active against TF. FVIIa-induced coagulation, and its activity was higher in those tissues that display less TF. The largest amount of TFPI and TF were detected in complicated arterial plaques. By immunofluorescence, TFPI colocalized with platelet- and fibrin-rich areas within the organized thrombi. Atherosclerotic vessel sections promote activation of factor X, which is dependent on the presence of TF and enhanced by preincubation of the sections with anti-TFPI IgG. Taken altogether, our results suggest that TFPI is largely expressed in the normal vessel wall and enhanced in the atherosclerotic vessel, in a manner suggesting a significant role of TFPI in the regulation of TF activity.

Arteriosclerosis↗

PubMed2000

Multiple connexin expression in regenerating arterial endothelial gap junctions.

Endothelial cells form gap junctions that, according to vessel type, may be composed of up to 3 types of connexin, connexin37, connexin40, and connexin43. Although changes in connexin expression have been linked to growth and injury in cultured endothelial cells, information on connexin expression in regenerating endothelium in situ is lacking. We investigated gap junction distribution and expression of all 3 endothelial connexins during healing in rat carotid artery after denudation injury. En face viewing of the vascular luminal surface by means of immunoconfocal microscopy was used to examine the spatial and temporal expression pattern of the endothelial connexins. Gap junction spots labeled by specific antisera against connexin37, connexin40, and connexin43 were quantified 7, 14, and 28 days after injury, and the relations among the connexins were examined by using colocalization analysis. Complementary electron microscopy was also conducted. After injury, the regenerating endothelium initially expressed small, sparse gap junctions, the numbers of which progressively increased to values equivalent to those of controls. Although connexin40 gap-junctional spot size and area returned to uninjured levels by 28 days after injury, connexin37 and connexin43 spot size and area exceeded those of the uninjured artery (P<0.05). Double-label analysis showed that even though colocalization of connexins to the same gap-junctional spot is a common feature, the extent of colocalization was time dependent (>80% in the intact artery at postinjury day 28 and <70% at postinjury days 7 and 14, P<0.01). We conclude that distinct alterations in expression of the 3 connexins are associated with regeneration of the arterial endothelium in situ, implying different intercellular communication requirements during the various phases of the healing process.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

Age-related alteration of gap junction distribution and connexin expression in rat aortic endothelium.

We investigated endothelial gap junctions and their three component connexins, connexin37 (Cx37), Cx40, and Cx43, during growth and senescence in rat aorta by en face immunoconfocal microscopy and electron microscopy. Gap junction spots labeled by specific antisera against Cx37, Cx40, and Cx43 were quantified at 1 day, 7 days, 28 days, 16 months, and > or =20 months of age, and the relationship between the connexins was examined by co-localization analysis. At birth, all three connexins were abundantly expressed; the number and total area of connexin spots then declined within 1 week (p<0.05 for each connexin). From 1 week, each connexin showed a distinct temporal expression pattern. Whereas Cx43 signal decreased progressively, Cx37 signal fluctuated in a downward trend. By contrast, Cx40 maintained an abundant level until > or =20 months of age (> or =20 months vs. 28 days, p<0.05 for number and total connexin signal area). These patterns were associated with changes in endothelial cell morphology. Double-label analysis showed that the extent of co-localization of connexins to the same gap junctional spot was age-dependent [>70% at birth and 28 days old; <70% at later stages (p<0.05)]. We conclude that expression of the three connexins in aortic endothelium is age-related, implying specific intercellular communication requirements during different stages after birth.

Aging↗

PubMed2000

Dephosphorylation agents depress gap junctional communication between rat cardiac cells without modifying the Connexin43 phosphorylation degree.

The functional state of gap junctional channels and the phosphorylation status of Connexine43 (Cx43), the major gap junctional protein in rat heart, were evaluated in primary cultures of neonatal rat cardiomyocytes. H7, able to inhibit a range of serine/threonine protein kinases, progressively reduced gap junctional conductance to approximately 13% of its initial value within 10 min except when protein phosphatase inhibitors were also present. The dephosphorylating agent 2,3-Butanedione monoxime (BDM) produced both a quick and reversible interruption of cell-to-cell communication as well as a parallel slow inhibition of junctional currents. The introduction of a non-hydrolysable ATP analogue (ATPgammaS) in the cytosol delayed the second component, suggesting that it was the consequence of protein dephosphorylation. Western blot analysis reveals 2 forms of Cx43 with different electrophoretic mobilities which correspond to its known phosphorylated and dephosphorylated forms. After exposure of the cells to H7 (1 mmol/l, 1h) or BDM (15 mmol/l, 15 min), no modification in the level of Cx43 phosphorylation was observed. The lack of direct correlation between the inhibition of cell-to-cell communication and changes in the phosphorylation status of Cx43 suggest that the functional state of junctional channels might rather be determined by regulatory proteins associated to Cx43.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Cardiovascular disease.

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 function between cells of the arterial wall. Altered gap junctional coupling is implicated in the genesis of arrhythmia, a major cause of death in heart disease. Two abnormalities in myocardial gap junctions distribution at the border zone of infarcts and reduced levels of connexin43 (Cx43; alpha 1)--may lead to heterogeneous wavefront propagation and lowered conduction velocity, key factors that precipitate arrhythmia. In the major arteries, endothelial cells express Cx40 (alpha 5) and Cx37 (alpha 4) and, in some instances, also Cx43, whereas underlying medial smooth muscle cells express only Cx43. Increased Cx43 expression between medial smooth muscle cells is intimately linked to phenotypic transformation to the synthetic state in both early human coronary phenotypic transformation to the synthetic state in both early human coronary atherosclerosis, and in the response of the arterial wall to injury. The accumulating evidence suggests that gap junctions in both their guises--as pathways for cell-to-cell signalling in the vessel wall and as pathways for impulse conduction in the heart--may have key roles in the initial pathogenesis and eventual clinical manifestation of human cardiovascular disease.

Amino Acid Sequence↗

PubMed1999

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.

Blotting, Northern↗

PubMed1999

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells.

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle alpha-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3-0.5% of cells were found to be desmin-positive, and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.

Actins↗

PubMed1999

The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium.

Gene transfer to the respiratory epithelium is currently suboptimal and may be helped by the identification of limiting biological barriers. We have, therefore, developed an ex vivo model which retains many of the characteristics of in vivo native airways including mucociliary clearance, mucus coverage and an intact cellular structure. Using this model we have demonstrated several barriers to gene transfer. Liposome-mediated gene transfer was inhibited by normal mucus, with removal of this layer increasing expression approximately 25-fold. In addition both liposome and adenovirus were inhibited by CF sputum. The apical membrane represented a significant barrier to both agents. Adenovirus-mediated expression could be significantly augmented by increasing contact time or by pre-treatment of tissues with a nominally calcium-free medium. The presence of these extracellular and plasma membrane barriers appeared to be the key parameters responsible for the approximately three log difference in gene expression found in vitro compared with our ex vivo model. Cytoskeletal elements and the cell cycle also influenced in vitro gene transfer, and represent further barriers which need to be overcome.

Adenoviridae↗

PubMed1999

Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteries of rabbit.

Lipoproteins play a major role in cardiovascular disease and atherosclerosis. In the vascular wall, they strongly influence the organization of extracellular matrix. The present study set out to investigate the changes in the extracellular matrix of the vessel wall induced by atherogenic diet, focusing on type VIII collagen, a vascular collagen that has not previously been investigated in detail. The influence of cholesterol diet on the expression, distribution, and deposition of type VIII collagen was examined in carotid arteries of New Zealand White rabbits. Carotid arteries of rabbits receiving diet supplemented with 1% cholesterol for 6 weeks and those on the same regimen followed by normal chow for 1 day, 10 days, 5 weeks, and 12 weeks were studied and compared with controls not exposed to the cholesterol diet. Carotid arteries of normocholesterolemic rabbits contained type VIII collagen-expressing cells in all layers, with focal accumulations of expressing cells in the subendothelial areas, the outer medial zone, and the adventitia. In response to cholesterol diet, type VIII collagen synthesis was reduced in media and adventitia and the distribution patterns changed. Expressing cells were found predominantly in the endothelium, and type VIII collagen accumulated in the intimal space. Immunogold labeling for electron microscopy revealed that type VIII collagen in the intima is associated with microfibrils extending from the internal elastic lamina. Withdrawal of cholesterol resulted in reestablishment of the normal distribution pattern. Northern and Western blot analyses supported the immunoconfocal and in situ hybridization data, demonstrating decreased type VIII collagen expression in response to cholesterol diet and progressive recovery to normal levels with time after withdrawal of cholesterol. Our study demonstrates that type VIII collagen is modulated in the presence of cholesterol. The data indicate that type VIII collagen is specifically remodeled during early experimental atherosclerosis, implying a role for this extracellular matrix component in neointimal growth.

Animals↗

PubMed1999

Differential expression of connexin43 and desmin defines two subpopulations of medial smooth muscle cells in the human internal mammary artery.

Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle alpha-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.

Aged↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

Mechanisms of acute eosinophil mobilization from the bone marrow stimulated by interleukin 5: the role of specific adhesion molecules and phosphatidylinositol 3-kinase.

Mobilization of bone marrow eosinophils is a critical early step in their trafficking to the lung during allergic inflammatory reactions. We have shown previously that the cytokine interleukin (IL)-5, generated during an allergic inflammatory reaction in the guinea pig, acts systemically to mobilize eosinophils from the bone marrow. Here, we have investigated the mechanisms underlying this release process. Examination by light and electron microscopy revealed the rapid migration of eosinophils from the hematopoietic compartment and across the bone marrow sinus endothelium in response to IL-5. Using an in situ perfusion system of the guinea pig hind limb, we showed that IL-5 stimulated a dose-dependent selective release of eosinophils from the bone marrow. Eosinophils released from the bone marrow in response to IL-5 expressed increased levels of beta2 integrin and a decrease in L-selectin, but no change in alpha4 integrin levels. A beta2 integrin-blocking antibody markedly inhibited the mobilization of eosinophils from the bone marrow stimulated by IL-5. In contrast, an alpha4 integrin blocking antibody increased the rate of eosinophil mobilization induced by IL-5. In vitro we demonstrated that IL-5 stimulates the selective chemokinesis of bone marrow eosinophils, a process markedly inhibited by two structurally distinct inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002. Wortmannin was also shown to block eosinophil release induced by IL-5 in the perfused bone marrow system. The parallel observations on the bone marrow eosinophil release process and responses in isolated eosinophils in vitro suggest that eosinophil chemokinesis is the driving force for release in vivo and that this release process is regulated by alpha4 and beta2 integrins acting in opposite directions.

Androstadienes↗

PubMed1998

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.

Adult↗

PubMed1998

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.

Animals↗