ARTICLE |
Correspondence to: Cheng-Ho Tsai, Dept. of Cardiac Medicine, Mackay Memorial Hospital, 92, Sec 2, North Chung San Road, Taipei 10449, Taiwan. E-mail: cht7678@ms2.mmh.org.tw
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Summary |
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We investigated the phenotypic features of cardiomyocytes, including the gap junctions, in the myocardial sleeve of thoracic veins. Single cardiomyocytes, isolated from the canine pulmonary veins (PV) and superior vena cava (SVC) using digestive enzymes, were examined by immunoconfocal microscopy using antisera against connexin43 (Cx43), Cx40, and other cell markers. The results showed that isolated cardiomyocytes displayed rod shapes of various sizes, ranging from <50 µm to >200 µm in length, and all the cells expressed -actinin and vinculin. Gap junctions made of various amounts of Cx43 and Cx40 were found at the cell borders. These two connexins were extensively co-localized. Comparison between the thoracic veins showed that cells of the SVC contained more Cx43 gap junctions (total Cx43 gap junctions area per cell surface area, 4.0 ± 0.2% vs 1.5 ± 0.2%; p<0.01). In addition, for single-nucleus cells, those from the PV were longer (103.7 ± 3.6 vs 85.0 ± 3.1 µm; p<0.01) but narrower (14.4 ± 0.5 vs 16.9 ± 0.9 µm; p<0.01). In conclusion, canine thoracic veins contain cardiomyocytes with differences in shape and gap junctions, suggesting that the electrical conduction properties may be different between the thoracic veins. (J Histochem Cytochem 51:259266, 2003)
Key Words: gap junctions, connexins, myocardial sleeve, single cardiomyocytes
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Introduction |
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Extension of myocardial tissue from the atria onto the connecting thoracic veins, known as myocardial sleeve, accompanied by the existence of electrical activity in the venous wall, have been reported in mammals for a long time (
Gap junctions, made of molecules belonging to the connexin multigene family, are clusters of cell membrane aqueous channels, which in the heart provide a low-resistance pathway for electrical coupling between the adjacent cardiomyocytes. In mammals, Cx40, Cx43, and Cx45 are present in the atrial cardiomyocytes (
In this study we investigated the phenotypes, including the morphology and gap junctions, of the cardiomyocytes isolated from canine PV and SVC. Our results show that a marked heterogeneity exists in the cardiomyocytes of the venous wall.
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Materials and Methods |
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Isolation of Cardiomyocytes
Cardiomyocytes were isolated from five adult mongrel dogs (2025 kg). Animals were anesthetized with sodium pentobarbital (30 mg/kg IV). After thoracotomy the hearts were rapidly removed and immersed in normal Tyrode solution (containing 137 mM NaCl, 4 mM KCl, 15 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 2.7 mM CaCl2, and 11 mM dextrose) equilibrated with a gas mixture (97% O2 and 3% CO2) at room temperature (RT). The left atrium (LA), PV (cut at lines about 5 mm proximal to the LAPV junction and 20 mm distal to the ending of myocardial extension onto PV), and SVC (cut at lines about 5 mm proximal to the RASVC junction and 20 mm distal to the ending of myocardial extension onto SVC), were dissected out. The tube-like isolated PV and SVC were turned inside out and the proximal end and side branches were ligated, leaving the distal end connected to a Langendorff perfusion column via a polyethylene tubing. Perfusion (500 ml/hr) started with oxygenated normal Tyrode solution (37C) and was then replaced with Ca2+-free oxygenated Tyrode solution containing 300 U/ml collagenase (Type I; Sigma, St Louis, MO) and 0.5 U/ml protease (Sigma; Type XIV). After softening, the atrial end of the samples was cut off at about 5 mm. The remaining samples were cut into fine pieces and gently shaken in 510 ml of Ca2+-free oxygenated Tyrode solution to free single cardiomyocytes. LA myocytes were also isolated through the perfusion of collagenase and protease. The isolated cells were fixed with 2% paraformaldehyde (pH 7.2) for 10 min, washed with Ca2+-free Tyrode solution, spun (250 rpm, 5 min) onto silanized slides (DAKO; Glostrup, Denmark) using a cytospin (Life Science International; Cheshire, UK), and stored at 30C in PBS containing 0.02% NaCN3. The work conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and with the ROC Animal Protection Law (Scientific Application of Animals), 1998.
Immunocytochemistry
Antibodies.
The polyclonal antisera against Cx43 and Cx40 were produced in rabbits [designated Cx43 (R530) and Cx40 (R2), respectively], a chicken [Cx43(Chk)], and a guinea pig [Cx40 (GP8)] against the synthetic peptides corresponding to residues 314322 [for Cx43] or 254268 (for Cx40) of the cytoplasmic C-terminal tail of rat connexins. The polyclonal antiserum against Cx45 was produced in a rabbit [Cx45 (R1)] and a rat [Cx45 (Rat9)] against a synthetic peptide corresponding to residues 354367 of the cytoplasmic C-terminal tail of human Cx45. Affinity-purification and confirmation of the specificity by Western blotting and immunoconfocal microscopy were conducted as previously reported (-actinin and vinculin (Sigma). Smooth muscle cells were identified using smooth muscle
-actin (Dako). For secondary antibodies, donkey anti-rabbit, anti-chicken, or anti-guinea pig immunoglobulin conjugated to either CY3 or CY5, and anti-mouse to CY5 (Chemicon; Temecula, CA) were used. For single labeling, CY3-conjugated antibodies were used.
Immunolabeling of Connexins and Cell Markers.
For single labeling, cells were blocked in 0.5% BSA (15 min) and incubated with the anti-Cx43 [1:100 for Cx43 (R530); 1:50 for Cx43 (Chk)], anti-Cx40 [1:200 for both Cx40 (R2) and Cx40 (GP8)], anti-Cx45 (1:50 for both Cx45 (R1) and Cx45 (Rat9)], anti--actinin (1:200), anti-vinculin (1:200), or anti-smooth muscle
-actin (1:50) at 37C for 2 hr. The samples were then treated with CY3-conjugated secondary antibody (1:500, RT, 1 hr). In experiments in which each of the connexins and
-actinin were simultaneously localized in the same samples, incubation was with a mixture of one of the polyclonal antisera (the same dilution as in the single-labeling experiment) plus anti-
-actinin (1:200), followed by incubation with a mixture of a CY3-conjugated antibody (for labeling connexin) and anti-mouse CY5 (for
-actinin). Double labeling of Cx40 and Cx43 was conducted using Cx40 (GP8) and Cx43 (R530), followed by incubation with a mixture of anti-guinea pig CY3 and anti-rabbit CY5. Between each step, the slides were thoroughly washed with PBS. Finally the cells were mounted. All experiments included cryosections of dog left atrium, fixed by methanol at -20C, and the isolated LA cells as positive controls and omission of primary antibody as negative controls. In parallel, cells were counterstained with bisbenzamide (1 µg/ml; Sigma) for 15 min to visualize their nuclei.
Confocal Laser Scanning Microscopy and Image Analysis
Stained samples were examined by confocal laser scanning microscopy using a Leica TCS SP equipped with argon/krypton and UV laser with the appropriate filter spectra adjusted for the detection of CY3, CY5, and bisbenzamide fluorescence. Consecutive optical sections taken at 0.5-µm intervals through the full thickness of cells were collected and transformed into projection views. The images from cells of double labeling were taken using sequential dual-channel scanning. Quantification of the cell size and Cx43-labeled spots was conducted using QWIN image analysis software (Leica; Heidelberg, Germany). The number of Cx43 gap junctions in the cardiomyocytes, as detected by the Cx43 (R530), is expressed as percentage of the cell surface area. Data, expressed as mean values (± SE), were compared statistically by t-test. A p value less than 0.05 was considered significant.
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Results |
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Characterization of Anti-connexin Antisera
Western blotting analysis showed that both anti-Cx43 antibodies recognized closely situated bands of about 43 kD in a cell membrane preparation of HeLa-43. Similarly, both anti-Cx40 antibodies recognized a single band of about 40 kD in the HeLa-40 cells, and both anti-Cx45 antibodies recognized a single band of about 45 kD in the HeLa-45 cells (Fig 1). For each group of experiments, the bands of Cx43, Cx40, and Cx45, each of which was absent in the other connexin-expressing HeLa cells and the wild-type cells (HeLa-W), were markedly inhibited by preincubation of the antibody with the peptide to which it was raised.
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Immunoconfocal microscopy of the HeLa cells incubated with each of the anti-connexin antisera demonstrated that, in each corresponding connexin-expressing cell, distinct punctate labeling existed at cellcell borders, typical of gap junctions. The labeling was abolished by peptide inhibition (Fig 2). No positive signal was apparent when the antibody was applied on the other HeLa transfectants and HeLa-W.
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Immunoconfocal Examination of Single Cardiomyocytes
Microscopic examination showed that cells with various morphological features were freed from the venous wall after treatment with the digestive enzymes. Among the isolated cells, those of rod shapes were generally larger compared to cells of other shapes. Immunolabeling showed that the rod-shaped cells isolated from the PV and SVC bore cross-striations positive for -actinin and vinculin (Fig 3). In general, the striation signals of
-actinin were much more conspicuous and better organized compared to those of vinculin, which also appeared at portions of cell borders. Such staining patterns of
-actinin and vinculin in the rod-shaped cells isolated from the venous wall were identical to the patterns seen in the LA working cardiomyocytes (Fig 3). In addition, no signal of smooth muscle
-actin was detected in any rod-shaped cells, thus confirming the cells with such features to be cardiomyocytes. On the other hand, all the striated cells were typically rod-shaped. Regarding the size, although cells as long as more than 200 µm and as short as less than 50 µm were observed, cells of extreme sizes were less common. Determinations of cell size should consider the possibility that cells in close attachment can be mistaken for single cells, especially for those possessing more than one nucleus, which appeared in about one third of the cells (Fig 3). Therefore, measurements were conducted on cells containing a single nucleus as unequivocally visualized in cells stained with bisbenzamide. Analysis of the cell size showed that the cardiomyocytes isolated from the PV were slightly but significantly longer and narrower than those from the SVC (p<0.01; Fig 4).
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Cx43-labeled spots were found clustered at domains of the cell borders (Fig 5), typical for the location of gap junctions. For the majority of cells, both ends of the long axis contained Cx43-labeled spots, although the amount varied widely among individual cells. Apart from the ends, Cx43 labeled spots were also detected at the lateral borders. Both Cx43 (R530) and Cx43 (Chk) antisera gave the same findings. For Cx40, detection with either Cx40 (R2) or Cx40 (GP8) showed a distribution pattern similar to that of Cx43 (Fig 5). Double labeling for both Cx40 and Cx43 demonstrated that more than 90% of the labeled spots were positive for both connexins.
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Comparison between the thoracic veins showed that in the PV cardiomyocytes the number of Cx43-labeled spots was less (10.1 ± 0.9 per cell) and the size of the spots was smaller (0.5 ± 0.1 µm2), compared to the SVC cells (23.7 ± 1.4 per cell and 0.9 ± 0.1 µm2, respectively; both p<0.01). Considering the variation in cell size and that in some cases, as mentioned above, closely attached cells could not be distinguished from single cells, to overcome such interferences with interpretation, we present the data as the value of the total area of Cx43-labeled spots divided by the cell surface area for each cell or each group of closely attached cells. The result showed that a significant difference exists between the PV and the SVC (p<0.01; Fig 6).
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For Cx45, neither antisera Cx45 (R1) nor Cx45 (Rat9) gave signals in the isolated cardiomyocytes.
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Discussion |
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The present study demonstrates that canine cardio-myocytes in the myocardial sleeve of the PV and SVC and those in the LA share a similar shape and immunocytochemically identified cross-striations. In addition, although situated in the venous wall, the cardiomyocytes differ phenotypically from the adjacent vascular smooth muscle cells. Furthermore, regarding the size of cardiomyocytes and the expression of gap junctions in the cells, a marked variation exists between the SVC and the PV. All these findings are based on the examination of cardiomyocytes freed from the tissues by enzyme digestion and by the application of a panel of affinity-purified anti-connexin antisera.
It should be noted that, in the present study, the two anti-Cx45 antisera, Cx45 (R1) and Cx45 (Rat9), give no signals in the isolated cells. Because the two anti-Cx45 are well characterized by Western blotting and immunocytochemistry using the connexin-expressing HeLa cells, one possible explanation for the absence of Cx45 signal in the isolated cells is the fixation method. The use of paraformaldehyde for the single cardiomyocytes in the present study may reduce the signal of Cx45, the expression level of which is known to be low in cardiac muscle (
The finding in the present study that cardiomyocytes isolated from the venous wall have similar expression patterns of Cx43 and Cx40 is consistent with our recent report that the majority of cardiomyocyte gap junctions in canine SVC contain both Cx40 and Cx43 (-actinin (indicative of an abundant amount of contractile elements in the cytoplasm) in all rod-shaped cells provides evidence that the cardiomyocytes in the venous wall are structurally different from the nodal cells, in which contractile elements are few (
One important finding from the present study is that differences exist between the PV and the SVC regarding the size of the cardiomyocytes and their expression of Cx43 gap junctions. Previous reports have shown that expression of connexins in the contractile myocardium is chamber-specific (
In conclusion, the canine PV and SVC contain cardiomyocytes with various features. The differential expression of Cx43 gap junctions and differences in cell size in the myocardial sleeve suggest that individual thoracic veins possess distinct electrical conduction properties.
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Acknowledgments |
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Supported by grants NSC-90-2314-B-195-018 from the National Science Council, Taiwan, and MMH-9112 from the Medical Research Department of the Mackay Memorial Hospital, Taiwan.
We thank Dr Fung J. Lin for assistance with statistics.
Received for publication June 3, 2002; accepted October 2, 2002.
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