1 Biology Department, Goddard Laboratories, University of Pennsylvania, Philadelphia, PA, USA
2 Division of Neonatology, Department of Pediatrics, Duke University, Durham, NC, USA
3 Laboratory of Developmental Biology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
4 Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC, USA
5 Department of Medicine, University of Pennsylvania Medical School, Philadelphia, PA, USA
*Author for correspondence (e-mail: clo{at}nhlbi.nih.gov)
Accepted 24 January 2002
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SUMMARY |
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Key words: Connexin43, Gap junctions, Coronary artery, Cell proliferation, Cell migration, Proepicardial cell
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INTRODUCTION |
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The connexin gene known as connexin43 or 1 connexin (referred to as Cx43
1; Gja1 Mouse Genome Informatics) plays a crucial role in cardiac development, as Cx43
1 knockout mice die shortly after birth due to pulmonary outflow obstruction (Reaume et al., 1995
). Typically, the Cx43
1 knockout mouse heart exhibits two prominent pouches at the base of the pulmonary outflow tract, a region known as the infundibulum of the heart. Studies using transgenic mouse models to manipulate Cx43
1 function indicate that the outflow obstruction probably involves the perturbation of cardiac neural crest cell migration. Thus, when Cx43
1-mediated gap junction communication in cardiac neural crest cells was up- or downregulated in the CMV43 (overexpression) and FC (dominant negative) transgenic mice, respectively, right ventricular cardiac defects associated with outflow obstruction were observed (Ewart et al., 1997
; Sullivan et al., 1998
). In the CMV43 transgenic mice, an increase in gap junction communication was associated with an increase in the rate of neural crest cell migration and an elevation in the abundance of neural crest cells in the heart outflow tract (Huang et al., 1998a
). By contrast, in the FC transgenic and Cx43
1 knockout mice, a reduction in coupling was associated with a reduction in cell migration rate and a lower abundance of neural crest cells in the heart (Huang et al., 1998a
). Although the FC and CMV43 transgenic mice both exhibited defects involving the heart outflow tract, the same region affected in the Cx43
1 knockout mice, neither animal model exhibited the pouches typically seen in Cx43
1 knockout mouse heart (Ewart et al., 1997
; Huang et al., 1998b
; Sullivan et al., 1998
). This was surprising, as the cardiac neural crest cells from the FC transgenic mice showed similar reductions in gap junction coupling and cell locomotion as that found in the Cx43
1 knockout mice (Huang et al., 1999a
; Xu et al., 2002
). Our present study suggests that this discrepancy may reflect the fact that formation of the pouches involves not only the cardiac neural crest cells, but also another extra-cardiac migratory cell population, the proepicardial cells.
The proepicardial cells originate from the proepicardial organ (PEO), a grape-like cluster of cells derived from the hepatic primordium and found at E9.0-E9.5 at the ventral region of the sinus venosus (Viragh and Challice, 1981; Viragh et al., 1993
). These cells rapidly migrate over the primitive tubular heart to form the epicardium, and subsequently, cells from the epicardium undergo an epithelial-mesenchymal transformation and infiltrate the heart, giving rise to the vascular smooth muscle cells of the coronary arteries, and also fibroblasts in the heart (Dettman et al., 1998
; Mikawa and Gourdie, 1996
; Vrancken-Peeters et al., 1997
; Vrancken-Peeters et al., 1999
). Although proepicardially derived cells also have been suggested to contribute endothelial cells in the heart, this possibility remains unresolved (Poelman et al., 1993
; Mikawa and Gourdie, 1996
). When the epicardium is not maintained or does not form, such as in the
4 integrin and VCAM-1 knoockout mice, coronary arteries fail to develop (Kwee et al., 1995
; Yang et al., 1996
). The phenotype of these knockout mice strongly suggests that cell-cell adhesion and cell-matrix interactions play an important role in the invasion and subsequent deployment of the epicardial cells.
Our present study suggests that cell-cell interactions mediated by Cx431 gap junctions also play an important role in the deployment of the proepicardial cells. The cardiac defects in the Cx43
1 knockout mice are more subtle than that seen in the
4 integrin or VCAM-1 knockout mice, as formation of the epicardium does not appear to be affected by the loss of Cx43
1 function. In the Cx43
1 knockout mouse heart, coronary arteries form but are abnormally patterned. Histological analysis suggested that the coronary artery anomalies and the infundibular pouches involve the abnormal deployment of the proepicardially derived vascular smooth muscle cells. Analysis of the proepicardially derived cells revealed that they are functionally well coupled, and they also have an abundance of Cx43
1 gap junction contacts. In the Cx43
1-deficient proepicardial cells, coupling is greatly reduced, but in contrast to Cx43
1-deficient neural crest cells, this was associated with an elevation in the rate of cell locomotion and cell proliferation. These and other findings suggest that Cx43
1 gap junctions play an essential role in normal coronary artery patterning. We propose that this may involve a dual role for Cx43
1 gap junctions in modulating the deployment of the proepicardial and neural crest cells. We further discuss the possible involvement of Cx43
1 in human congenital cardiovascular anomalies and cardiovascular disease.
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MATERIALS AND METHODS |
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Immunohistochemical analysis
Hearts from mouse fetuses and neonates were fixed in 4% paraformaldehyde (PF) overnight, and after dehydration in a graded series of alcohol, they were paraffin embedded, cut into serial sections, de-paraffinized and subjected to immunostaining as previously described (Waldo et al., 1999; Epstein et al., 2000
). Smooth muscle cells were identified using antibodies to a smooth muscle cytoskeletal protein, SM22
(Zhang et al., 2001
) or smooth muscle myosin heavy chain (Biomedical Technologies, Stoughton, MA). Anti-proliferating cell nuclear antigen (PCNA) antibody (Zymed Laboratories, San Francisco, CA) were used to examine cell proliferation, and to examine apoptosis, a TUNEL assay was carried out using the APOPTAG kit from Intergen Company (Purchase, NY).
For immunohistochemical analysis of proepicardial organ (PEO) explant cultures, fixation and subsequent immunostaining were carried out as previously described (Li and Nagy, 2000). For whole-mount Cx43
1 immunostaining, E9.5 wild-type mouse embryos were incubated with primary and secondary antibodies for 48 hours and 20 hours, respectively. After washing, the embryos were dehydrated, plastic embedded, cut into 5 µm sections and mounted on slides using a Vectorshield mounting medium (Vector Laboratory, Inc.).
Proepicardial organ and heart explants
PEO explants were obtained from E9.0-E9.5 mouse embryos, with E0.5 designated as the morning a vaginal plug was found. Only the top part of the protruding PEO cluster was removed, in order to minimize contamination from hepatic primordium. The explants were plated on glass coverslips coated with Type I rat tail collagen and maintained at 37°C in Dulbeccos modified essential medium (high glucose) supplemented with 10% fetal bovine serum, unless otherwise noted. To obtain epicardial explants, E11.5 mouse hearts were similarly plated onto collagen coated coverslips. A polyclonal anti-cytokeratin antibody (Dako) was used to examine the identity and purity of cultured PEO explants. For the study of proepicardial cell differentiation, a cocktail of two monoclonal antibodies against smooth muscle calponin was used (clone CP93 and hCP, Sigma).
Analysis of dye coupling and cell motility in the PEO explants
To monitor dye coupling, coverslips containing 24 hours PEO explants were transferred into phosphate buffered L-15 medium (Sigma) supplemented with 10% fetal bovine serum, and maintained at 37°C on a heated stage of a Leitz Diavert microscope equipped for epifluorescent illumination. Single proepicardial cells were impaled with a glass microelectrode filled with 5% carboxyfluorescein and iontophoretic dye injection was carried out for 2 minutes using 1 nA hyperpolarizing current pulses of 0.5 second duration at 1 Hz. The number of dye-filled cells at the end of 2 minutes of iontophoresis was recorded. To analyze proepicardial cell motility, images of the PEO explant cultures were recorded at 5 minute intervals for 24 hours. Time lapse movies generated in this manner were used to trace the migration paths of individual proepicardial cells, and from which the speed and directionality of cell movement were determined using the Dynamic Imaging Analysis Software (Solltech, Oakdale, IA).
Cell proliferation analysis with bromodeoxyuridine (BrdU) labeling
To examine the proliferation rate of proepicardial cells, 24 hours explant cultures were incubated with 10 µM BrdU for 1 hour, fixed in 4% PF for 30 minutes and immunostained with an anti-BrdU antibody (Calbiochem, San Diego, CA) and further counterstained using Hematoxylin. Images of the stained PEO explant cultures were captured digitally, and the labeled nuclei were quantitated using the Openlab software (Improvision, Coventry, UK).
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RESULTS |
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Cx431 gap junctions in the mouse PEO and fetal epicardium
To determine if Cx431 is expressed in the PEO, we carried out whole-mount immunostaining of E9.5 mouse embryos using a rabbit polyclonal Cx43
1 antibody. Semi-thin plastic sections of the immunostained embryos were examined by darkfield immunofluorescence microscopy. This analysis revealed that the PEO consists of mesenchymal cells with a dendritic morphology (Fig. 5A). They exhibit punctate cell surface immunolabeling typical of gap junctions. In some regions, particularly along cell processes, very long profiles of Cx43
1 immunostaining were observed (Fig. 5B,C). As the PEO is a transient structure that quickly delaminates to form the epicardium, we further examined the epicardium of E12.5 mouse embryos, and found the continued expression of Cx43
1 gap junctions in the epicardial cells (purple in Fig. 6A,B). A similar analysis of the Cx43
1 knockout mouse heart showed that the epicardium is present and exhibits normal tissue morphology, including expression of cytokeratin (blue in Fig. 6C,D), a marker specific for epicardial cells (Dettman et al., 1998
).
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We observed that the migration of proepicardial cells usually began with the extension of lamellipodia, and occasionally filopodia were seen. After emergence from the explants, the proepicardial cells migrated as a coherent sheet, with cells at the periphery maintaining their leading position (Fig. 9). A significant elevation in the speed of cell locomotion was observed in both the heterozygous and homozygous Cx431 knockout proepicardial cells. It is important to note that the same increases in speed were observed in the heterozygous and homozygous Cx43
1 knockout proepicardial cells, whether cell motility was examined over the first 4 hours or the entire 20 hour interval of the experiment (Table 2).
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DISCUSSION |
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It is important to consider these findings in light of previous work showing that formation of the coronary arteries is also dependent on neural crest cells (Bartelings et al., 1993; Hood and Rosenquist, 1992
; Hyer et al., 1999
; Waldo et al., 1994
). Unlike the proepicardial cells, neural crest cells do not contribute structurally to the coronary arteries and thus their role is presumably a regulatory one. In fact, our earlier studies had indicated the involvement of neural crest perturbations in the cardiac anomalies of the Cx43
1 knockout mouse (Ewart et al., 1997
; Huang et al., 1998a
; Huang et al., 1998b
). We observed neural crest cells migrate into the outflow tract of the Cx43
1 knockout mouse heart, but in reduced abundance. In contrast to our findings with the proepicardial cells, significant alteration in cell locomotory behavior was observed only in the homozygous knockout neural crest cells (Xu et al., 2002
). Together, these findings suggest that the coronary artery anomalies in the Cx43
1 knockout mouse cannot be solely dependent on neural crest perturbations, but rather may arise from the combined effects of perturbations involving the neural crest and proepicardial cells.
Neural crest and proepicardial cells and the regulation of coronary artery development
Given that neural crest and proepicardial cells are both found at the base of the heart where the coronary artery stems form by tunneling through the base of the aorta, perhaps signaling mediated through interactions between the neural crest and proepicardial cells may play a role in modulating the initial patterning of coronary arteries. The altered motility of the proepicardial and neural crest cells in the Cx431 knockout mouse potentially could lead to an excess of proepicardial cells and a reduction in neural crest cells. Such an imbalance between the two cell populations may alter cell signaling that is important in the patterning of coronary arteries. This signaling could involve gap junction-mediated communication between crest and proepicardial cells, or paracrine signaling not requiring direct cell-cell contact. It is also possible that the role of neural crest may be mediated via secondary interactions involving the myocardium or endocardium. Various hypotheses have been put forward regarding the role of neural crest cells in coronary artery development, including ensuring the survival of persisting coronary arteries by laying down the cardiac ganglia at the base of the heart (Waldo et al., 1994
). Another possibility is that neural crest cells involved in peripheral innervation in the heart may provide signals that can modulate proepicardial cell deployment and thus help pattern coronary artery development. Regardless of the precise mechanism of proepicardial and neural crest interactions, our findings suggest that normal coronary artery development may be dependent on a precise balance of cardiac neural crest versus proepicardial cells, not merely whether neural crest or proepicardial cells are present in the heart.
Infundibular pouches in the Cx431 knockout mouse heart
We also investigated formation of the pouches in the Cx431 knockout mouse heart. Such pouches are not observed in neural crest-ablated embryos, and have never been seen in transgenic mouse models with Cx43
1 perturbations targeted to neural crest cells. We observed that these pouches are lined by a tissue deficient in the expression of cardiomyocyte markers. Although the precise identity of cells in this tissue is not known, we have previously shown that it expresses smooth muscle
-actin (Huang et al., 1998b
; Lo and Wessels, 1999), which normally is turned off in the working myocardium at this stage of development. This would suggest that the pouch comprises cells that are in early stages of vascular smooth muscle cell differentiation. Consistent with this possibility, we observed a subendocardial layer of cells expressing vascular smooth muscle markers. However, subjacent to these smooth muscle-like cells, we also detected the presence of fibroblast cells as indicated by heavy trichrome staining. These observations suggest that formation of the pouches may involve the proepicardial cells, which are progenitors of all vascular smooth muscle and fibroblast cells in the heart. Thus, the enhanced rate of proepicardial cell migration and proliferation potentially could result in an excess of proepicardial cells at the base of the heart, thereby playing a role in pouch formation.
Consistent with this model is our observation that the FC transgenic mice never exhibit pouches, but have outflow obstruction like that of the Cx431 knockout mice. Previous studies of neural crest cells from FC transgenic embryos have shown reductions in dye coupling and migration rate to levels like that of the Cx43
1 knockout mouse. By contrast, FC transgenic proepicardial cells showed no change in dye coupling, nor in the rate of cell migration or proliferation. These observations suggest that Cx43
1 deficiency in the proepicardial cells is necessary for pouch formation.
Nevertheless, it is likely that other cells or tissues are involved in pouch formation. This is indicated by the fact that although predominantly only homozygous knockout animals have pouches, proepicardial cells from both the heterozygous and homozygous Cx431 knockout mice showed similar cell migration and proliferation alterations. Our finding that distal coronary artery branches were directly continuous with the pouches in some Cx43
1 knockout animals suggests that coronary artery anomalies have a role in pouch formation. We hypothesize that the infundibular pouches, like the coronary artery anomalies, may arise from perturbations involving both the cardiac neural crest and proepicardial cells. We note that pouch formation probably does not arise from the perturbation of endothelial cells, as cardiac malformations were not observed when tie2 promoter Cre was used to target the deletion of a floxed Cx43
1 allele in endothelial cells (Theis et al., 2001
).
Cx431 gap junctions and the modulation of cell motility
The present study together with our previous work suggests that Cx431 gap junctions play an important role in modulating the motility of two migratory cell populations essential for cardiovascular development, the neural crest and proepicardial cells. In contrast to neural crest cells, which showed a decrease in the apparent rate of cell migration in the absence of Cx43
1 function, the opposite was observed for the Cx43
1-deficient proepicardial cells. The proepicardial cells also showed increased cell proliferation, which was not observed in the Cx43
1-deficient cardiac neural crest cells. These differences perhaps reflect different signal transduction pathways functioning in neural crest versus the proepicardial cells, or the differential permeability of Cx43
1 gap junction channels to different signaling molecules active in these cell signaling pathways. In the proepicardial cells, although dye coupling was found to decrease in line with the loss of one versus both Cx43
1 alleles, cell migration and cell proliferation were identically altered in the heterozygous and homozygous knockout proepicardial cells. These observations raise the possibility that the role of Cx43
1 in cell motility may not be mediated simply by its channel-forming capacity, a possibility that is also suggested by our recent study of neural crest cell motility in other knockout mouse models (Xu et al., 2002
).
Cx431 and congenital coronary arterial anomalies
The infundibular pouches observed in the Cx431 knockout mice share many features in common with congenital aneurysm of an aortic sinus of Valsalva in humans (Boutefeu et al., 1978
; Barragry et al., 1988
). Multiple other coronary anomalies observed in the Cx43
1 knockout mice also have been described in humans. For example, a high anterior origin of the right coronary artery (as seen in 8048, 8462, 8463 and 8065 in Fig. 3) is observed in 2% to 6% of individuals undergoing routine coronary angiography. Anomalous origin of the left coronary artery from the pulmonary artery (similar to Fig. 1E-H) is associated with increased morbidity and mortality (Levin et al., 1978
). Anomalous origin of the right coronary artery from the pulmonary artery also has been observed in humans but is relatively rare. Anomalous origin of the left coronary artery from the right coronary artery or a single right coronary artery, similar to that observed in the Cx43
1 knockout mice (see Fig. 3, mouse 7949), is associated with a high incidence of sudden cardiac death in young individuals (Cheitlin et al., 1974
; Liberthson et al., 1979
; Roberts, 1986
; Kragel and Roberts, 1988
). In light of these findings, we propose that Cx43
1 should be evaluated as a candidate gene for mutations that may play a role in human coronary artery anomalies.
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ACKNOWLEDGMENTS |
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