Summary
The contractile force of the cardiomyocyte is transmitted through the adherens junction, a component of the intercalated disc, enabling the myocardium to function as a syncytium. The cadherin family of cell adhesion receptors, located in the adherens junction, interact homophilically to mediate strong cell-cell adhesion. Ectopic expression of cadherins is associated with changes in tumor cell behavior and pathology. To examine the effect of cadherin specificity on cardiac structure and function, we expressed either the epithelial cadherin, E-cadherin, or N-cadherin in the heart of transgenic mice. E-cadherin was localized to the intercalated disc structure in these animals similar to endogenous N-cadherin. Both N- and E-cadherin transgenic animals developed dilated cardiomyopathy. However, misexpression of E-cadherin led to earlier onset and increased mortality compared with N-cadherin mice. A dramatic decrease in connexin 43 was associated with the hypertrophic response in E-cadherin transgenic mice. Myofibril organization appeared normal although, vinculin, which normally localizes to the intercalated disc, was redistributed to the cytoplasm in the E-cadherin transgenic mice. Furthermore, E-cadherin induced cyclin D1, nuclear reduplication, and karyokinesis in the absence of cytokinesis, resulting in myocytes with two closely opposed nuclei. By contrast, N-cadherin overexpressing transgenic mice did not exhibit an increase in cyclin D1, suggesting that E-cadherin may provide a specific growth signal to the myocyte. This study demonstrates that modulation of cadherin-mediated adhesion can lead to dilated cardiomyopathy and that E-cadherin can stimulate DNA replication in myocytes normally withdrawn from the cell cycle.
Introduction
The structural integrity of the heart is maintained by the end to end connection between the myocytes, called the intercalated disc. The intercalated disc of adult cardiac muscle consists of three main junctional complexes; zonula adherens, desmosome, and gap junction, each with defined functions ( Forbes and Sperelakis, 1985). The adherens junction provides strong cell-cell adhesion mediated by the cadherin/catenin complex via linkage to the actin cytoskeleton ( Tepass et al., 2000). It is also the site of attachment of the myofibrils, and thus enables transmission of the contractile force across the plasma membrane. The desmosome provides structural support through interactions of desmosomal cadherins with the intermediate filament system [i.e. desmin ( Green and Gaudry, 2000)]. The gap junction provides intercellular communication via electrical stimulus and small molecules that pass through a channel generated by a family of proteins called connexins ( Severs et al., 2001). The different junctional complexes must be properly organized in the intercalated disc to mediate normal cell-cell interactions between myocytes. The expression and distribution of many of these junctional components are often perturbed in cardiovascular disease ( Dupont et al., 2001; Fujio et al., 1995; Peters et al., 1993; Schaper et al., 1991; Wang and Gerdes, 1999); however, it is unclear whether these changes are involved in the etiology of the disease.
Classical cadherins are a family of cell surface glycoproteins that mediate calcium-dependent cell-cell adhesion primarily in a homophilic manner ( Vleminckx and Kemler, 1999). The classical cadherins are single pass transmembrane proteins comprising five extracellular domains, a transmembrane domain and a cytoplasmic domain. The cadherins form cisdimers in the plasma membrane that interact in an anti-parallel fashion with like cadherins on a neighboring cell, creating an adhesion zipper between the cells ( Shapiro et al., 1995). Cadherin adhesive activity is regulated by a group of proteins belonging to the catenin family that bind to the conserved cytoplasmic domain of the cadherin ( Gumbiner, 2000).β -Catenin or γ-catenin (plakoglobin) bind directly to the C-terminal region of the cadherin, whereas p120 interacts with the juxtamembrane region ( Anastasiadis and Reynolds, 2000). α-Catenin binds to β- orγ -catenin, which links the cadherin/catenin complex either directly ( Rimm et al., 1995) or indirectly ( Knudsen et al., 1995; Watabe-Uchida et al., 1998) to the actin cytoskeleton. Recently, a novel catenin,α T-catenin, was found to be expressed at high levels in the heart, where it localized to the intercalated disc ( Janssens et al., 2001).
Cadherin family members have distinct spatial and temporal patterns of expression during embryonic development and in the adult ( Takeichi, 1995). N-cadherin is widely expressed in the early postimplantation embryo ( Radice et al., 1997) including the precardiac mesoderm and continues to be expressed at high levels in embryonic, fetal and adult myocardium ( Angst et al., 1997). N-cadherin is found in other tissues such as skeletal muscle, which expresses multiple cadherin subtypes including R- and M-cadherin ( Kaufmann et al., 1999). By contrast, E-cadherin is not expressed in muscle, but mainly found in epithelia throughout the body. In adult myocardium, N-cadherin/catenin complex is primarily localized to adherens junctions in intercalated discs where it serves as an attachment site for myofibrils. In addition, N-cadherin is found in extrajunctional sites localized to periodic bands along the lateral membrane referred to as costameres ( Goncharova et al., 1992). The addition of function blocking antibodies to chick myocyte culture has demonstrated the importance of N-cadherin in cell-cell interaction and myofibril organization ( Goncharova et al., 1992; Peralta Soler and Knudsen, 1994). Furthermore, injection of cDNA encoding a truncated N-cadherin molecule lacking its extracellular domain (i.e. dominant-negative) caused cells to lose contact with their neighbors and myofibril organization was disrupted ( Hertig et al., 1996). N-cadherin has been implicated in several aspects of cardiac development including sorting out of the precardiac mesoderm ( Linask et al., 1997), establishment of left-right asymmetry ( Garcia-Castro et al., 2000), cardiac looping morphogenesis ( Shiraishi et al., 1993), and trabeculation of the myocardial wall ( Ong et al., 1998). Using gene targeting technology, we previously demonstrated that loss of N-cadherin resulted in embryonic lethality associated with multiple developmental abnormalities including a severe cardiovascular defect ( Radice et al., 1997). Recently, we generated chimeric embryos with N-cadherin double-knockout ES cells demonstrating that N-cadherin-mediated adhesion was essential for maintaining myocyte interactions during the morphological transition from an epithelial to compacted myocardial cell layer ( Kostetskii et al., 2001). Taken together, these data indicate that N-cadherin plays a critical role in myocardial development and function.
There is limited information pertaining to the consequences of ectopic or overexpression of cadherins on tissue homeostasis. Importantly, recent data indicate that misexpression of cadherins influences cellular behavior of tumor cells. Ectopic expression of N-cadherin in squamous cell carcinoma cell lines ( Islam et al., 1996) as well as breast cancer cells ( Hazan et al., 2000; Nieman et al., 1999) either in the presence or absence of endogenous E-cadherin leads to increased invasiveness in vitro and in vivo. Furthermore, misexpression of E-cadherin in retinal pigment epithelial (RPE) cell lines affects the distribution of polarized proteins such as Na+,K+-ATPase and the expression of cytoskeletal proteins ( Marrs et al., 1995). In a different experimental system, overexpression of E-cadherin in the epithelial intestinal crypts of chimeric mice decreased the cellular migration up the villus ( Hermiston et al., 1996). Taken together, these studies indicate that cadherin function is dependent on cellular context as well as levels of expression.
To address the role of cadherin subtype specificity on cardiac development and function, we generated transgenic mice expressing either N- or E-cadherin in the heart ( Luo et al., 2001). Ectopic expression of E-cadherin in the myocardium did not interfere with normal cardiac development consistent with its ability to restore myocyte adhesion and cardiac morphogenesis in N-cadherin-null embryos ( Luo et al., 2001). However, both overexpression of N-cadherin or misexpression of E-cadherin in the adult myocardium caused dilated cardiomyopathy, which was probably caused by perturbation of normal intercalated disc function. Ectopic expression of the epithelial cadherin resulted in earlier onset of the phenotype and increased mortality compared with mice that overexpress N-cadherin. Furthermore, misexpression of E-cadherin induced DNA synthesis in the absence of cytokinesis in myocytes normally withdrawn from the cell cycle; this resulted in an increased number of `binucleated' myocytes in the transgenic heart. Our results indicate that modulation of cadherin-mediated adhesion leads to dilated cardiomyopathy in mice with pathological features similar to those found in human patients with end stage heart failure.
Materials and Methods
Mouse husbandry
The generation and genotyping of the αMHC/chicken N-cadherin andα MHC/human E-cadherin transgenic mice were described previously ( Luo et al., 2001). Briefly, tail DNA was obtained from the mice and subjected to PCR using primers specific for the transgenes. The mice were maintained on an SJL/C57BL/6J genetic background.
Immunohistochemistry and histological analysis
Hearts were fixed overnight in freshly prepared formalin in PBS, pH 7.4. After rinsing in PBS, the hearts were dehydrated and embedded in paraffin wax according to standard procedures. Sections (6 μm) were cut, mounted, dewaxed in xylene, rehydrated through an ethanol series, and then heated in 1× Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) in a microwave oven (350 watts) for 10 minutes to unmask the epitope. The sections were washed in PBS before blocking with 5% nonfat milk/PBS for 30 minutes. Antibodies were diluted in 5% nonfat milk/PBS as follows: mouse monoclonal anti N-cadherin, 1:100 (3B9; Zymed, So. San Francisco, CA); mouse monoclonal anti E-cadherin, 1:100 (4A2C7; Zymed, So. San Francisco, CA); mouse monoclonal anti-β-catenin, 1:100 (CAT-5H10; Zymed); rabbit polyclonal anti-connexin 43, 1:100 (Zymed); mouse monoclonal anti-cyclin D1, 1:50 (sc-450; Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-vinculin, 1:500 (a kind gift from Joe Sanger, University of Pennsylvania), rat monoclonal anti-chicken N-cadherin, 1:100 (NCD-2, a kind gift from Masatoshi Takeichi, Kyoto University, Kyoto, Japan). Samples were incubated overnight at 4°C with primary antibodies. After washing in PBS, sections were incubated with biotinylated anti-mouse or anti-rat IgG or FITC-conjugated goat anti-mouse, 1:500 (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature. After washing in PBS, if necessary, sections were incubated with Cy3-conjugated strepavidin for 30 minutes at room temperature. The sections were washed in PBS, and mounted for analysis with a confocal microscope. For nuclear staining, the slide mounting solution contained DAPI (4′, 6-diamidino-2-phenylindole, 1:500, Sigma, St Louis, MO). Quantification of binucleated cells was performed with Openlab software (Improvision).
For histological analysis, hearts were isolated at different ages, fixed in formalin, processed for paraffin sectioning, and stained with either Hematoxylin and Eosin or Alizarin Red. Ultrastructural analysis of myocardium was performed by transmission electron microscopy as previously described ( Lavker et al., 1991).
Western immunoblotting
Freshly isolated hearts were washed in cold PBS and then homogenized in extraction buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.2, 1% Triton, 0.1% SDS, 0.5% deoxycholic acid, sodium salt), Homogenates were subjected to SDS-PAGE, and the resolved proteins were transferred to nitrocellulose by a semi-dry transfer system. After blocking with 5% nonfat milk for 2 hours, the blots were washed in TBS-T pH 7.6, and incubated for 1 hour at room temperature with primary antibodies. Antibodies were diluted in 5% nonfat milk/PBS as follows: mouse monoclonal anti-N-cadherin, 1:1000; mouse monoclonal anti E-cadherin, 1:1000; mouse monoclonal anti-β-catenin, 1:1000; rabbit polyclonal anti-connexin 43, 1:1000; mouse monoclonal anti-vinculin, 1:5000; rat monoclonal anti-chicken N-cadherin, 1:500; mouse monoclonal anti-rabbit GAPDH, 1:4000 (RDI, Flanders, NJ). The blots were washed in TBS-T and incubated with secondary antibodies: alkaline phosphatase-conjugated anti-mouse, anti-rat or anti-rabbit IgG (1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA), for 1 hour at room temperature. After washing with TBS-T, the bound antibodies were detected using enhanced chemifluorescence (Vistra ECF kit; Amersham, Arlington Heights, IL). Chick embryonic heart and human keratinocyte lysates were used as positive controls for anti-chick N-cadherin and anti-human E-cadherin antibodies, respectively. Variations in sample loading were normalized relative to GAPDH signal. Labeled blots were analyzed using Storm 860 Imaging system and Imagequant software (Molecular Dynamics, Sunnyvale, CA).
Northern blot analysis
Total heart RNA was collected from transgenic and nontransgenic littermates and purified with Tri Reagent (Sigma). RNA (15 μg) was separated on a 1.5% formadehyde agarose gel, transferred to Zeta-Probe (Bio-Rad) blotting membrane in 20× SSC solution overnight. After transferring, the blot was crosslinked and prehybridized with QuickHyb solution (Stratagene) at 68°C for 20 minutes. The rat ANF cDNA probe (a kind gift from Jason Rogers, Washington University, St Louis, MO) and GAPDH cDNA probe (Clontech) were labeled with 32P using DNA labeling beads (Amersham) and purified. The blot was hybridized with the labeled probes, washed, and analyzed using the Storm 860 Imaging system and Imagequant software.
Results
Altered cadherin expression in the myocardium leads to cardiomyopathy
To determine the effects of misexpression of cadherins on heart function, the well-characterized mouse αmyosin heavy chain (MHC) promoter was used to express either the chicken N-cadherin (αMHC/Ncad) or human E-cadherin (αMHC/Ecad) gene specifically in the heart. Theα MHC promoter is initially active in the primitive heart, its expression later becomes restricted to the atrial chambers during development, and ventricular expression commences in the neonatal heart ( Subramaniam et al., 1991). The epithelial cadherin, E-cadherin, was chosen for these experiments to determine if a nonmuscle cadherin is compatible with normal formation and function of the intercalated disc structure. We previously demonstrated that E-cadherin can functionally substitute for N-cadherin in the embryonic myocardium of N-cadherin-null embryos ( Luo et al., 2001); however these partially rescued embryos die before intercalated disc formation. Both the αMHC/Ncad and αMHC/Ecad transgenes are capable of restoring myocyte adhesion and cardiac morphogenesis in N-cadherin-null embryos indicating that these exogenous cadherins probably mediate cell adhesion in N-cadherin-expressing myocardium as well. In this study, we compare the phenotypic effects of overexpression of N-cadherin and ectopic expression of E-cadherin in the adult myocardium. The human E-cadherin and chicken N-cadherin genes were chosen for these experiments because both homologs are well conserved across species (90% and 92% amino acid similarity, respectively), and species-specific antibodies are available to distinguish exogenous and endogenous proteins. Furthermore, mouse N-cadherin can bind to chicken N-cadherin in a cell aggregation assay confirming its functional similarity ( Miyatani et al., 1989). In contrast, N-cadherin does not interact with either mouse E- or P-cadherin ( Miyatani et al., 1989). Since cadherin-mediated adhesion occurs primarily in a homophilic manner (i.e. cadherins on one cell interact with like molecules on an adjacent cell), we predicted that using cadherin transgenes from different species would not affect their adhesive function in mice.
The generation of the αMHC/Ncad and αMHC/Ecad transgenic lines was described previously ( Luo et al., 2001). Comparison of protein expression in the heart of different transgenic mice indicated high expression in several lines ( Fig. 1A). An additional smaller band (approximately 105 kDa) observed in the higher expressingα MHC/Ecad lines has been observed previously ( Yoshida-Noro et al., 1984) and may correspond to a degradation product. To confirm that expression of the transgene was restricted to the heart, western blot analysis was performed on the following noncardiac tissues: brain, kidney, liver, lung and spleen, from the αMHC/Ncad4 and αMHC/Ecad10 lines. No expression was observed in noncardiac tissues (data not shown) consistent with previous reports with the αMHC promoter ( Gulick et al., 1991). Mice hemizygous for either the αMHC/Ncad orα MHC/Ecad transgenes were mated with wild-type animals. A number of pups from αMHC/Ecad matings died between one and two weeks after birth. In comparison, no significant neonatal lethality was observed in theα MHC/Ncad lines. The loss of αMHC/Ecad pups was further substantiated by examining the genotypes of the animals that survived to weaning age. Mendelian inheritance predicts that 50% of the mice will inherit the transgene, which was the case for the αMHC/Ncad transgene (line N4, 125 transgenic:126 nontransgenic). By contrast, mice expressing comparable levels of E-cadherin were under-represented at weaning (line E33, 75 transgenic:126 nontransgenic, P<0.02). Furthermore, severalα MHC/Ecad mice died suddenly during handling.
Cadherin misexpression and cardiac hypertrophy in transgenic mice. (A) Theα MHC promoter was used to express either chicken N-cadherin or human E-cadherin specifically in the myocardium. Western blot analysis was performed on heart lysates from αNcad and αEcad transgenic mice using species-specific antibodies. GAPDH signal shows loading of samples between lanes. (B) Wholemount images of hearts removed from 4-week-old nontransgenic (left) and transgenic (right) littermates. Note the increased size of theα MHC/Ecad heart in comparison to αMHC/Ncad. (C) Bar graph illustrating the heart weight:body weight ratios of four transgenic lines compared with wild-type. C, chick heart; H, human keratinocyte; NT, nontransgenic. Bars, 2.5 mm (B).
Both transgenic animals exhibited aberrant cardiac morphology; however, the phenotype was more pronounced in the surviving αMHC/Ecad mice, in comparison to the αMHC/Ncad mice, as demonstrated by increased dilation of the atria and ventricles ( Fig. 1B). Comparison of the heart weight to body weight ratios ofα MHC/Ncad and surviving αMHC/Ecad mice illustrates this point further (line N4 versus line E33, Fig. 1C). Mice (<100 days old) expressing high levels of exogenous cadherin in the heart displayed a significant increase in heart weight:body weight ratio compared with wild-type mice consistent with a hypertrophic response ( Fig. 1C). This phenotype was associated with increased postnatal lethality especially among the αMHC/Ecad mice. Females positive for either the αMHC/Ecad orα MHC/Ncad transgene had difficulty during pregnancy and nursing; therefore, the transgene was transmitted through the transgenic males. Cardiac hypertrophy was not observed in the lower expressing αMHC/Ncad andα MHC/Ecad mice indicating an association between transgene expression levels and the cardiac phenotype. Taken together, our data are consistent with a more severe dilated phenotype observed in the surviving αMHC/Ecad mice in comparison with the αMHC/Ncad.
E-cadherin localizes to the intercalated disc in cardiomyocytes
To determine the cellular localization of cadherins in the myocardium, immunohistochemistry was performed on hearts from 4-week-old transgenic mice. Consistent with previous reports ( Angst et al., 1997), endogenous N-cadherin localizes predominantly to the intercalated disc in wild-type hearts with little cytoplasmic staining ( Fig. 2A). N-cadherin immunostaining in the αMHC/Ncad transgenic heart represents either chicken N-cadherin alone ( Fig. 2B) or mouse and chicken N-cadherin together ( Fig. 2C). As predicted, the cadherin staining patterns were identical with both antibodies ( Fig. 2B,C), as shown by the merged images ( Fig. 2D). Similar to endogenous N-cadherin, the chicken protein also localized to the intercalated disc ( Fig. 2B). In addition, excess exogenous N-cadherin was found distributed throughout the cytoplasm ( Fig. 2B). The wild-type heart was negative for E-cadherin expression ( Fig. 2E), as was the case for endogenous E-cadherin (data not shown). By contrast, human E-cadherin, like endogenous N-cadherin ( Fig. 2G), localized to the intercalated disc in the αMHC/Ecad transgenic heart ( Fig. 2F). The merged images demonstrate that both exogenous E-cadherin and endogenous N-cadherin were colocalized to the intercalated disc ( Fig. 2H). Both N- and E-cadherin were observed in the cytoplasm of these transgenic hearts (αNcad4 and αEcad33), whereas lower expressing transgenic lines showed only intercalated disc staining (data not shown) similar to the endogenous N-cadherin pattern. This suggests that the intercalated disc was saturated with cadherin protein in the high expressing lines leading to its accumulation in the cytoplasm. These data show that the epithelial cadherin, E-cadherin, is correctly localized to the intercalated disc structure in heart muscle along with endogenous N-cadherin.
E-cadherin localizes to the intercalated disc in the transgenic heart. Heart sections from 4-week-old animals were analyzed by indirect immunofluorescence for distribution of N-cadherin and E-cadherin. Endogenous N-cadherin was localized to the intercalated disc in wild-type heart (A). For comparison, a αMHC/Ncad4 heart section was double-stained for chicken N-cadherin (B) and both mouse and chicken N-cadherin (C). No antibody was available that recognizes only mouse and not chicken; therefore, the staining patterns in (B) and (C) were identical in the transgenic heart, which was confirmed in the merged image (D). Chicken N-cadherin was located in the intercalated disc (B) similar to endogenous N-cadherin observed in wild-type (A). In addition, exogenous N-cadherin was distributed throughout the cytoplasm (B). In contrast, little cytoplasmic staining was observed for N-cadherin in wild-type heart (A). As expected, E-cadherin was absent from wild-type heart (E). A αMHC/Ecad33 heart section was double-stained for E-cadherin (F) and N-cadherin (G) and merged (H) demonstrating the co-localization of these cadherin subtypes in the intercalated disc (arrows). Excess E-cadherin was observed in the cytoplasm. Bar, 25 μm.
E-cadherin stimulates DNA replication in terminally differentiated myocytes
Histologic analysis of hearts from 4-week-old mice demonstrated the severity of the phenotype observed in the transgenic hearts ( Fig. 3A-C). An enlarged left ventricle was observed in the αMHC/Ncad heart, whereas both ventricles were severely dilated in the αMHC/Ecad heart. In addition, dilated thin-walled atria were observed in the pumpkin-shaped αMHC/Ecad heart ( Fig. 3C). Both theα MHC/Ncad and αMHC/Ecad transgenic hearts contained enlarged hyperchromatic myocyte nuclei with increased myofibrillar width consistent with cardiac hypertrophy ( Fig. 3D-F). At two months of age, left atrial thrombosis was observed in the transgenic mice ( Fig. 3G). Furthermore, white spots or streaks were often observed in the atria and ventricles of the transgenic hearts (not shown). Alizarin Red staining indicated that these white spots correspond to regions of calcification in the heart ( Fig. 3H). Limited regions of fibrosis were observed in older transgenic animals (data not shown). The thrombosis and calcification were observed in both αMHC/Ncad (not shown) and αMHC/Ecad transgenic mice; however, misexpression of E-cadherin often caused earlier onset and a more severe phenotype compared with overexpression of N-cadherin.
Histological analysis of wild-type and transgenic hearts. Heart sections from wild-type (A,D), αMHC/Ncad (B,E), and αMHC/Ecad (C,F,G,H) indicated that both transgenes can elicit a hypertrophic response; however, the αMHC/Ecad was consistently more severe. The αMHC/Ncad heart (B) was enlarged with dilated left ventricle and thin ventricular wall. In comparison, both ventricles and atria were severely dilated in theα MHC/Ecad heart (C). Enlarged myocytes with hyperchromatic nuclei were observed in both transgenic hearts (E,F). Many `binucleated' myocytes were seen in the αMHC/Ecad heart (F, arrows) in comparison to αMHC/Ncad (E). At 2 months of age, left atrial thrombosis (arrow) was observed in theα MHC/Ecad heart (G). Alizarin Red staining showed regions of calcification (arrows) in the ventricle and pericardium of the αMHC/Ecad heart (H). Thrombosis and calcification were observed in the αMHC/Ncad hearts (not shown). Bars, 2.5 mm (A,B,C,G); 50 μm (D,E,F); 500 μm (H).
The molecular mechanism(s) that control cell cycle withdrawal in terminally differentiated myocytes is poorly understood. In the case of αMHC/Ecad hearts, we observed many hypertrophic myocytes with two enlarged nuclei side by side ( Fig. 3F), which suggested that DNA replication and nuclear division had occurred but cytokinesis had not taken place. The consistent proximal positioning of the two nuclei suggested that this phenomena did not result from fusion of two myocytes, but that the nuclei originated from the same cell. The phenotype, referred to here as `binucleated', was easily visualized by DAPI staining the heart sections for nuclear DNA ( Fig. 4). Karyokinesis often was not complete, resulting in myocytes with two nuclei not completely separated ( Fig. 4C). By contrast, no significant increase in `binucleated' myocytes was observed in transgenic hearts expressing N-cadherin (4% versus 27%) indicating that the phenotype was caused specifically by E-cadherin ( Fig. 4D). The `binucleated' phenotype was observed in both atrial and ventricular myocytes beginning postnatally at 1 week of age coincident with increased E-cadherin expression (data not shown). This phenotype was observed in the three highest expressing αMHC/Ecad transgenic lines (E10, E22, E33). In comparison, the number of `binucleated' myocytes did not increase significantly in the lower expressing lines (E9, E12) indicating that the E-cadherin effect was dosage dependent. The cell-cycle-dependent kinase, cyclin D1, plays an important role in the initiation of DNA synthesis, therefore we examined its expression in the transgenic hearts. Consistent with the `binucleated' phenotype, an increase in cyclin D1 expression was observed in the αMHC/Ecad heart ( Fig. 4F) in comparison to αMHC/Ncad ( Fig. 4E) and wild-type (data not shown) hearts. To determine whether E-cadherin affected myocyte proliferation in the embryonic myocardium, we examined the cell proliferation rate at E10.5 using BrdU labeling in utero. No significant overall difference in BrdU incorporation was observed between transgenic and wild-type animals (data not shown). The effect of E-cadherin on myocyte proliferation appears to be restricted to postnatal myocytes, which normally withdraw from the cell cycle.
Assessment of the `binucleated' phenotype in αMHC/Ecad transgenic mice. DAPI staining of sections from 4-week-old wild-type (A) and transgenic (B,C) hearts indicated an increase in `binucleated' hypertrophic nuclei (arrowheads) in the αMHC/Ecad mice. Many normal size nuclei were found in wild-type heart (A). In comparison, far fewer nuclei were observed in similar areas of hypertrophic myocardium in αNcad4 (B) and αEcad33 (C). The percentage of `binucleated' myocytes was assessed by counting several different fields containing a total of 150 hypertrophic myocytes (D). The `binucleated' phenotype was observed in other αMHC/Ecad transgenic lines such as E10. Indirect immunofluorescence of heart sections from 2-week-old animals indicated an increase in cyclin D1 (arrowheads) in αMHC/Ecad (F), but not αMHC/Ncad (E), consistent with the `binucleated' phenotype. Bar, 25 μm (A,B,C); 50 μm (E,F).
Downregulation of connexin 43 and redistribution of vinculin inα MHC/Ecad transgenic hearts
The intercalated disc consists of different junctions, including the adherens junction, desmosome and gap junction, which function cooperatively to maintain normal cell-cell interactions between working myocytes. We examined the expression and cellular localization of several components of these junctions in hearts of 4-week-old transgenic mice. β-Catenin localizes predominantly to the intercalated disc in wild-type hearts with very little cytoplasmic staining ( Fig. 5A). In comparison, increased staining for β-catenin in the intercalated disc and cytoplasm was observed in both αMHC/Ncad ( Fig. 5B) and αMHC/Ecad ( Fig. 5C) transgenic hearts. The increase in β-catenin was consistent with the increase in total cadherin observed in the transgenic hearts ( Fig. 2). This coordinated regulation of cadherin and catenin has been observed by others in vitro ( Nagafuchi et al., 1991; Redfield et al., 1997) and in vivo ( Hermiston et al., 1996). A small increase in α-catenin and no significant increase inγ -catenin (plakoglobin), which are additional components of the cadherin/catenin adhesion complex, were observed in the transgenic hearts (data not shown). In addition, the desmosome marker, desmoplakin, was not changed significantly in the transgenic hearts (data not shown). The gap junction protein, connexin 43 (Cx43), is normally localized to the intercalated disc in the mature myocardium, as shown in wild-type hearts ( Fig. 5D). In the transgenic hearts, the punctate Cx43 staining was reduced in both αMHC/Ncad ( Fig. 5E) and to an even greater extent in αMHC/Ecad ( Fig. 5F) hearts. Vinculin was associated with the intercalated disc and Z bands in the wild-type heart ( Fig. 5G). A similar pattern was observed in the αMHC/Ncad heart except that the intercalated disc staining was more intense ( Fig. 5H) consistent with more functional cadherin/catenin adhesion complexes. However, in theα MHC/Ecad heart, less vinculin was found in the intercalated disc; instead, it was redistributed to the cytoplasm and showed a striated and diffuse pattern ( Fig. 5I). Both downregulation of Cx43 and increased vinculin in the cytoplasm are associated with cardiac hypertrophy and heart failure in humans ( Dupont et al., 2001; Peters et al., 1993; Schaper et al., 1991) and animal models ( Wang and Gerdes, 1999).
Expression and cellular localization of components of the intercalated disc. Heart sections from wild-type (A,D,G), αMHC/Ncad (B,E,H), andα MHC/Ecad (C,F,I) animals 4-weeks old were analyzed by indirect immunofluorescence for distribution of β-catenin (A,B,C), connexin 43 (D,E,F) and vinculin (G,H,I). β-Catenin staining increased significantly in the intercalated disc and cytoplasm of both transgenic lines (B,C) in comparison to wild-type heart (A). In contrast, connexin 43 staining decreased in both lines with greater reduction seen in αMHC/Ecad (F) compared withα MHC/Ncad (E). Vinculin staining appeared more concentrated in the intercalated disc of αMHC/Ncad (H) compared with wild-type (G) hearts. By contrast, redistribution of vinculin to the cytoplasm resulted in less intercalated disc staining in the αMHC/Ecad heart (I). Bar, 25μ m.
Changes in protein expression associated with cardiac hypertrophy in the cadherin transgenic mice
To determine how changes in protein expression correlated with the cadherin-induced hypertrophy response, western blot analysis was performed on hearts from 3-day- to 4-week-old wild-type and transgenic mice ( Fig. 6). The time course of expression of the αMHC/Ncad and αMHC/Ecad transgenes was consistent with previous reports using this cardiac promoter increasing postnatally in the ventricular myocardium ( Palermo et al., 1996; Subramaniam et al., 1991). As expected, the mouse and chicken N-cadherin proteins migrate together; therefore quantitation relative to endogenous protein was performed by comparing cadherin levels in wild-type and transgenic littermates. Since N-cadherin and E-cadherin were distinguishable on the blot, the relative levels of endogenous and exogenous cadherin were compared directly. Quantitation of the Western blots indicated that both transgenes were expressed at approximately the same levels (2.3, αNcad4 versus 2.6,α Ecad10) relative to endogenous N-cadherin, demonstrating that quantitative differences in exogenous cadherin were most probably not responsible for the more severe cardiac phenotype observed in theα MHC/Ecad mice. The expression of E-cadherin had no significant effect on endogenous N-cadherin levels. In both αMHC/Ncad and αMHC/Ecad hearts, β-catenin levels increase concomitantly with exogenous cadherin. By contrast, β-catenin levels normally decrease with age in wild-type littermates. Since plakoglobin is capable of binding to the cytoplasmic domain of N-cadherin, we examined plakoglobin levels in the transgenic hearts; however, no significant change in expression was observed (data not shown). The gap junction protein, Cx43, showed a remarkable reduction inα MHC/Ecad mice in comparison to αMHC/Ncad mice at 4 weeks of age, consistent with the more severe hypertrophic response. The degree of downregulation of Cx43 correlated with the severity of the hypertrophy and varied among animals from the same transgenic line. By contrast, vinculin levels appeared similar in wild-type and transgenic hearts indicating that the increased cytoplasmic staining in the αMHC/Ecad hearts probably represented a redistribution away from the cell surface, which has been observed in human cardiac hypertrophy ( Schaper et al., 1991). In addition, F-actin protein levels did not change in the transgenic mice (data not shown) suggesting that increased cadherin expression did not alter the actin cytoskeleton significantly. Taken together, the more severe perturbations of intercalated disc proteins in the αMHC/Ecad mice were consistent with an earlier onset and increased mortality observed inα MHC/Ecad animals compared with αMHC/Ncad mice.
Changes in protein expression associated with cardiac hypertrophy in transgenic hearts. Western blot analysis of heart lysates from 3-day- to 4-week-old wild-type and αMHC/Ncad (A) and αMHC/Ecad (B) transgenic mice shows the time course of the expression of the transgene in relation to other intercalated disc proteins. Note the dramatic decrease in Cx43 protein in the 4-week-old αEcad heart compared with αNcad, consistent with the severe hypertrophy response caused by E-cadherin. GAPDH signal shows loading of samples between lanes. tg, transgene; d, days; w, weeks.
Modulation of cadherin expression in the heart leads to upregulation of ANF
To determine the time of onset and severity of the cardiac phenotype inα MHC/Ncad and αMHC/Ecad mice, northern blot analysis was performed on heart samples to examine expression of the hypertrophy marker, ANF ( Vikstrom et al., 1998), in 3-day- to 4-week-old wild-type and transgenic mice ( Fig. 7A). Both αMHC/Ncad and αMHC/Ecad transgenic mice displayed increased ANF expression compared with wild-type. However, ANF increased earlier and to a greater extent in αMHC/Ecad mice consistent with the severity of the hypertrophic response in these animals. The increase in ANF expression was already significant by one week after birth and was consistent with the early postnatal lethality observed in the αMHC/Ecad line ( Fig. 7B).
Time course of ANF expression in wild-type and transgenic hearts. Northern blot analysis of ANF mRNA in αMHC/Ncad and αMHC/Ecad hearts from 3-day- to 4-week-old animals (A). GAPDH signal shows loading of samples between lanes. (B) Graphic comparison of changes in ANF mRNA levels between wild-type and transgenic littermates. Note the increase in ANF expression inα Ecad heart at one week of age compared with αNcad. ANF mRNA levels were normalized to GAPDH mRNA levels. tg, transgene; d, days; w, weeks.
Electron microscopic analysis shows well-organized myofibrillar structures in the E-cadherin transgenic hearts
To examine myofibril organization and cell-cell contacts more closely, transmission electron microscopy was performed on hearts from wild-type and transgenic 4-week-old mice. Ultrastructural analysis showed that wild-type,α MHC/Ecad, and αMHC/Ncad (data not shown) hearts had well-organized myofibrils with clearly defined Z bands ( Fig. 8). The intercalated disc structures appeared well formed, and insertion of the myofibrils into the adherens junction appeared normal. In summary, the misexpression of cadherins did not result in any gross morphological abnormalities in the myocardium of the transgenic animals.
Transmission electron microscopy of wild-type and transgenic hearts. Ventricular myocardium of 4-week-old wild-type (A,B) and αMHC/Ecad (C,D) mice appeared remarkably similar at the ultrastructural level. The myocytes displayed well-organized myofibrils that inserted into the adherens junctions of the E-cadherin transgenic heart similar to wild-type. The intercalated disc structures also appeared similar in both hearts. Bars, 2 μm (A,C); 500 nm (B,D).
Discussion
The inappropriate expression of different cadherin subtypes during cancer progression is thought to be a critical step in the disease process. The ability of N-cadherin to alter cell morphology and behavior has been documented in E-cadherin-expressing epithelial tumor cell lines. Here we examine the reverse situation, namely, the effect of misexpression of the epithelial cadherin, E-cadherin, in N-cadherin-expressing cardiac muscle in transgenic mice. We previously demonstrated that transgenic expression of either N- or E-cadherin rescued myocyte adhesion and cardiac morphogenesis in N-cadherin-null embryos ( Luo et al., 2001). Furthermore, culture of myocytes derived from rescued embryos indicate that E-cadherin can support myofibrillogenesis (Y. Luo and G. Radice, unpublished). In the present study, we examine the effects of misexpressing cadherins in the myocardium of adult transgenic mice. Cardiac-specific expression of either N- or E-cadherin led to dilated cardiomyopathy in the transgenic animals; however, the phenotype was more severe in the E-cadherin-expressing mice. Ectopic expression of E-cadherin led to earlier onset and increased mortality compared with N-cadherin mice.
The cadherin superfamily can be divided into several subfamilies including the classical cadherins, which consist of approximately 20 members in mammals. Structural and biochemical studies suggest that cadherins form cis-dimers that interact in an anti-parallel fashion with a like cadherin pair on an adjacent cell. This configuration is repeated forming a zipper-like structure thus establishing an adherens junction between the cells. It is thought that tissue-specific expression of different cadherin subtypes is critical for normal tissue development and function. Most cell types including skeletal muscle express multiple cadherin subtypes; however, cardiac muscle depends primarily on one classical cadherin, N-cadherin. By contrast, E-cadherin is normally not expressed in muscle, but found in most epithelia throughout the body. The chick and mouse N-cadherin proteins show high overall amino acid similarity (92%) including the nearly identical (99%) transmembrane and cytoplasmic domains. The ability of these N-cadherin proteins to interact in trans was demonstrated in a cell aggregation assay using L-cells transfected with cDNA encoding either chick or mouse N-cadherin ( Miyatani et al., 1989). In addition, it is likely that these cadherin homologs generate cis-dimers, which produce an interspecies chimeric zipper structure. The cis interaction is likely given the extent of amino acid similarity (92%) and the fact that N- and R-cadherin, which are less well conserved (74% similarity), interact to form lateral dimers ( Shan et al., 2000). By contrast, mouse N- and E-cadherin show 49% amino acid similarity overall and in vitro studies indicate that N- and E-cadherin do not interact in either cis or trans ( Miyatani et al., 1989; Shan et al., 2000).
In adult myocardium, N-cadherin/catenin complex is primarily localized to adherens junctions in intercalated discs where it serves as an attachment site for myofibrils, in addition to its structural role in maintaining myocyte adhesion. Increased cadherin expression in transgenic hearts was accompanied by increased steady-state levels of β-catenin, also depicted by increased immunolocalization of cadherin/catenin complexes to the intercalated disc. How might cadherin misexpression effect myocyte interactions? Several possible factors affecting intercalated disc function are considered. We speculate that excess cadherin/catenin complexes compared with myofibrils may alter the contractile dynamics by changing the stoichiometry of the cadherin/myofibril connection leading to less efficient force transduction across the plasma membrane ( Fig. 9). The working heart is under tremendous mechanical load, therefore it is difficult to rule out the possibility that subtle differences between mouse and chick N-cadherin may structurally perturb the adhesion zipper. In comparison, the adherens junction in αMHC/Ecad mice consists of two different cadherin/catenin complexes that may further perturb the contractile dynamics since E-cadherin cannot interact with N-cadherin and differences in the cytoplasmic domain may alter myofibril connections. In both cases, the dissipation of the contractile force across the plasma membrane leads to a compensatory response (i.e. hypertrophy) with the greater effect caused by introduction of the epithelial cadherin. Since both transgenes are expressed at approximately similar levels, a qualitative verses quantitative difference in cadherin-mediated adhesion is probably responsible for the severe cardiac hypertrophy observed in the E-cadherin transgenic mice.
Model of how misexpression of cadherins in the heart may interfere with normal intercalated disc function. Schematic diagrams representing adherens junctions comprised of different cadherin subtypes through which the contractile force is transduced across the plasma membrane (A-C). In wild-type heart muscle (A), N-cadherin dimers (black bars) interact to form a zipper structure critical for strong cell-cell adhesion. In αMHC/Ncad mice (B), the mouse (black) and chicken (gray) N-cadherin are very homologous and interact, at least in trans, to generate a chimeric zipper structure. Mouse and chicken N-cadherin are nearly identical in the cytoplasmic and transmembrane domains; therefore we predict normal interaction(s) with the submembranous myofibril connection. However, the excess cadherin/catenin complexes compared with myofibrils alters the contractile dynamics leading to less efficient force transduction across the plasma membrane. Inα MHC/Ecad mice (C), in addition to excess cadherin/catenin complexes the contractile dynamics may be further perturbed due to the presence of E-cadherin (stipple), since it cannot interact with N-cadherin and differences in the cytoplasmic domain may alter myofibril connections. In both models (B,C), the dissipation of the contractile force across the plasma membrane leads to a compensatory response (i.e. hypertrophy) with the greater effect caused by introduction of the epithelial cadherin.
The increased penetrance, earlier onset, and increased severity of the cardiac phenotype in αMHC/Ecad mice suggests that the epithelial cadherin may further perturb the transmission of the contractile force across the intercalated disc structure. In contrast to chick N-cadherin, the less similar E-cadherin is more likely to interfere with adherens junction organization by intercalating into the cadherin zipper, thus disrupting the normal homogeneous clustering of N-cadherin/catenin complexes ( Fig. 9). Mouse and chicken N-cadherin are nearly identical in the cytoplasmic and transmembrane domains; therefore, we predict normal interaction(s) with the submembranous myofibril connection. In comparison, the cytoplasmic domain of E-cadherin is less similar (61%) and somewhat shorter (10 amino acids) than N-cadherin. Although TEM did not indicate any obvious defect in myofibril insertion at the cell membrane, it is possible that subtle molecular differences in the E-cadherin/myofibril connection may lead to contractile dysfunction in theα MHC/Ecad heart. In summary, E-cadherin may be acting as a dominant-negative cadherin in heart muscle by altering the cadherin zipper structure leading to less efficient contractile force transduction across the plasma membrane.
Terminally differentiated cardiac myocytes normally withdraw from the cell cycle after birth. Therefore, we were surprised that E-cadherin misexpression in the postnatal mouse heart led to `binucleated' myocytes; this phenotype was not observed in the N-cadherin transgenic animals. In addition to its role in cell adhesion, β-catenin in concert with the TCF/LEF family of transcription factors is capable of regulating cell cycle progression by transcriptionally activating genes such as cyclin D1 ( Tetsu and McCormick, 1999). However, increased expression of β-catenin is probably not responsible for cyclin D1 expression in our in vivo model since β-catenin is upregulated in both N- and E-cadherin transgenic mice, and the `binucleated' phenotype is observed only in the E-cadherin animals. Although E-cadherin was expressed throughout the myocardium, a relatively small fraction of the myocytes exhibited the `binucleated' phenotype. This suggests that expression of other factors required for DNA replication may be limited to a subset of cardiomyocytes present in the postnatal heart. A multinucleation phenotype without hypertrophy was observed previously in αMHC/cyclin D1 transgenic mice ( Soonpaa et al., 1997) consistent with the role of cyclin D1 in cell cycle progression; however, it is unclear how E-cadherin induces cyclin D1 expression in myocytes. In contrast to our findings, epithelial cell proliferation decreased when E-cadherin was overexpressed in the intestinal crypts of transgenic mice ( Hermiston et al., 1996) and in mammary carcinoma cells ( St Croix et al., 1998) demonstrating that cadherin `signaling' activity is dependent on cellular context. Interestingly, in the adult, normally N-cadherin protein levels are highest in the heart and brain, two tissues that express little or no E-cadherin. Normally, N-cadherin-expressing myocytes and neurons withdraw from the cell cycle following terminal differentiation, whereas E-cadherin-expressing epithelial cells maintain their capacity to undergo cell division in the adult. The ability of E-cadherin to stimulate DNA synthesis in myocytes, in contrast to N-cadherin, suggests that N-cadherin may normally provide growth inhibitory signals to regulate cell cycle progression in the postnatal heart. It is intriguing to speculate that different cadherin subtypes may regulate cell cycle progression in differentiated tissues. A 69-amino acid region of EC4 of N-cadherin was recently shown to be sufficient to promote both an epithelial to mesenchyme transition in squamous epithelial cells and increased cell motility ( Kim et al., 2000). In future studies, it will be of interest to determine whether the cytoplasmic or extracellular domain of E-cadherin is responsible for the `binucleated' phenotype.
The intercalated disc acts as an organizing center for the adjoining myocytes with different junctional complexes performing specific inter-related functions. Structural perturbation of the intercalated disc accompanied by downregulation of N-cadherin was observed in a hereditary hamster model of dilated cardiomyopathy ( Fujio et al., 1995). Furthermore, in a chronic pressure overload model,β -catenin was redistributed from the plasma membrane to the cytoplasm; however, there was no change in N-cadherin ( Wang and Gerdes, 1999). The downregulation of Cx43 and increased cytoplasmic localization of vinculin in the E-cadherin transgenic mice are consistent with observations made in patients with chronic heart failure ( Peters et al., 1993; Schaper et al., 1991). The dramatic effect on Cx43 expression is probably not caused by cadherin overexpression per se, but due to the severe hypertrophy response caused by ectopic expression of E-cadherin in the myocardium.
The ability of cancer cells to alter their cadherin profile suggests that other diseases may also be affected by inappropriate cadherin expression. In the future, it will be interesting to examine cardiac tissue from patients to determine whether pressure overload, viral infection, and other insults may lead to inappropriate expression of E-cadherin. For example, misexpression may result from demethylation of the E-cadherin promoter and its subsequent transcriptional activation in cardiomyocytes. Furthermore, the bacterium Listeria monocytogenes uses human E-cadherin as a receptor. Recently, a transgenic mouse model for Listeria infection was generated by expressing human E-cadherin in the intestinal epithelium ( Lecuit et al., 2001). It will be interesting to determine whether our animal model may provide a novel paradigm to examine bacterial infection in the heart.
Cell-cell and cell-extracellular matrix interactions are important for tissue homeostasis. Altering cadherin-mediated adhesion, as demonstrated here, had a pronounced effect on cardiac pathology. In contrast, similar transgenic experiments expressing wild-type α5 integrin, a fibronectin receptor, did not interfere with heart function. However, in the same study an α5 integrin molecule with its cytoplasmic domain deleted (i.e. gain of function) caused severe cardiac hypertrophy ( Valencik and McDonald, 2001). Furthermore, proteins involved in regulation of the actin cytoskeleton, such as the small GTP-binding protein rac1 and the muscle LIM protein MLP, have been implicated in cardiomyopathy in mice ( Arber et al., 1997; Sussman et al., 2000) and humans ( Zolk et al., 2000). Taken together, the data suggest that dysregulation of either cadherin or the actin cytoskeleton can lead to impaired force transmission and dilated cardiomyopathy.
In summary, we provide the first evidence that altering the cadherin composition of the intercalated disc can lead to cardiomyopathy and that E-cadherin, but not N-cadherin, can stimulate DNA synthesis in cardiomyocytes normally withdrawn from the cell cycle.
Acknowledgements
We thank Karen Knudsen for her comments on the manuscript, Jean Richa and the University of Pennsylvania Transgenic Core Facility, and Neelima Shah and the Biomedical Imaging Core Facility for performing the electron microscopy analysis. This work was supported by grants from the National Institutes of Health (HL57554) and American Heart Association Pennsylvania/Delaware Affiliate (0051086U) to G.L.R.
- Accepted January 21, 2002.
- © The Company of Biologists Limited 2002
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