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Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart

M. Celeste Ferreira-Cornwell^1,*,, Yang Luo^1,*, Navneet Narula², Jennifer M. Lenox¹, Melanie Lieberman¹ and Glenn L. Radice^1,

¹ Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, 1355 Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104, USA
² Department of Pathology, University of Pennsylvania School of Medicine, 1355 Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104, USA
^* These authors contributed equally to this work
Present address: GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, USA

View larger version (40K): [in a new window]   Fig. 1. Cadherin misexpression and cardiac hypertrophy in transgenic mice. (A) The 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 Ncad and Ecad 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 the MHC/Ecad heart in comparison to MHC/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).

View larger version (62K): [in a new window]   Fig. 2. 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 MHC/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 MHC/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.

View larger version (88K): [in a new window]   Fig. 3. Histological analysis of wild-type and transgenic hearts. Heart sections from wild-type (A,D), MHC/Ncad (B,E), and MHC/Ecad (C,F,G,H) indicated that both transgenes can elicit a hypertrophic response; however, the MHC/Ecad was consistently more severe. The MHC/Ncad heart (B) was enlarged with dilated left ventricle and thin ventricular wall. In comparison, both ventricles and atria were severely dilated in the MHC/Ecad heart (C). Enlarged myocytes with hyperchromatic nuclei were observed in both transgenic hearts (E,F). Many `binucleated' myocytes were seen in the MHC/Ecad heart (F, arrows) in comparison to MHC/Ncad (E). At 2 months of age, left atrial thrombosis (arrow) was observed in the MHC/Ecad heart (G). Alizarin Red staining showed regions of calcification (arrows) in the ventricle and pericardium of the MHC/Ecad heart (H). Thrombosis and calcification were observed in the MHC/Ncad hearts (not shown). Bars, 2.5 mm (A,B,C,G); 50 µm (D,E,F); 500 µm (H).

View larger version (75K): [in a new window]   Fig. 4. Assessment of the `binucleated' phenotype in MHC/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 MHC/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 Ncad4 (B) and Ecad33 (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 MHC/Ecad transgenic lines such as E10. Indirect immunofluorescence of heart sections from 2-week-old animals indicated an increase in cyclin D1 (arrowheads) in MHC/Ecad (F), but not MHC/Ncad (E), consistent with the `binucleated' phenotype. Bar, 25 µm (A,B,C); 50 µm (E,F).

View larger version (118K): [in a new window]   Fig. 5. Expression and cellular localization of components of the intercalated disc. Heart sections from wild-type (A,D,G), MHC/Ncad (B,E,H), and MHC/Ecad (C,F,I) animals 4-weeks old were analyzed by indirect immunofluorescence for distribution of ß-catenin (A,B,C), connexin 43 (D,E,F) and vinculin (G,H,I). ß-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 MHC/Ecad (F) compared with MHC/Ncad (E). Vinculin staining appeared more concentrated in the intercalated disc of MHC/Ncad (H) compared with wild-type (G) hearts. By contrast, redistribution of vinculin to the cytoplasm resulted in less intercalated disc staining in the MHC/Ecad heart (I). Bar, 25 µm.

View larger version (52K): [in a new window]   Fig. 6. 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 MHC/Ncad (A) and MHC/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 Ecad heart compared with Ncad, consistent with the severe hypertrophy response caused by E-cadherin. GAPDH signal shows loading of samples between lanes. tg, transgene; d, days; w, weeks.

View larger version (26K): [in a new window]   Fig. 7. Time course of ANF expression in wild-type and transgenic hearts. Northern blot analysis of ANF mRNA in MHC/Ncad and MHC/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 in Ecad heart at one week of age compared with Ncad. ANF mRNA levels were normalized to GAPDH mRNA levels. tg, transgene; d, days; w, weeks.

View larger version (207K): [in a new window]   Fig. 8. Transmission electron microscopy of wild-type and transgenic hearts. Ventricular myocardium of 4-week-old wild-type (A,B) and MHC/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).

View larger version (23K): [in a new window]   Fig. 9. 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 MHC/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. In 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.