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

M. Celeste Ferreira-Cornwell 0 2 Yang Luo 2 Navneet Narula 1 Jennifer M. Lenox 2 Melanie Lieberman 2 Glenn L. Radice ) 2 0 Present address: GlaxoSmithKline Pharmaceuticals , Collegeville, PA 19426 , USA 1 Department of Pathology, University of Pennsylvania School of Medicine, 1355 Biomedical Research Building II/III , 421 Curie Blvd., Philadelphia, PA 19104 , USA 2 Center for Research on Reproduction and Women's Health - 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 Ncadherin 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. 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 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. 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). b Catenin or g -catenin (plakoglobin) bind directly to the Cterminal region of the cadherin, whereas p120 interacts with the juxtamembrane region (Anastasiadis and Reynolds, 2000). a -Catenin binds to b - or g -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, a Tcatenin, 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, Ncadherin 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 Ncadherin 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-c (...truncated)