Jifen Li

Induced Deletion of the N-Cadherin Gene in the Heart Leads to Dissolution of the Intercalated Disc Structure

The structural integrity of the heart is maintained by the end-to-end connection between the myocytes called the intercalated disc. The intercalated disc contains different junctional complexes that enable the myocardium to function as a syncytium. One of the junctional complexes, the zonula adherens or adherens junction, consists of the cell adhesion molecule, N-cadherin, which mediates strong homophilic cell–cell adhesion via linkage to the actin cytoskeleton. To determine the function of N-cadherin in the working myocardium, we generated a conditional knockout containing loxP sites flanking exon 1 of the N-cadherin (Cdh2) gene. Using a cardiac-specific tamoxifen-inducible Cre transgene, N-cadherin was deleted in the adult myocardium. Loss of N-cadherin resulted in disassembly of the intercalated disc structure, including adherens junctions and desmosomes. The mutant mice exhibited modest dilated cardiomyopathy and impaired cardiac function, with most animals dying within two months after tamoxifen administration. Decreased sarcomere length and increased Z-line thickness were observed in the mutant hearts consistent with loss of muscle tension because N-cadherin was no longer available to anchor myofibrils at the plasma membrane. Ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous ventricular tachycardia, confirming that the deaths were arrhythmic in nature. A significant decrease in the gap junction protein, connexin 43, was observed in the N-cadherin–depleted hearts. This animal model provides the first demonstration of the hierarchical relationship of the structural components of the intercalated disc in the working myocardium, thus establishing N-cadherin’s paramount importance in maintaining the structural integrity of the heart.

Cardiac-Specific Loss of N-Cadherin Leads to Alteration in Connexins With Conduction Slowing and Arrhythmogenesis

The remodeling of ventricular gap junctions, as defined by changes in size, distribution, or function, is a prominent feature of diseased myocardium. However, the regulation of assembly and maintenance of gap junctions remains poorly understood. To investigate N-cadherin function in the adult myocardium, we used a floxed N-cadherin gene in conjunction with a cardiac-specific tamoxifen-inducible Cre transgene. The mutant animals appeared active and healthy until their sudden death ≈2 months after deleting N-cadherin from the heart. Electrophysiologic analysis revealed abnormal conduction in the ventricles of mutant animals, including diminished QRS complex amplitude consistent with loss of electrical coupling in the myocardium. A significant decrease in the gap junction proteins, connexin-43 and connexin-40, was observed in N-cadherin–depleted myocytes. Perturbation of connexin function resulted in decreased ventricular conduction velocity, as determined by optical mapping. Our data suggest that perturbation of the N-cadherin/catenin complex in heart disease may be an underlying cause, leading to the establishment of the arrythmogenic substrate by destabilizing gap junctions at the cell surface.

Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling.

ABSTRACT Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/β-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase β-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3β. Finally, β-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects β-catenin activity in the heart and its implications for disease pathogenesis.Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/β-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase β-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3β. Finally, β-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects β-catenin activity in the heart and its implications for disease pathogenesis.

N-cadherin haploinsufficiency affects cardiac gap junctions and arrhythmic susceptibility.

Cardiac-specific deletion of the murine gene (Cdh2) encoding the cell adhesion molecule, N-cadherin, results in disassembly of the intercalated disc (ICD) structure and sudden arrhythmic death. Connexin 43 (Cx43)-containing gap junctions are significantly reduced in the heart after depleting N-cadherin, therefore we hypothesized that animals expressing half the normal levels of N-cadherin would exhibit an intermediate phenotype. We examined the effect of N-cadherin haploinsufficiency on Cx43 expression and susceptibility to induced arrhythmias in mice either wild-type or heterozygous for the Cx43 (Gja1)-null allele. An increase in hypophosphorylated Cx43 accompanied by a modest decrease in total Cx43 protein levels was observed in the N-cadherin heterozygous mice. Consistent with these findings N-cadherin heterozygotes exhibited increased susceptibility to ventricular arrhythmias compared to wild-type mice. Quantitative immunofluorescence microscopy revealed a reduction in size of large Cx43-containing plaques in the N-cadherin heterozygous animals compared to wild-type. Gap junctions were further decreased in number and size in the N-cad/Cx43 compound heterozygous mice with increased arrhythmic susceptibility compared to the single mutants. The scaffold protein, ZO-1, was reduced at the ICD in N-cadherin heterozygous cardiomyocytes providing a possible explanation for the reduction in Cx43 plaque size. These data provide further support for the intimate relationship between N-cadherin and Cx43 in the heart, and suggest that germline mutations in the human N-cadherin (Cdh2) gene may predispose patients to increased risk of cardiac arrhythmias.

Loss of Cadherin-Binding Proteins β-Catenin and Plakoglobin in the Heart Leads to Gap Junction Remodeling and Arrhythmogenesis

ABSTRACT Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome, including plakoglobin (JUP). We previously reported that cardiac tissue-specific plakoglobin (PG) knockout (PG CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin (CTNNB1), showed increased association with the gap junction protein connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and β-catenin specifically in the heart (i.e., double knockout [DKO]). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD, consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and β-catenin, are indispensable for maintaining mechanoelectrical coupling in the heart.

Loss of αT-catenin alters the hybrid adhering junctions in the heart and leads to dilated cardiomyopathy and ventricular arrhythmia following acute ischemia.

It is generally accepted that the intercalated disc (ICD) required for mechano-electrical coupling in the heart consists of three distinct junctional complexes: adherens junctions, desmosomes and gap junctions. However, recent morphological and molecular data indicate a mixing of adherens junctional and desmosomal components, resulting in a ‘hybrid adhering junction’ or ‘area composita’. The α-catenin family member αT-catenin, part of the N-cadherin–catenin adhesion complex in the heart, is the only α-catenin that interacts with the desmosomal protein plakophilin-2 (PKP2). Thus, it has been postulated that αT-catenin might serve as a molecular integrator of the two adhesion complexes in the area composita. To investigate the role of αT-catenin in the heart, gene targeting technology was used to delete the Ctnna3 gene, encoding αT-catenin, in the mouse. The αT-catenin-null mice are viable and fertile; however, the animals exhibit progressive cardiomyopathy. Adherens junctional and desmosomal proteins were unaffected by loss of αT-catenin, with the exception of the desmosomal protein PKP2. Immunogold labeling at the ICD demonstrated in the αT-catenin-null heart a preferential reduction of PKP2 at the area composita compared with the desmosome. Furthermore, gap junction protein Cx43 was reduced at the ICD, including its colocalization with N-cadherin. Gap junction remodeling in αT-catenin-knockout hearts was associated with an increased incidence of ventricular arrhythmias after acute ischemia. This novel animal model demonstrates for the first time how perturbation in αT-catenin can affect both PKP2 and Cx43 and thereby highlights the importance of understanding the crosstalk between the junctional proteins of the ICD and its implications for arrhythmogenic cardiomyopathy.

Alpha-Catenins Control Cardiomyocyte Proliferation by Regulating Yap Activity

Rationale: Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional coactivator Yes-associated protein (Yap) is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators and signaling pathways that control Yap activity in the heart are poorly understood. Objective: To investigate the role of &agr;-catenins in the heart using cardiac-specific &agr;E- and &agr;T-catenin double knockout mice. Methods and Results: We used 2 cardiac-specific Cre transgenes to delete both &agr;E-catenin (Ctnna1) and &agr;T-catenin (Ctnna3) genes either in the perinatal or in the adult heart. Perinatal depletion of &agr;-catenins increased cardiomyocyte number in the postnatal heart. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the &agr;-catenin double knockout hearts. Fetal genes were increased in the &agr;-catenin double knockout hearts indicating a less mature cardiac gene expression profile. Knockdown of &agr;-catenins in neonatal rat cardiomyocytes also resulted in increased proliferation, which could be blocked by knockdown of Yap. Finally, inactivation of &agr;-catenins in the adult heart using an inducible Cre led to increased nuclear Yap and cardiomyocyte proliferation and improved contractility after myocardial infarction. Conclusions: These studies demonstrate that &agr;-catenins are critical regulators of Yap, a transcriptional coactivator essential for cardiomyocyte proliferation. Furthermore, we provide proof of concept that inhibiting &agr;-catenins might be a useful strategy to promote myocardial regeneration after injury.

A New Perspective on Intercalated Disc Organization: Implications for Heart Disease

Adherens junctions and desmosomes are intercellular adhesive junctions and essential for the morphogenesis, differentiation, and maintenance of tissues that are subjected to high mechanical stress, including heart and skin. The different junction complexes are organized at the termini of the cardiomyocyte called the intercalated disc. Disruption of adhesive integrity via mutations in genes encoding desmosomal proteins causes an inherited heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC). Besides plakoglobin, which is shared by adherens junctions and desmosomes, other desmosomal components, desmoglein-2, desmocollin-2, plakophilin-2, and desmoplakin are also present in ultrastructurally defined fascia adherens junctions of heart muscle, but not other tissues. This mixed-type of junctional structure is termed hybrid adhering junction or area composita. Desmosomal plakophilin-2 directly interacts with adherens junction protein alphaT-catenin, providing a new molecular link between the cadherin-catenin complex and desmosome. The area composita only exists in the cardiac intercalated disc of mammalian species suggesting that it evolved to strengthen mechanical coupling in the heart of higher vertebrates. The cross-talk among different junctions and their implication in the pathogenesis of ARVC are discussed in this review.

Beyond cell adhesion: the role of armadillo proteins in the heart.

Plakoglobin (PG, γ-Catenin, JUP), a member of the armadillo protein family and close homolog of β-catenin, functions to link cell surface cadherin molecules with the cytoskeleton. PG is the only junctional component found in both desmosomes and adherens junctions and thus plays a critical role in the regulation of cell-cell adhesion. Similar to β-catenin, PG is able to interact with components of the Wnt signaling pathway and directly affect gene expression by binding with LEF/TCF transcription factors. In addition, it has been proposed that PG functions primarily as a competitive inhibitor of β-catenin transcriptional activity by sequestering LEF/TCF. Compared to β-catenin, the contribution of PG as a transcriptional regulator in either physiological or pathological conditions is poorly understood. There is increasing clinical interest in PG as both a structural protein as well as a signaling molecule as mutations have been identified in the human PG gene that cause Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) and cutaneous syndromes. This review will discuss the connection between altered cell adhesion and gene expression and its contribution to disease pathogenesis.

N-cadherin haploinsufficiency increases survival in a mouse model of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is often detected at a late stage, hence the identification of new therapies that have potential to block tumor progression is critical for this lethal disease. N-cadherin upregulation has been observed in many cancers including PDA, however, a causal role for this cell adhesion receptor in disease progression has yet to be defined. The concomitant expression of oncogenic KrasG12D and mutant p53 (Trp53R172H) in the murine pancreas results in metastatic PDA that recapitulates the cognate features of human pancreatic cancer providing an excellent animal model to identify genes required for tumor progression. Here we determine the consequences of genetically manipulating N-cadherin expression in a mouse model of PDA. Remarkably, mice with reduced N-cadherin expression (that is, Ncad−/+) survived 25% longer (177 vs 142 days, P<0.05) than animals expressing two wild-type N-cadherin (Cdh2) alleles. The survival benefit is likely due to a cumulative effect of N-cadherins role in different aspects of tumorigenesis including tumor-cell survival, growth, migration and invasion. Interestingly, reduced hedgehog signaling may contribute to the better prognosis for the Ncad−/+ mice. Moreover, the matrix metalloproteinase MMP-7, associated with poor prognosis in PDA, was reduced in Ncad−/+ tumors. Finally, Ncad−/+ tumor cells exhibited decreased FGF-stimulated ERK1/2 activation consistent with N-cadherins ability to promote FGFR signaling. These data support a critical role for N-cadherin in PDA and its potential prognostic value. Additionally, this study provides in vivo genetic evidence that the cell-surface protein N-cadherin represents a promising therapeutic target for the treatment of pancreatic cancer.