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1.  Arrhythmogenic right ventricular dysplasia, a cell adhesion cardiomyopathy: insights into disease pathogenesis from preliminary genotype—phenotype assessment 
Heart  2006;92(12):1720-1723.
Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetically determined heart muscle disorder presenting clinically with even lethal ventricular arrhythmias, particularly in the young and athletes. It is reported familial with recessive and most commonly dominant inheritance. Disease‐causing genes are increasingly recognised among desmosomal proteins plakoglobin, desmoplakin, plakophilin2, and desmoglein2 displaying phenotypic heterogeneity. Mutations in the plakoglobin and desmoplakin genes have been identified to underlie recessive ARVC associated with woolly hair and palmoplantar keratoderma (Naxos disease), while mutations in plakophilin2, desmoglein2 as well as desmoplakin have been identified to underlie the dominant non‐syndromic form. Preliminary genotype–phenotype assessment indicates that mutations affecting the outer dense plaque of desmosome (desmoglein2, plakoglobin, plakophilin2 and the N‐terminal of desmoplakin) result in ARVC with the ordinary described phenotype. However, mutations at the inner dense plaque, particularly affecting the desmin‐binding site of desmoplakin, may result in ARVC with predominantly left ventricular involvement and clinical overlapping with dilated cardiomyopathy. The interesting finding of abnormal distribution of plakoglobin, independently of the primarily affected protein, might suggest a common pathway for plakoglobin in ARVC pathogenesis.
PMCID: PMC1861281  PMID: 16698823
arrhythmogenic right ventricular dysplasia/cardiomyopathy; Naxos disease; cell‐adhesions; desmosomal proteins; sudden death
2.  Altered Desmosomal Proteins in Granulomatous Myocarditis and Potential Pathogenic Links to Arrhythmogenic Right Ventricular Cardiomyopathy 
Immunoreactive signal for the desmosomal protein plakoglobin (γ-catenin) is reduced at cardiac intercalated disks in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a highly arrhythmogenic condition caused by mutations in genes encoding desmosomal proteins. Previously, we observed a “false positive” case in which plakoglobin signal was reduced in a patient initially thought to have ARVC but who actually had cardiac sarcoidosis. Sarcoidosis can masquerade clinically as ARVC, but has not previously been associated with altered desmosomal proteins.
Methods and Results
We observed marked reduction in immunoreactive signal for plakoglobin at cardiac myocyte junctions in patients with sarcoidosis and giant cell myocarditis, both highly arrhythmogenic forms of myocarditis associated with granulomatous inflammation. In contrast, plakoglobin signal was not depressed in lymphocytic (non-granulomatous) myocarditis. To determine whether cytokines might promote dislocation of plakoglobin from desmosomes, we incubated cultures of neonatal rat ventricular myocytes with selected inflammatory mediators. Brief exposure to low concentrations of IL-17, TNFα and IL-6, cytokines implicated in granulomatous myocarditis, caused translocation of plakoglobin from cell-cell junctions to intracellular sites, whereas other potent cytokines implicated in non-granulomatous myocarditis had no effect, even at much high concentrations. We also observed myocardial expression of IL-17 and TNFα, and elevated serum levels of inflammatory mediators including IL-6R, IL-8, MCP1 and MIP1β in ARVC patients (all p<0.0001 compared with controls).
These results suggest novel disease mechanisms involving desmosomal proteins in granulomatous myocarditis and implicate cytokines, perhaps derived in part from the myocardium, in disruption of desmosomal proteins and arrhythmogenesis in ARVC.
PMCID: PMC3203520  PMID: 21859801
plakoglobin; desmosome; sarcoidosis; giant cell myocarditis; cytokines
3.  Desmosomal Cadherins Are Decreased in Explanted Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Patient Hearts 
PLoS ONE  2013;8(9):e75082.
Arrhythmogenic right ventricular Dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited cardiomyopathy associated with ventricular arrhythmia, heart failure and sudden death. Genetic studies have demonstrated the central role of desmosomal proteins in this disease, where 50% of patients harbor a mutation in a desmosmal gene. However, clinical diagnosis of the disease remains difficult and molecular mechanisms appears heterogeneous and poorly understood. The aim of this study was to characterize the expression profile of desmosomal proteins in explanted ARVD/C heart samples, in order to identify common features of the disease.
Methods and Results
We examined plakophilin-2, desmoglein-2, desmocollin-2, plakoglobin and β-catenin protein expression levels from seven independent ARVD/C heart samples compared to two ischemic, five dilated cardiomyopathy and one healthy heart sample as controls. Ventricular and septum sections were examined by immunoblot analysis of total heart protein extracts and by immunostaining.
Immunoblots indicated significant decreases in desmoglein-2 and desmocollin-2, independent of any known underlying mutations, whereas immune-histochemical analysis showed normal localization of all desmosomal proteins. Quantitative RT-PCR revealed normal DSG2 and DSC2 mRNA transcript levels, suggesting increased protein turn-over rather than transcriptional down regulation.
Reduced cardiac desmoglein-2 and desmocollin-2 levels appear to be specifically associated with ARVD/C, independent of underlying mutations. These findings highlight a key role of desmosomal cadherins in the pathophysiology of ARVD/C. Whether these reductions could be considered as specific markers for ARVD/C requires replication analysis.
PMCID: PMC3781033  PMID: 24086444
4.  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.
PMCID: PMC2879923  PMID: 20585598
5.  Cardiac Tissue-Restricted Deletion of Plakoglobin Results in Progressive Cardiomyopathy and Activation of β-Catenin Signaling▿ †  
Molecular and Cellular Biology  2011;31(6):1134-1144.
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.
PMCID: PMC3067899  PMID: 21245375
6.  Remodeling of the cardiac sodium channel, Connexin43 and Plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy 
Arrhythmogenic cardiomyopathy (AC) is tightly associated with desmosomal mutations in the majority of patients. Arrhythmogenesis in AC patients is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. The aim of the present study was to assess the immunoreactive signal levels of the sodium channel protein NaV1.5, as well as Connexin43 and Plakoglobin, in myocardial specimens obtained from AC patients.
Left and right ventricular free wall (LVFW/RVFW) post-mortem material was obtained from 5 AC patients and 5 age and sex-matched controls. RV septal biopsies (RVSB) were taken from another 15 AC patients. All patients fulfilled the 2010 revised Task Force Criteria for AC diagnosis. Immunohistochemical analyses were performed using antibodies against Connexin43 (Cx43), Plakoglobin, NaV1.5, Plakophilin-2 and N-Cadherin.
N-Cadherin and Desmoplakin immunoreactive signals and distribution were normal in AC patients compared to control. Plakophilin-2 signals were unaffected unless a PKP2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to control. Immunoreactive signal levels of PKG, Cx43 and NaV1.5 were disturbed in 74%, 70% and 65% of the patients, respectively.
Reduced immunoreactive signal of PKG, Cx43 and NaV1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Nav1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies.
PMCID: PMC3608196  PMID: 23178689
7.  Arrhythmogenic right ventricular cardiomyopathy/dysplasia 
Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a heart muscle disease clinically characterized by life-threatening ventricular arrhythmias. Its prevalence has been estimated to vary from 1:2,500 to 1:5,000. ARVC/D is a major cause of sudden death in the young and athletes. The pathology consists of a genetically determined dystrophy of the right ventricular myocardium with fibro-fatty replacement to such an extent that it leads to right ventricular aneurysms. The clinical picture may include: a subclinical phase without symptoms and with ventricular fibrillation being the first presentation; an electrical disorder with palpitations and syncope, due to tachyarrhythmias of right ventricular origin; right ventricular or biventricular pump failure, so severe as to require transplantation. The causative genes encode proteins of mechanical cell junctions (plakoglobin, plakophilin, desmoglein, desmocollin, desmoplakin) and account for intercalated disk remodeling. Familiar occurrence with an autosomal dominant pattern of inheritance and variable penetrance has been proven. Recessive variants associated with palmoplantar keratoderma and woolly hair have been also reported. Clinical diagnosis may be achieved by demonstrating functional and structural alterations of the right ventricle, depolarization and repolarization abnormalities, arrhythmias with the left bundle branch block morphology and fibro-fatty replacement through endomyocardial biopsy. Two dimensional echo, angiography and magnetic resonance are the imaging tools for visualizing structural-functional abnormalities. Electroanatomic mapping is able to detect areas of low voltage corresponding to myocardial atrophy with fibro-fatty replacement. The main differential diagnoses are idiopathic right ventricular outflow tract tachycardia, myocarditis, dialted cardiomyopathy and sarcoidosis. Only palliative therapy is available and consists of antiarrhythmic drugs, catheter ablation and implantable cardioverter defibrillator. Young age, family history of juvenile sudden death, QRS dispersion ≥ 40 ms, T-wave inversion, left ventricular involvement, ventricular tachycardia, syncope and previous cardiac arrest are the major risk factors for adverse prognosis. Preparticipation screening for sport eligibility has been proven to be effective in detecting asymptomatic patients and sport disqualification has been life-saving, substantially declining sudden death in young athletes.
PMCID: PMC2222049  PMID: 18001465
8.  Mutations with pathogenic potential in proteins located in or at the composite junctions of the intercalated disk connecting mammalian cardiomyocytes: a reference thesaurus for arrhythmogenic cardiomyopathies and for Naxos and Carvajal diseases 
Cell and Tissue Research  2012;348(2):325-333.
In the past decade, an avalanche of findings and reports has correlated arrhythmogenic ventricular cardiomyopathies (ARVC) and Naxos and Carvajal diseases with certain mutations in protein constituents of the special junctions connecting the polar regions (intercalated disks) of mature mammalian cardiomyocytes. These molecules, apparently together with some specific cytoskeletal proteins, are components of (or interact with) composite junctions. Composite junctions contain the amalgamated fusion products of the molecules that, in other cell types and tissues, occur in distinct separate junctions, i.e. desmosomes and adherens junctions. As the pertinent literature is still in an expanding phase and is obviously becoming important for various groups of researchers in basic cell and molecular biology, developmental biology, histology, physiology, cardiology, pathology and genetics, the relevant references so far recognized have been collected and are presented here in the following order: desmocollin-2 (Dsc2, DSC2), desmoglein-2 (Dsg2, DSG2), desmoplakin (DP, DSP), plakoglobin (PG, JUP), plakophilin-2 (Pkp2, PKP2) and some non-desmosomal proteins such as transmembrane protein 43 (TMEM43), ryanodine receptor 2 (RYR2), desmin, lamins A and C, striatin, titin and transforming growth factor-β3 (TGFβ3), followed by a collection of animal models and of reviews, commentaries, collections and comparative studies.
PMCID: PMC3349860  PMID: 22450909
Arrhythmogenic ventricular cardiomyopathy; Carvajal disease; Composite junction; Desmosomes; Intercalated disk; Naxos disease
9.  Compound and Digenic Heterozygosity Contributes to Arrhythmogenic Right Ventricular Cardiomyopathy 
To define the genetic basis of arrhythmogenic right ventricular cardiomyopathy.
Arrhythmogenic right ventricular cardiomyopathy (ARVC), characterized by right ventricular fibrofatty replacement and arrhythmias, causes sudden death. Autosomal dominant Inheritance, reduced penetrance, and 7 desmosome-encoding causative genes are known. The basis of low penetrance is poorly understood.
ARVC probands and family members were enrolled, blood obtained, lymphoblastoid cell lines immortalized, DNA extracted, PCR amplification of desmosome-encoding genes performed, PCR products sequenced and diseased tissue samples studied for intercellular junction protein distribution using confocal immunofluorescence microscopy and antibodies against key proteins.
We identified 21 variants in plakophilin-2 (PKP2) in 38 of 198 probands (19%), including missense, nonsense, splice site, and deletion/insertion mutations. Pedigrees showed wide intra-familial variability (severe early-onset disease to asymptomatic individuals). In 9/38 probands, PKP2 variants were identified that were encoded in trans (compound heterozygosity). The 38 probands hosting PKP2 variants were screened for other desmosomal genes mutations; second variants (digenic heterozygosity) were identified in 16/38 subjects with PKP2 variants (42%) including desmoplakin (DSP, n=6), desmoglein-2 (DSG2, n=5), plakophilin-4 (PKP4, n=1), and desmocollin-2 (DSC2, n=1). Heterozygous mutations in non-PKP 2desmosomal genes occurred in 14/198 subjects (7%), including DSP (n=4), DSG2 (n=5), DSC2 (n=3), and junctional plakoglobin (JUP, n=2). All variants occurred in conserved regions; none were identified in 700 ethnic-matched controls.
Immunohistochemical analysis demonstrated abnormalities of protein architecture.
These data suggest that the genetic basis of ARVC includes reduced penetrance with compound and digenic heterozygosity. Disturbed junctional cytoarchitecture in subjects with desmosomal mutations confirms that ARVC is a disease of the desmosome and cell junction.
PMCID: PMC2852685  PMID: 20152563
Arrhythmias; Cardiomyopathies; Desmosomes; Intercalated Disks; Genetic Mutations
10.  Arrhythmogenic Right Ventricular Cardiomyopathy is A Disease of Cardiac Stem Cells 
Current Opinion in Cardiology  2010;25(3):222-228.
To review recent developments in clinical aspects, molecular geneticsand pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC).
Recent findings
ARVC is a primary disease of the myocardium characterized by fibro-adipocytic replacement of myocytes, predominantly in the right ventricle.
Phenotypic expression of ARVC is variable and a significant number of patients may exhibit a subtle phenotype, particularly in the early stages of the disease. Mutations in DSP, JUP, PKP2, DSG2 and DSC2; encoding desmosomal proteins desmoplakin (DP), plakoglobin (PG), plakophilin 2 (PKP2), desmoglein 2 (DSG2), and desmocollin 2 (DSC2), respectively, cause ARVC. Thus, ARVC, at least in a subset, is a disease of desmosomes. In addition, mutations in TMEM43 and TGFB1 have been associated with ARVC. Mechanistic studies indicate that suppressed canonical Wnt signaling, imposed by nuclear PG, is the responsible mechanism for the pathogenesis of ARVC. It leads to the differentiation of a subset of second heart field cardiac progenitor cells at the epicardium to adipocytes due to enhanced expression of adipogenic factors. This mechanism explains the predominant involvement of the right ventricle in ARVC. Hence, ARVC is the first identified disease of disrupted differentiation of cardiac progenitor cells.
Advances in molecular genetics and the pathogenesis of ARVC could afford the opportunity for a genetic-based diagnosis and development of novel diagnostic markers and therapeutic targets aimed to prevent, attenuate and reverse the evolving phenotype.
PMCID: PMC2980568  PMID: 20124997
Cardiomyopathy; Genetics; Sudden death; Heart failure; Stem cells
11.  Desmoglein 2 mutant mice develop cardiac fibrosis and dilation 
Basic Research in Cardiology  2011;106(4):617-633.
Desmosomes are cell–cell adhesion sites and part of the intercalated discs, which couple adjacent cardiomyocytes. The connection is formed by the extracellular domains of desmosomal cadherins that are also linked to the cytoskeleton on the cytoplasmic side. To examine the contribution of the desmosomal cadherin desmoglein 2 to cardiomyocyte adhesion and cardiac function, mutant mice were prepared lacking a part of the extracellular adhesive domain of desmoglein 2. Most live born mutant mice presented normal overall cardiac morphology at 2 weeks. Some animals, however, displayed extensive fibrotic lesions. Later on, mutants developed ventricular dilation leading to cardiac insufficiency and eventually premature death. Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed. Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue. Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure. Taken together, the desmoglein 2-mutant mice display features of dilative cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy, an inherited human heart disease with pronounced fibrosis and ventricular arrhythmias that has been linked to mutations in desmosomal proteins including desmoglein 2.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-011-0175-y) contains supplementary material, which is available to authorized users.
PMCID: PMC3105238  PMID: 21455723
Desmoglein 2; Desmosome; Mouse model; Cardiomyopathy; ARVC
12.  A novel desmocollin-2 mutation reveals insights into the molecular link between desmosomes and gap junctions 
Heart Rhythm  2011;8(5):711-718.
Cellular adhesion mediated by cardiac desmosomes is a prerequisite for proper electric propagation mediated by gap junctions in the myocardium. However, the molecular principles underlying this interdependence are not fully understood.
The purpose of this study was to determine potential causes of right ventricular conduction abnormalities in a patient with borderline diagnosis of arrhythmogenic right ventricular cardiomyopathy.
To assess molecular changes, the patient's myocardial tissue was analyzed for altered desmosomal and gap junction (connexin43) protein levels and localization. In vitro functional studies were performed to characterize the consequences of the desmosomal mutations.
Loss of plakoglobin signal was evident at the cell junctions despite expression of the protein at control levels. Although the distribution of connexin43 was not altered, total protein levels were reduced and changes in phosphorylation were observed. The truncation mutant in desmocollin-2a is deficient in binding plakoglobin. Moreover, the ability of desmocollin-2a to directly interact with connexin43 was abolished by the mutation. No pathogenic potential of the desmoglein-2 missense change was identified.
The observed abnormalities in gap junction protein expression and phosphorylation, which precede an overt cardiac phenotype, likely are responsible for slow myocardial conduction in this patient. At the molecular level, altered binding properties of the desmocollin-2a mutant may contribute to the changes in connexin43. In particular, the newly identified interaction between the desmocollin-2a isoform and connexin43 provides novel insights into the molecular link between desmosomes and gap junctions.
PMCID: PMC3085091  PMID: 21220045
Cardiomyopathy; Conduction; Connexin43; Desmocollin-2; Desmoglein-2; Desmosome; Functional studies; Gap junction; Mutation; Plakoglobin; ARVC, arrhythmogenic right ventricular cardiomyopathy; Cx43, connexin43; DAPI, 4′,6-diamidino-2-phenylindole; DSC2, desmocollin-2; DSG2, desmoglein-2; DSP, desmoplakin; GFP, green fluorescent protein; GST, glutathione-S-transferase; ICS, intracellular cadherin segment; PG, plakoglobin; PKP2, plakophilin-2; RV, right ventricle; YFP, yellow fluorescent protein
13.  Autoimmune and infectious skin diseases that target desmogleins 
Desmosomes are intercellular adhesive junctions of epithelial cells that contain two major transmembrane components, the desmogleins (Dsg) and desmocollins (Dsc), which are cadherin-type cell–cell adhesion molecules and are anchored to intermediate filaments of keratin through interactions with plakoglobin and desmoplakin. Desmosomes play an important role in maintaining the proper structure and barrier function of the epidermis and mucous epithelia. Four Dsg isoforms have been identified to date, Dsg1–Dsg4, and are involved in several skin and heart diseases. Dsg1 and Dsg3 are the two major Dsg isoforms in the skin and mucous membranes, and are targeted by IgG autoantibodies in pemphigus, an autoimmune disease of the skin and mucous membranes. Dsg1 is also targeted by exfoliative toxin (ET) released by Staphylococcus aureus in the infectious skin diseases bullous impetigo and staphylococcal scalded skin syndrome (SSSS). ET is a unique serine protease that shows lock and key specificity to Dsg1. Dsg2 is expressed in all tissues possessing desmosomes, including simple epithelia and myocardia, and mutations in this gene are responsible for arrhythmogenic right ventricular cardiomyopathy/dysplasia. Dsg4 plays an important adhesive role mainly in hair follicles, and Dsg4 mutations cause abnormal hair development. Recently, an active disease model for pemphigus was generated by a unique approach using autoantigen-deficient mice that do not acquire tolerance against the defective autoantigen. Adoptive transfer of Dsg3−/− lymphocytes into mice expressing Dsg3 induces stable anti-Dsg3 IgG production with development of the pemphigus phenotype. This mouse model is a valuable tool with which to investigate immunological mechanisms of harmful IgG autoantibody production in pemphigus. Further investigation of desmoglein molecules will continue to provide insight into the unsolved pathophysiological mechanisms of diseases and aid in the development of novel therapeutic strategies with minimal side effects.
PMCID: PMC3108298  PMID: 20467217
cadherin; pemphigus; impetigo; SSSS; mouse model; ELISA
14.  Quantitative Immunohistochemistry of Desmosomal Proteins (Plakoglobin, Desmoplakin and Plakophilin), Connexin-43, and N-cadherin in Arrhythmogenic Cardiomyopathy: An Autopsy Study 
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder related to mutations in desmosomal proteins. The current study tests the hypothesis that immunohistochemical staining for desmosomal proteins is of diagnostic utility by studying autopsy-confirmed cases of ARVC.
Methods and Results:
We studied 23 hearts from patients dying suddenly with ARVC. Control subject tissues were 21 hearts from people dying from non-cardiac causes (n=15), dilated cardiomyopathy (n=3) and coronary artery disease (n=3).
Areas free of fibrofatty change or scarring were assessed on 50 sections from ARVC (24 left ventricle, 26 right ventricle) and 28 sections from controls. Immunohistochemical stains against plakoglobin, plakophilin, desmoplakin, connexin-43, and N-cadherin were applied and area expression analyzed by computerized morphometry. Desmin was stained as a control for fixation and similarly analyzed.
The mean area of desmin expression was similar in controls and ARVC (86% vs. 85%, p=0.6). Plakoglobin expression was 4.9% ± 0.3% in controls, vs. 4.6% ± 0.3% in ARVC (p=0.3). Plakophilin staining was 4.8% ± 0.3% in controls vs. 4.4% ± 03% in ARVC (p=0.3). Desmoplakin staining was 3.4% in controls vs. 3.2 ± 0.2% in ARVC (p=0.6). There were no significant differences when staining was compared between right and left ventricles (all p > 0.1).
For non-desmosomal proteins, the mean area of connexin-43 staining showed no significant difference by presence of disease.
The small and insignificant decrease in junction protein expression in ARVC suggests that immunohistochemistry is not a useful tool for the diagnosis.
PMCID: PMC3680985  PMID: 23802019
ARVC; arrhytmogenic cardiomyopaty; sudden death; autopsy.
15.  Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations 
Cardiovascular Research  2010;90(1):77-87.
Recent immunohistochemical studies observed the loss of plakoglobin (PG) from the intercalated disc (ID) as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting a final common pathway for this disease. However, the underlying molecular processes are poorly understood.
Methods and results
We have identified novel mutations in the desmosomal cadherin desmocollin 2 (DSC2 R203C, L229X, T275M, and G371fsX378). The two missense mutations (DSC2 R203C and T275M) have been functionally characterized, together with a previously reported frameshift variant (DSC2 A897fsX900), to examine their pathogenic potential towards PG's functions at the ID. The three mutant proteins were transiently expressed in various cellular systems and assayed for expression, processing, localization, and binding to other desmosomal components in comparison to wild-type DSC2a protein. The two missense mutations showed defects in proteolytic cleavage, a process which is required for the functional activation of mature cadherins. In both cases, this is thought to cause a reduction of functional DSC2 at the desmosomes in cardiac cells. In contrast, the frameshift variant was incorporated into cardiac desmosomes; however, it showed reduced binding to PG.
Despite different modes of action, for all three variants, the reduced ability to provide a ligand for PG at the desmosomes was observed. This is in agreement with the reduced intensity of PG at these structures observed in ARVC patients.
PMCID: PMC3058729  PMID: 21062920
Arrhythmogenic right ventricular cardiomyopathy; Desmocollin-2; Desmosome; Functional studies; Mutation
16.  The Amino-terminal Domain of Desmoplakin Binds to Plakoglobin and Clusters Desmosomal Cadherin–Plakoglobin Complexes  
The Journal of Cell Biology  1997;139(3):773-784.
The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.
PMCID: PMC2141713  PMID: 9348293
17.  The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential 
The Journal of Cell Biology  1996;133(2):359-369.
Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C- domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so- called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.
PMCID: PMC2120792  PMID: 8609168
18.  Genome-wide association identifies a deletion in the 3’ untranslated region of Striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy 
Human genetics  2010;128(3):315-324.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins.
We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine-mapping and direct DNA sequencing identified an eight base pair deletion in the 3’ untranslated region (UTR) of the striatin (STRN) gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3’ UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, plakophilin- 2, plakoglobin and desmoplakin, all involved in the pathogenesis of ARVC in human beings.
We suggest that striatin may serve as a novel candidate gene for human ARVC.
PMCID: PMC2962869  PMID: 20596727
19.  Molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia/cardiomyopathy 
Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. Here, we studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (boxer dogs).
We studied hearts from 12 boxers with confirmed ARVD/C and two controls. Ventricular sections from four animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for western blot analysis. Proteins investigated were N-cadherin, plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin43 (Cx43).
In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted boxers displayed preservation of N-Cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis revealed no mutations in desmoplakin, plakoglobin, Cx43, or plakophilin 2.
Mutation(s) responsible for ARVD/C in boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell-cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help advance our understanding of the molecular basis, pathophysiology and potential therapeutic approach to patients with ARVD/C.
PMCID: PMC2080779  PMID: 17765621
20.  Loss of Cadherin-Binding Proteins β-Catenin and Plakoglobin in the Heart Leads to Gap Junction Remodeling and Arrhythmogenesis 
Molecular and Cellular Biology  2012;32(6):1056-1067.
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.
PMCID: PMC3295003  PMID: 22252313
21.  Geographical distribution of plakophilin-2 mutation prevalence in patients with arrhythmogenic cardiomyopathy 
Netherlands Heart Journal  2012;20(5):234-239.
Arrhythmogenic cardiomyopathy (AC) is characterised by myocardial fibrofatty tissue infiltration and presents with palpitations, ventricular arrhythmias, syncope and sudden cardiac death. AC is associated with mutations in genes encoding the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), desmoglein-2 (DSG2), desmocollin-2 (DSC2) and junctional plakoglobin (JUP). In the present study we compared 28 studies (2004–2011) on the prevalence of mutations in desmosomal protein encoding genes in relation to geographic distribution of the study population. In most populations, mutations in PKP2 showed the highest prevalence. Mutation prevalence in DSP, DSG2 and DSC2 varied among the different geographic regions. Mutations in JUP were rarely found, except in Denmark and the Greece/Cyprus region.
PMCID: PMC3346879  PMID: 22527912
Cardiomyopathy; Plakophilin-2; Mutation; Desmosome; Prevalence; Geography; Medicine & Public Health; Medicine/Public Health, general
22.  Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes 
Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent upon the plakoglobin binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyper-adhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.
PMCID: PMC3961504  PMID: 24056861
23.  Clinical usefulness of immunohistochemistry for plakoglobin, N-cadherin, and connexin-43 in the diagnosis of arrhythmogenic right ventricular cardiomyopathy 
Diagnosing arrhythmogenic right ventricular cardiomyopathy (ARVC) is often challenging because no single diagnostic tool is available to detect the disease. We evaluated whether analysis of plakoglobin, N-cadherin, and connexin-43 immunoreactivity can be used as a significant test in diagnosis of ARVC. We selected subjects with suspicion of ARVC (n=22) in patients who underwent endomyocardial biopsy (EMB) in Kyungpook National University Hospital (n=1326). The patients (n=22) were classified into definite ARVC patients (n=17) and borderline ARVC (n=5). We selected control subjects (n=20) who were autopsied and died of non-cardiac disease. Hematoxylin-eosin, Masson’s trichrome, and immunohistochemical stains for plakoglobin, N-cadherin, and connexin-43 were used for all specimens. Reduced immunoreactivity of plakoglobin was observed in 13 (76%) of the 17 patients with a definite ARVC and in 4 (80%) of the 5 patients with a borderline ARVC. All subjects displayed no significant reduction of the immunoreactivity for connexin-43 as well as for N-cadherin. Our investigation revealed that the immunohistochemical analysis for plakoglobin had an accuracy of 81%, 76% sensitivity, and 84% specificity in diagnosis of ARVC. Results of our study showed that the immunohistochemical analysis of plakoglobin had a relatively high sensitivity and specificity in ARVC, but immunohistochemistry for plakoglobin alone could not be relied upon as a diagnostic test for ARVC. We confirmed that N-cadherin and connexin-43 had no diagnostic value in ARVC.
PMCID: PMC3843274  PMID: 24294380
Plakoglobin; N-cadherin; connexin-43; arrhythmogenic right ventricular cardiomyopathy
24.  Genetic bases of arrhythmogenic right ventricular Cardiomyopathy 
Heart International  2006;2(1):17.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heart muscle disease in which the pathological substrate is a fibro-fatty replacement of the right ventricular myocardium. The major clinical features are different types of arrhythmias with a left branch block pattern. ARVC shows autosomal dominant inheritance with incomplete penetrance. Recessive forms were also described, although in association with skin disorders.
Ten genetic loci have been discovered so far and mutations were reported in five different genes. ARVD1 was associated with regulatory mutations of transforming growth factor beta-3 (TGFβ3), whereas ARVD2, characterized by effort-induced polymorphic arrhythmias, was associated with mutations in cardiac ryanodine receptor-2 (RYR2). All other mutations identified to date have been detected in genes encoding desmosomal proteins: plakoglobin (JUP) which causes Naxos disease (a recessive form of ARVC associated with palmoplantar keratosis and woolly hair); desmoplakin (DSP) which causes the autosomal dominant ARVD8 and plakophilin-2 (PKP2) involved in ARVD9. Desmosomes are important cell-to-cell adhesion junctions predominantly found in epidermis and heart; they are believed to couple cytoskeletal elements to plasma membrane in cell-to-cell or cell-to-substrate adhesions.
PMCID: PMC3184660  PMID: 21977247
Arrhythmias; Sudden death; Molecular genetics; Desmosomes
25.  Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 in cultured keratinocytes 
Experimental dermatology  2012;21(8):10.1111/j.1600-0625.2012.01520.x.
Mutations in the ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by western analysis. The expression of selected desmosomal, adherens and tight junction (TJ) proteins was then studied in the SPCA1-deficient and control keratinocytes cultured in low (0.06 mM) or high (1.2 mM) calcium concentration. The mRNA and protein levels of most TJ components were up-regulated in non-treated control keratinocyte cultures upon switch from low to high calcium concentration. In contrast, SPCA1-deficient keratinocytes displayed high levels of TJ proteins claudin 1 and 4 even in low calcium. ZO-1 did not however follow similar expression patterns. Protein levels of occludin, betacatenin, E-cadherin, desmoplakin, desmogleins 1-3, desmocollin 2/3 and plakoglobin did not show marked changes in SPCA1 deficient keratinocytes. Indirect immunofluorescence labeling revealed delayed translocation of desmoplakin and desmoglein 3 in desmosomes, and increased intracellular pools of TJ and desmosomal components in SPCA1 inhibited keratinocytes. The results show that SPCA1 regulates the levels of claudins 1 and 4, but does not affect desmosomal protein levels, indicating that TJ proteins are differently regulated. The results also suggest a potential role for claudins in HHD.
PMCID: PMC3879075  PMID: 22639968
Hailey-Hailey disease; tight junction; keratinocyte; ATP2C1; SPCA1

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