Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), an autosomal-dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement. We screened 112 ARVC/D probands for mutations in desmocollin-2 (DSC2) gene and detected two different amino-acid substitutions (p.E102K, p.I345T) and a frameshift variation (p.A897KfsX4) in 7 (6.2%) patients. DSC2a variant p.A897KfsX4, previously reported as a p.E896fsX900 mutation, was identified in five unrelated probands. Four of them were found to carry one or two mutations in different ARVC/D genes. Unexpectedly, p.A897KfsX4 variation was also found in 6 (1.5%) out of 400 control chromosomes. In vitro functional studies showed that, unlike wild-type DSC2a, this C-terminal mutated protein was localised in the cytoplasm. p.A897KfsX4 variation affects the last five amino acids of the DSC2a isoform but not of DSC2b. In contrast with what we found in other human tissues, in the heart DSC2b is more expressed than DSC2a, suggesting that relative deficiency of DSC2a might be compensated by isoform b. In conclusion, DSC2 gene mutations are not frequently involved in ARVC/D. The p.A897KfsX4 variation, identified in several Italian healthy control subjects, which affects only one of the two DSC2 isoforms, may be considered a rare variant, though possibly affecting phenotypic expression of concomitant ARVC/D mutations.
sudden death; ARVC/D; desmosome; DSC2; polymorphism
To review recent developments in clinical aspects, molecular geneticsand pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC).
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.
Cardiomyopathy; Genetics; Sudden death; Heart failure; Stem cells
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.
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.
Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement.
We screened 54 ARVC probands for mutations in desmocollin-2 (DSC2), the only desmocollin isoform expressed in cardiac tissue.
Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing.
To evaluate the pathogenic potentials of the DSC2 mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells.
We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions.
In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm.
The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.
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.
Arrhythmogenic ventricular cardiomyopathy; Carvajal disease; Composite junction; Desmosomes; Intercalated disk; Naxos disease
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.
plakoglobin; desmosome; sarcoidosis; giant cell myocarditis; cytokines
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.
Cardiomyopathy; Plakophilin-2; Mutation; Desmosome; Prevalence; Geography; Medicine & Public Health; Medicine/Public Health, general
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.
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.
ARVC; arrhytmogenic cardiomyopaty; sudden death; autopsy.
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.
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
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.
Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype with a yet-to-be-defined molecular mechanism. We established atrial myocyte cell lines expressing siRNA against desmoplakin (DP), responsible for human ARVC. We show suppression of DP expression leads to nuclear localization of the desmosomal protein plakoglobin and a 2-fold reduction in canonical Wnt/β-catenin signaling through Tcf/Lef1 transcription factors. The ensuing phenotype is increased expression of adipogenic and fibrogenic genes and accumulation of fat droplets. We further show that cardiac-restricted deletion of Dsp, encoding DP, impairs cardiac morphogenesis and leads to high embryonic lethality in the homozygous state. Heterozygous DP-deficient mice exhibited excess adipocytes and fibrosis in the myocardium, increased myocyte apoptosis, cardiac dysfunction, and ventricular arrhythmias, thus recapitulating the phenotype of human ARVC. We believe our results provide for a novel molecular mechanism for the pathogenesis of ARVC and establish cardiac-restricted DP-deficient mice as a model for human ARVC. These findings could provide for the opportunity to identify new diagnostic markers and therapeutic targets in patients with ARVC.
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.
arrhythmogenic right ventricular dysplasia/cardiomyopathy; Naxos disease; cell‐adhesions; desmosomal proteins; sudden death
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable myocardial disorder associated with fibrofatty replacement of myocardium and ventricular arrhythmia. A subset of ARVC is categorized as Naxos disease, which is characterized by ARVC and a cutaneous disorder. A homozygous loss-of-function mutation of the Plakoglobin (Jup) gene, which encodes a major component of the desmosome and the adherens junction, had been identified in Naxos patients, although the underlying mechanism remained elusive. We generated Jup mutant mice by ablating Jup in cardiomyocytes. Jup mutant mice largely recapitulated the clinical manifestation of human ARVC: ventricular dilation and aneurysm, cardiac fibrosis, cardiac dysfunction and spontaneous ventricular arrhythmias. Ultra-structural analyses revealed that desmosomes were absent in Jup mutant myocardia, whereas adherens junctions and gap junctions were preserved. We found that ventricular arrhythmias were associated with progressive cardiomyopathy and fibrosis in Jup mutant hearts. Massive cell death contributed to the cardiomyocyte dropout in Jup mutant hearts. Despite the increase of β-catenin at adherens junctions in Jup mutant cardiomyoicytes, the Wnt/β-catenin-mediated signaling was not altered. Transforming growth factor-beta-mediated signaling was found significantly elevated in Jup mutant cardiomyocytes at the early stage of cardiomyopathy, suggesting an important pathogenic pathway for Jup-related ARVC. These findings have provided further insights for the pathogenesis of ARVC and potential therapeutic interventions.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease characterized by fibrofatty replacement of the myocardium and ventricular arrhythmias, associated with mutations in the desmosomal genes. Only a missense mutation in the DES gene coding for desmin, the intermediate filament protein expressed by cardiac and skeletal muscle cells, has been recently associated with ARVC. We screened 91 ARVC index cases (53 negative for mutations in desmosomal genes and an additional 38 carrying desmosomal gene mutations) for DES mutations. Two rare missense variants were identified. The heterozygous p.K241E substitution was detected in 1 patient affected with a severe form of ARVC who also carried the p.T816RfsX10 mutation in plakophilin-2 gene. This DES substitution, showing an allele frequency of <0.01 in the control population, is predicted to cause an intolerant amino acid change in a highly conserved protein domain. Thus, it can be considered a rare variant with a possible modifier effect on the phenotypic expression of the concomitant mutation. The previously known p.A213V substitution was identified in 1 patient with ARVC who was negative for mutations in the desmosomal genes. Because a greater prevalence of p.A213V has been reported in patients with heart dilation than in control subjects, the hypothesis that this rare variant could have an unfavorable effect on cardiac remodeling cannot be ruled out. In conclusion, our data help to establish that, in the absence of skeletal muscle involvement suggestive of a desminopathy, the probability of DES mutations in ARVC is very low. These findings have important implications in the mutation screening strategy for patients with ARVC.
We sought to quantify the number and length of desmosomes, gap junctions, and adherens junctions in arrhythmogenic right ventricular cardiomyopathy (ARVC) and non-ARVC dogs, and to determine if ultrastructural changes existed.
Hearts from 8 boxer dogs afflicted with histopathologically confirmed ARVC and 6 dogs without ARVC were studied.
Quantitative transmission electron microscopy (TEM) and Western blot semi-quantification of α-actinin were used to study the intercalated disc and sarcomere of the right and left ventricles.
When ARVC dogs were compared to non-ARVC dogs reductions in the number of desmosomes (P = 0.04), adherens junctions (P = 0.04) and gap junctions (P = 0.02) were found. The number of gap junctions (P = 0.04) and adherens junctions (P = 0.04) also were reduced in the left ventricle, while the number of desmosomes was not (P = 0.88). A decrease in the length of desmosomal complexes within LV samples (P=0.04) was found. These findings suggested disruption of proteins providing attachment of the cytoskeleton to the intercalated disc. Immunoblotting did not demonstrate a quantitative reduction in the amount of α-actinin in ARVC afflicted samples. All boxers with ARVC demonstrated the presence of electron dense material originating from the Z band and extending into the sarcomere, apparently at the expense of the cytoskeletal structure.
These results emphasize the importance of structural integrity of the intercalated disc in the pathogenesis of ARVC. In addition, observed abnormalities in sarcomeric structure suggest a novel link between ARVC and the actin-myosin contractile apparatus.
Canine; ARVC; Boxer; electron microscopy; desmosome; intercalated disc; cardiac ultrastructure
The diagnosis of arrhythmogenic right ventricular cardiomyopathy can be challenging. Disease-causing mutations in desmosomal genes have been identified. A novel diagnostic feature, loss of immunoreactivity for plakoglobin from the intercalated disks, recently was proposed.
The purpose of this study was to identify two novel mutations in the intracellular cadherin segment of desmoglein-2 (G812S and C813R in exon 15). Co-segregation of the G812S mutation with disease expression was established in a large Caucasian family. Endomyocardial biopsies of two individuals showed reduced plakoglobin signal at the intercalated disk.
To understand the pathologic changes occurring in the diseased myocardium, functional studies on three mutations in exon 15 of desmoglein-2 (G812C, G812S, C813R) were performed.
Localization studies failed to detect any differences in targeting or stability of the mutant proteins, suggesting that they act via a dominant negative mechanism. Binding assays were performed to probe for altered binding affinities toward other desmosomal proteins, such as plakoglobin and plakophilin-2. Although no differences were observed for the mutated proteins in comparison to wild-type desmoglein-2, binding to plakophilin-2 depended on the expression system (i.e., bacterial vs mammalian protein expression). In addition, abnormal migration of the C813R mutant protein was observed in gel electrophoresis.
Loss of plakoglobin immunoreactivity from the intercalated disks appears to be the endpoint of complex pathologic changes, and our functional data suggest that yet unknown posttranslational modifications of desmoglein-2 might be involved.
Arrhythmogenic right ventricular cardiomyopathy; Desmoglein-2; Desmosome; Genetics; Missense mutation; Plakophilin-2; ARVC, arrhythmogenic right ventricular cardiomyopathy; Cx43, connexin43; 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
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.
Arrhythmias; Cardiomyopathies; Desmosomes; Intercalated Disks; Genetic Mutations
Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of heart and skin diseases. This has led to the hypothesis that defective cell-cell adhesion is the underlying cause of injury in tissues that repeatedly bear high mechanical loads. In this study, we examined the effects of two different mutations in plakoglobin on cell migration, stiffness, and adhesion. One is a C-terminal mutation causing Naxos disease, a recessive syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal skin and hair. The other is an N-terminal mutation causing dominant inheritance of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin mutations on a broad range of cell mechanical behavior, we characterized a model system consisting of stably transfected HEK cells which are particularly well suited for analyses of cell migration and adhesion. Both mutations increased the speed of wound healing which appeared to be related to increased cell motility rather than increased cell proliferation. However, the C-terminal mutation led to dramatically decreased cell-cell adhesion, whereas the N-terminal mutation caused a decrease in cell stiffness. These results indicate that different mutations in plakoglobin have markedly disparate effects on cell mechanical behavior, suggesting complex biomechanical roles for this protein.
cell mechanics; cell adhesion; junctional proteins
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.
Arrhythmias; Sudden death; Molecular genetics; Desmosomes
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.
Mutations in genes encoding desmosomal proteins have been identified as the major cause of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC), in which the right ventricle is “replaced” by fibrofatty tissue, resulting in lethal arrhythmias. In this issue of the JCI, Garcia-Gras et al. demonstrate that cardiac-specific loss of the desmosomal protein desmoplakin is sufficient to cause nuclear translocation of plakoglobin, upregulation of adipogenic genes in vitro, and a shift from a cardiomyocyte to an adipocyte cell fate in vivo (see the related article beginning on page 2012). This evidence for potential Wnt/β-catenin signaling defects sets the scene for a comprehensive exploration of the contributions of this pathway to the pathophysiology of ARVC, not only through perturbation of cardiac patterning and development, but also through effects on myocardial differentiation and physiology.
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.
Plakoglobin; N-cadherin; connexin-43; arrhythmogenic right ventricular cardiomyopathy
This study provides evidence that decreased expression of the desmosomal cadherin desmocollin-2 enhances intestinal epithelial cell proliferation and promotes tumor formation via an Akt/β-catenin pathway.
Desmocollin-2 (Dsc2) and desmoglein-2 (Dsg2) are transmembrane cell adhesion proteins of desmosomes. Reduced expression of Dsc2 has been reported in colorectal carcinomas, suggesting that Dsc2 may play a role in the development and/or progression of colorectal cancer. However, no studies have examined the mechanistic contribution of Dsc2 deficiency to tumorigenesis. Here we report that loss of Dsc2 promotes cell proliferation and enables tumor growth in vivo through the activation of Akt/β-catenin signaling. Inhibition of Akt prevented the increase in β-catenin–dependent transcription and proliferation following Dsc2 knockdown and attenuated the in vivo growth of Dsc2-deficient cells. Taken together, our results provide evidence that loss of Dsc2 contributes to the growth of colorectal cancer cells and highlight a novel mechanism by which the desmosomal cadherins regulate β-catenin signaling.