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1.  Altered Desmosomal Proteins in Granulomatous Myocarditis and Potential Pathogenic Links to Arrhythmogenic Right Ventricular Cardiomyopathy 
Background
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).
Conclusions
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.
doi:10.1161/CIRCEP.111.964890
PMCID: PMC3203520  PMID: 21859801
plakoglobin; desmosome; sarcoidosis; giant cell myocarditis; cytokines
2.  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.
doi:10.1128/MCB.01025-10
PMCID: PMC3067899  PMID: 21245375
3.  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.
doi:10.1136/hrt.2005.081679
PMCID: PMC1861281  PMID: 16698823
arrhythmogenic right ventricular dysplasia/cardiomyopathy; Naxos disease; cell‐adhesions; desmosomal proteins; sudden death
4.  Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations 
Cardiovascular Research  2010;90(1):77-87.
Aims
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.
Conclusion
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.
doi:10.1093/cvr/cvq353
PMCID: PMC3058729  PMID: 21062920
Arrhythmogenic right ventricular cardiomyopathy; Desmocollin-2; Desmosome; Functional studies; Mutation
5.  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.
doi:10.1155/2010/207835
PMCID: PMC2879923  PMID: 20585598
6.  Arrhythmogenic Right Ventricular Cardiomyopathy is A Disease of Cardiac Stem Cells 
Current Opinion in Cardiology  2010;25(3):222-228.
Purpose
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.
Summary
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.
doi:10.1097/HCO.0b013e3283376daf
PMCID: PMC2980568  PMID: 20124997
Cardiomyopathy; Genetics; Sudden death; Heart failure; Stem cells
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.
doi:10.1186/1750-1172-2-45
PMCID: PMC2222049  PMID: 18001465
8.  Desmosomal Cadherins Are Decreased in Explanted Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Patient Hearts 
PLoS ONE  2013;8(9):e75082.
Aims
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.
Conclusion
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.
doi:10.1371/journal.pone.0075082
PMCID: PMC3781033  PMID: 24086444
9.  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
10.  Restrictive loss of plakoglobin in cardiomyocytes leads to arrhythmogenic cardiomyopathy 
Human Molecular Genetics  2011;20(23):4582-4596.
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.
doi:10.1093/hmg/ddr392
PMCID: PMC3209829  PMID: 21880664
11.  Genetic Variation in Titin in ARVC-Overlap Syndromes 
Circulation  2011;124(8):876-885.
Background
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks.
Methods and Results
All 312 titin exons known to be expressed in human cardiac titin and the complete 3’ untranslated region were sequenced in 38 ARVC families. Eight unique TTN variants were detected in 7 families including a prominent Thr2896Ile mutation that showed complete segregation with the ARVC phenotype in one large family. The Thr2896IIe mutation maps within a highly conserved immunoglobulin-like fold (Ig10 domain), located in titin’s spring region. Native gel electrophoresis, NMR, intrinsic fluorescence, and proteolysis assays of wildtype and mutant Ig10 domains revealed that the Thr2896IIe exchange reduces the structural stability and increases the propensity towards degradation of the Ig10 domain. The phenotype of TTN variant carriers was characterized by history of sudden death (5/7 families), progressive myocardial dysfunction causing death or heart transplant (8/14 cases), frequent conduction disease (11/14), and incomplete penetrance (86%).
Conclusions
Our data provide evidence that titin mutations can cause ARVC, a finding that further expands the origin of the disease beyond desmosomal proteins. Structural impairment of the titin spring is a likely cause of ARVC and constitutes a novel mechanism underlying myocardial remodeling and sudden cardiac death.
doi:10.1161/CIRCULATIONAHA.110.005405
PMCID: PMC3167235  PMID: 21810661
cardiomyopathy; arrhythmia; death; sudden; genetics; arrhythmogenic right ventricular cardiomyopathy
12.  A novel desmocollin-2 mutation reveals insights into the molecular link between desmosomes and gap junctions 
Heart Rhythm  2011;8(5):711-718.
Background
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.
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1016/j.hrthm.2011.01.010
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.  Quantitative Immunohistochemistry of Desmosomal Proteins (Plakoglobin, Desmoplakin and Plakophilin), Connexin-43, and N-cadherin in Arrhythmogenic Cardiomyopathy: An Autopsy Study 
Background:
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.
Conclusions:
The small and insignificant decrease in junction protein expression in ARVC suggests that immunohistochemistry is not a useful tool for the diagnosis.
doi:10.2174/1874192401307010028
PMCID: PMC3680985  PMID: 23802019
ARVC; arrhytmogenic cardiomyopaty; sudden death; autopsy.
14.  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.
doi:10.1007/s12471-012-0274-x
PMCID: PMC3346879  PMID: 22527912
Cardiomyopathy; Plakophilin-2; Mutation; Desmosome; Prevalence; Geography; Medicine & Public Health; Medicine/Public Health, general
15.  Molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia/cardiomyopathy 
Background/Objective
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).
Methods
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).
Results
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.
Conclusions
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.
doi:10.1016/j.hrthm.2007.05.025
PMCID: PMC2080779  PMID: 17765621
16.  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.
doi:10.4081/hi.2006.17
PMCID: PMC3184660  PMID: 21977247
Arrhythmias; Sudden death; Molecular genetics; Desmosomes
17.  Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation 
The Journal of Cell Biology  1996;133(6):1367-1382.
Desmogleins are members of the cadherin superfamily which form the core of desmosomes. In vitro studies indicate that the cytoplasmic domain of desmogleins associates with plakoglobin; however, little is known about the role of this domain in desmosome recognition or assembly in vivo, or about the possible relation of desmoglein mutations to epidermal differentiation and disease. To address these questions we used transgenic mouse technology to produce an NH2-terminally truncated desmoglein (Pemphigus Vulgaris Antigen or Dsg3) in cells known to express its wild-type counterpart. Within 2 d, newborn transgenic animals displayed swelling of their paws, flakiness on their back, and blackening of the tail tip. When analyzed histologically and ultrastructurally, widening of intercellular spaces and disruption of desmosomes were especially striking in the paws and tail. Desmosomes were reduced dramatically in number and were smaller and often peculiar in structure. Immunofluorescence and immunoelectron microscopy revealed no major abnormalities in localization of hemidesmosomal components, but desmosomal components organized aberrantly, resulting in a loss of ultrastructure within the plaque. In regions where desmosome loss was prevalent but where some adhesive structures persisted, the epidermis was thickened, with a marked increase in spinous and stratum corneum layers, variability in granular layer thickness, and parakeratosis in some regions. Intriguingly, a dramatic increase in cell proliferation was also observed concomitant with biochemical changes, including alterations in integrin expression, known to be associated with hyperproliferation. An inflammatory response was also detected in some skin regions. Collectively, these findings demonstrate that a mutation in a desmoglein can perturb epidermal cell-cell adhesion, triggering a cascade of changes in the skin.
PMCID: PMC2120897  PMID: 8682871
18.  Myocyte necrosis underlies progressive myocardial dystrophy in mouse dsg2-related arrhythmogenic right ventricular cardiomyopathy 
The Journal of Experimental Medicine  2009;206(8):1787-1802.
Mutations in the cardiac desmosomal protein desmoglein-2 (DSG2) are associated with arrhythmogenic right ventricular cardiomyopathy (ARVC). We studied the explanted heart of a proband carrying the DSG2-N266S mutation as well as transgenic mice (Tg-NS) with cardiac overexpression of the mouse equivalent of this mutation, N271S-dsg2, with the aim of investigating the pathophysiological mechanisms involved. Transgenic mice recapitulated the clinical features of ARVC, including sudden death at young age, spontaneous ventricular arrhythmias, cardiac dysfunction, and biventricular dilatation and aneurysms. Investigation of transgenic lines with different levels of transgene expression attested to a dose-dependent dominant-negative effect of the mutation. We demonstrate for the first time that myocyte necrosis is the key initiator of myocardial injury, triggering progressive myocardial damage, including an inflammatory response and massive calcification within the myocardium, followed by injury repair with fibrous tissue replacement, and myocardial atrophy. These observations were supported by findings in the explanted heart from the patient. Insight into mechanisms initiating myocardial damage in ARVC is a prerequisite to the future development of new therapies aimed at delaying onset or progression of the disease.
doi:10.1084/jem.20090641
PMCID: PMC2722163  PMID: 19635863
19.  Disparate Effects of Different Mutations in Plakoglobin on Cell Mechanical Behavior 
Cell motility and the cytoskeleton  2008;65(12):964-978.
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.
doi:10.1002/cm.20319
PMCID: PMC2650236  PMID: 18937352
cell mechanics; cell adhesion; junctional proteins
20.  Desmoglein as a target in skin disease and beyond 
Much of the original research on desmosomes and their biochemical components was through analysis of skin and mucous membranes. The identification of desmogleins 1 and 3, desmosomal adhesion glycoproteins, as targets in pemphigus, a fatal autoimmune blistering disease of the skin and mucous membranes, provided the first link between desmosomes, desmogleins, and human diseases. The clinical and histological similarities of staphylococcal scalded skin syndrome or bullous impetigo and pemphigus foliaceus led us to identify desmoglein 1 as the proteolytic target of staphylococcal exfoliative toxins. Genetic analysis of striate palmoplantar keratoderma and hypotrichosis identified their responsible genes as desmogleins 1 and 4, respectively. More recently these fundamental findings in cutaneous biology were extended beyond the skin. Desmoglein 2, which is expressed earliest among the four isoforms of desmoglein in development and found in all desmosome-bearing epithelial cells, was found to be mutated in arrythmogenic right ventricular cardiomyopathy and has also been identified as a receptor for a subset of adenoviruses that cause respiratory and urinary tract infections. The story of desmoglein research illuminates how dermatologic research originally focused on one skin disease, pemphigus, has contributed to understanding biology and pathophysiology of many seemingly unrelated tissues and diseases.
doi:10.1038/jid.2011.390
PMCID: PMC3279627  PMID: 22189787
pemphigus; impetigo; hypotrichosis; cardiomyopathy; cadherin
21.  A Central Role for the Armadillo Protein Plakoglobin in the Autoimmune Disease Pemphigus Vulgaris 
The Journal of Cell Biology  2001;153(4):823-834.
In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG−/−) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG−/− cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG−/− keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.
PMCID: PMC2192383  PMID: 11352942
catenins; desmosomes; mouse keratinocytes; epithelial differentiation; blistering disease
22.  Desmin Mutations and Arrhythmogenic Right Ventricular Cardiomyopathy 
The American Journal of Cardiology  2013;111(3):400-405.
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.
doi:10.1016/j.amjcard.2012.10.017
PMCID: PMC3554957  PMID: 23168288
23.  Remodeling of the cardiac sodium channel, Connexin43 and Plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.hrthm.2012.11.018
PMCID: PMC3608196  PMID: 23178689
24.  Arrhythmogenic right ventricular cardiomyopathy: moving toward mechanism 
Journal of Clinical Investigation  2006;116(7):1825-1828.
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.
doi:10.1172/JCI29174
PMCID: PMC1483166  PMID: 16823481
25.  Arrhythmogenic ventricular cardiomyopathy: A paradigm shift from right to biventricular disease 
World Journal of Cardiology  2014;6(4):154-174.
Arrhythmogenic ventricular cardiomyopathy (AVC) is generally referred to as arrhythmogenic right ventricular (RV) cardiomyopathy/dysplasia and constitutes an inherited cardiomyopathy. Affected patients may succumb to sudden cardiac death (SCD), ventricular tachyarrhythmias (VTA) and heart failure. Genetic studies have identified causative mutations in genes encoding proteins of the intercalated disk that lead to reduced myocardial electro-mechanical stability. The term arrhythmogenic RV cardiomyopathy is somewhat misleading as biventricular involvement or isolated left ventricular (LV) involvement may be present and thus a broader term such as AVC should be preferred. The diagnosis is established on a point score basis according to the revised 2010 task force criteria utilizing imaging modalities, demonstrating fibrous replacement through biopsy, electrocardiographic abnormalities, ventricular arrhythmias and a positive family history including identification of genetic mutations. Although several risk factors for SCD such as previous cardiac arrest, syncope, documented VTA, severe RV/LV dysfunction and young age at manifestation have been identified, risk stratification still needs improvement, especially in asymptomatic family members. Particularly, the role of genetic testing and environmental factors has to be further elucidated. Therapeutic interventions include restriction from physical exercise, beta-blockers, sotalol, amiodarone, implantable cardioverter-defibrillators and catheter ablation. Life-long follow-up is warranted in symptomatic patients, but also asymptomatic carriers of pathogenic mutations.
doi:10.4330/wjc.v6.i4.154
PMCID: PMC3999336  PMID: 24772256
Arrhythmogenic right ventricular dysplasia/cardiomyopathy; Arrhythmias; Ventricular tachycardia; Sudden cardiac death; Implantable cardioverter defibrillator

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