Cell Tissue Res DOI 10.1007/s00441-014-2015-5

REVIEW

Arrhythmogenic cardiomyopathy: a disease of intercalated discs Martina Calore & Alessandra Lorenzon & Marzia De Bortoli & Giulia Poloni & Alessandra Rampazzo

Received: 29 July 2014 / Accepted: 18 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Arrhythmogenic cardiomyopathy (ACM) is an acquired progressive disease having an age-related penetrance and showing clinical manifestations usually during adolescence and young adulthood. It is characterized clinically by a high incidence of severe ventricular tachyarrhythmias and sudden cardiac death and pathologically by degeneration of ventricular cardiomyocytes with replacement by fibro-fatty tissue. Whereas, in the past, the disease was considered to involve only the right ventricle, more recent clinical studies have established that the left ventricle is frequently involved. ACM is an inherited disease in up to 50 % of cases, with predominantly an autosomal dominant pattern of transmission, although recessive inheritance has also been described. Since most of the pathogenic mutations have been identified in genes encoding desmosomal proteins, ACM is currently defined as a disease of desmosomes. However, on the basis of the most recent description of the intercalated disc organization and of the identification of a novel ACM gene encoding for an area composita protein, ACM can be considered as a disease of the intercalated disc, rather than only as a desmosomal disease. Despite increasing knowledge of the genetic basis of ACM, we are just beginning to understand early molecular events leading to cardiomyocyte degeneration, fibrosis and fibro-fatty substitution. This review summarizes recent advances in our comprehension of the link between the molecular genetics and pathogenesis of ACM and of the novel role of cardiac intercalated discs. Research in the authors’ laboratory is supported by the Strategic Program of the University of Padua, the Ricerca Sanitaria Finalizzata (Veneto Region), the University of Padua research project (PRAT) CPDA133979 and the Cariparo Foundation (Padova and Rovigo, Italy). M. Calore : A. Lorenzon : M. De Bortoli : G. Poloni : A. Rampazzo (*) Department of Biology, University of Padua, Via G. Colombo 3, 35131 Padua, Italy e-mail: [email protected]

Keywords Cardiomyopathy . Suddendeath . Cardiac cell-cell junctions . Intercalated disc

Introduction Arrhythmogenic cardiomyopathy (ACM) is a genetically determined heart muscle disease involving predominantly the right ventricle with progressive loss of myocytes and fibrofatty tissue replacement, resulting in regional or global abnormalities (Thiene et al. 1988; Nava et al. 1988). ACM is a recognized cause of sudden cardiac death in the young and is considered the most common cause of sudden death in competitive athletes in Italy (Corrado et al. 2006). Prevalence is estimated globally between 1:2000 and 1:5000 and men are affected more frequently than women (Bauce et al. 2008). ACM is clinically characterized by functional abnormalities of the right ventricle, electrocardiographic depolarization/ repolarization changes, syncope and ventricular arrhythmias that can lead to sudden death (Nava et al. 2000). Clinical manifestations develop most often between the second and fourth decade of life and are related to ventricular tachycardia or ventricular fibrillation possibly leading to sudden death, primarily in young people. Ventricular arrhythmias are worse during or immediately after exercise and participation in competitive sports has been associated with an increased risk of sudden death (Corrado et al. 2011). Clinical diagnosis of ACM is often difficult because of the non-specific nature of the disease features and the broad spectrum of phenotypic manifestations, ranging from severe to concealed forms. A scoring system to establish the diagnosis of ACM has been developed on the basis of the fulfillment of major and minor criteria encompassing structural, histological, electrocardiographic, arrhythmic and genetic features of the disease (McKenna et al. 1994; Marcus et al. 2010).

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The most striking pathologic features of ACM are the diffuse or segmental loss of the myocardium of the right ventricular free wall and its replacement by fibro-fatty tissue with a wave of progression from the epicardium to the endocardium. The phenotypic spectrum encompasses the right form and left-dominant and biventricular subtypes (Basso et al. 2011). In the classic right form, fibro-fatty replacement is limited to the right ventricle, typically located at the inferior, apical and infundibular walls (the so-called triangle of dysplasia). According to Corrado et al. (1997), left ventricular involvement is found in 76 % of ACM cases, typically affecting the posterior lateral wall. The left ventricle has been shown to be affected not only at the end stage of the disease but also as a primary target. The existence of various clinico-pathological subtypes of this cardiomyopathy supports the adoption of the more comprehensive term “arrhythmogenic cardiomyopathy” for this disease. In advanced stages, architectural changes with degenerated myocytes entrapped within fibro-fatty tissue can account for delayed activation and a re-entry mechanism triggering ventricular tachycardia/fibrillation. However, recent data suggest that electrical instability precedes structural abnormalities; arrhythmias have been described early in the disease process, before significant structural changes of the myocardium (Kaplan et al. 2004). This phenomenon might be attributable to abnormalities that occur in electrical coupling between cardiomyocytes, which are present even before the onset of cardiomyopathic changes (Rizzo et al. 2012). This review focuses on recent progress in the molecular genetics and pathogenesis of ACM and takes into account recent evidence supporting the concept that arrhythmogenic cardiomyopathy can be considered as a disease of intercalated discs (IDs).

Molecular genetics of ACM Familial occurrence of ACM is rather common (Nava et al. 1988). Inheritance is mainly autosomal dominant with incomplete penetrance and variable expressivity. Human genetic studies over the last 10 years have offered insights into the potential causes of ACM. Early work demonstrated substantial genetic heterogeneity: at least 15 independent loci and 13 disease genes have now been identified (Table 1). Nearly half of ACM probands harbor one or more mutations in genes encoding major components of cardiac desmosomes, including desmoplakin (DSP; Rampazzo et al. 2002), plakophilin-2 (PKP2; Gerull et al. 2004), desmoglein-2 (DSG2; Pilichou et al. 2006), desmocollin-2 (DSC2; Syrris et al. 2006) and plakoglobin (JUP; Asimaki et al. 2007; Fig. 1, Table 1). Desmosomes are important cell-cell adhesion junctions

that anchor stress-bearing intermediate filaments (IF) at sites of strong intercellular adhesion. The resulting scaffold plays a key role in providing mechanical integrity to tissues, such as the epidermis and heart, which are subjected to mechanical stress (Garrod and Chidgey 2008). The most commonly mutated gene among ACM patients Table 1 Genes and encoded proteins associated with arrhythmogenic cardiomyopathy (ACM) Disease gene

Protein function

Mechanical junction components PKP2 Plakophilin-2: desmosomal and area composita armadillo protein connecting desmoplakin to desmosomal cadherins DSP Desmoplakin : desmosomal and area composita protein linking intermediate filaments to desmosomal plaque DSG2 Desmoglein-2: desmosomal cadherin linking neighboring cells through its extracellular domains DSC2 Desmocollin-2: desmosomal cadherin linking neighboring cells through its extracellular domains JUP Plakoglobin: desmosomal and area composita armadillo protein connecting desmoplakin to desmosomal cadherins CTNNA3 αT-catenin: area composita protein binding plakophilin-2 in the intercalated disc TMEM43 Transmembrane protein-43 (LUMA): area composita protein previously thought to be a tetraspan transmembrane protein of the nuclear envelope Non-junctional components RYR2 Ryanodyne receptor-2: ryanodynic cardiac receptor calcium channel of the cardiac sarcoplasmic reticulum, essential for calcium intracellular homeostasis TGFB3 Transforming growth factor β3: cytokine involved in extracellular matrix deposition, cell adhesion and cellular signaling DES Desmin: intermediate filament protein of cardiomyocytes LMNA Lamin A/C: protein of the inner nuclear membrane involved in nuclear stability TTN Titin: sarcomeric protein acting as a molecular spring between sarcomeric Z and M lines PLN Phospholamban: integral membrane protein of the sarcoplasmic reticulum regulating the SERCA2a calcium ATPase

encodes for PKP2, with estimated prevalence ranging from 7 to 51 % and spikes of 70 % (Gerull et al. 2004; Basso et al. 2011; Fressart et al. 2010). PKP2, together with DSP and DSG2, belongs to the group of the “big three” ACM genes, as about 90 % of ACM mutations are located in these genes.

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Fig. 1 Molecular organization of mechanical junctions at an intercalated disc. a Immuno-electron micrographs of a cross-section of the myocardium of human heart, showing dense desmoplakin immunolabeling over the entirety of the intercalated disc, both in the desmosome and in area composita. This image was originally published in a study by Franke et al. (2006). Bar 0.5 μm. b Model composition of mechanical junctions in a cardiac intercalated disc. Major components of classical desmosomes are desmosomal cadherins desmoglein-2 (DSG2) and desmocollin-2 (DSC2) that connect neighboring cells through their extracellular domains and are linked via the armadillo proteins plakophilin-2 (PKP2)

and plakoglobin (PG) to desmoplakin (DSP), which in turn anchors the desmosomal plaque to desmin intermediate filaments (IF). The area composita is a heart-specific hybrid reinforced junction arising by the post-natal amalgamation of proteins of both genuine desmosomes and of genuine adherens junctions, bound both to F-actin cytoskeleton and to desmin intermediate filaments. Cardiac N-cadherin connects, via βcatenin (β-cat) or plakoglobin, to αT-catenin (αT-cat) or αE-catenin (αE-cat), which binds the F-actin cytoskeleton directly or indirectly via vinculin. Most of the mutations causing arrhythmogenic cardiomyopathy affect proteins belonging to the intercalated disc (stars)

Heterozygous mutations are commonly detected in ACM patients but homozygous mutations have also been reported in PKP2, DSP, DSC2, DSG2 and JUP genes in patients showing only ACM or ACM combined with cutaneous abnormalities (Basso et al. 2011; Sato et al. 2011). Recessive mutations in the JUP and DSP genes were originally reported in patients affected with Naxos disease and Carvajal

syndrome, respectively (McKoy et al. 2000; Norgett et al. 2000). Both syndromes are characterized by wooly hair, palmoplantar keratoderma and ACM in Naxos patients or biventricular dilated cardiomyoapthy in Carvajal patients (Protonotarios et al. 1988; Carvajal-Huerta 1998). The large majority of identified ACM mutations consists of point mutations, either missense or splice-site variations or

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short indels (Rampazzo 2006). However, heterozygous deletions of PKP2 exons and of the entire PKP2 gene have been reported by various groups (Roberts et al. 2013; Li Mura et al. 2013). Most recently, a novel mixed-type junctional structure, the so-called area composita, composed of both desmosome and adherens junction (AJs) proteins, has been described but only in the heart (Franke et al. 2006). The CTNNA3 gene encodes for αT-catenin, a protein highly expressed in cardiac AJs and also found in the area composita where it interacts specifically with the desmosomal protein PKP2 (Goossens et al. 2007). Recently, two heterozygous mutations in this gene have been identified in two ACM probands (van Hengel et al. 2013) suggesting, for the first time, a causal relationship between CTNNA3 mutations and ACM pathology. ACM mutations have also been found in the TMEM43 (transmembrane protein-43) gene, encoding for LUMA, widely speculated to be a tetraspan transmembrane protein of the nuclear envelope (Merner et al. 2008). A recent paper reporting immunolocalization experiments showed that, in mammals, LUMA localizes in the area composita, suggesting that this protein should be considered as a prominent component of (or associated with) cardiac IDs (Franke et al. 2014). Less frequently, ACM pathogenic mutations have been detected in genes encoding for non-junctional proteins, such as the ryanodyne receptor 2 (RYR2; Tiso et al. 2001), transforming growth factor β3 (TGFB3; Beffagna et al. 2005), desmin (DES; Klauke et al. 2010), titin (TTN; Taylor et al. 2011), lamin A/C (LMNA; Quarta et al. 2012) and phospholamban (PLN; van der Zwaag et al. 2012) ( Table 1). Emerging evidence indicates that ACM has a complex genetic background; this might explain the incomplete penetrance and the extremely variable expressivity of the disease phenotype, even among family members carrying the same mutation. More than one genetic variation might be required for clinical disease expression, thus justifying the marked intrafamilial and interfamilial phenotypic diversity. Compound heterozygous mutations in a single gene and digenic mutations (variants in two or more genes) are often necessary to develop the ACM phenotype (Bauce et al. 2010; Xu et al. 2010). Compared with patients with a single ACM mutation, multiple mutation carriers show a more severe phenotypic expression, including major right ventricular dilatation, a higher prevalence of left ventricular involvement, an increased risk of life-threatening arrhythmic events, ventricular tachycardia and syncope, or personal history of sudden cardiac death (aborted or not) (Fressart et al. 2010; Bauce et al. 2010; Rigato et al. 2013). Modifier genes, still-unknown disease-causing genes, common sequence variants and environmental and endogenous factors (such as age, sex, strenuous exercise, drugs, hormones, infection/inflammation and emotional stress) are also hypothesized to account for

much of the phenotypic variation between individuals (Sen-Chowdhry et al. 2010). In the last revision of ACM diagnostic criteria, the identification of a pathogenic mutation was considered as a major criterion (Marcus et al. 2010). This underlines the importance of assessing mutation pathogenicity, especially in the case of missense mutations. In a comprehensive study of genetic variation for ACM susceptibility genes, the overall yield of mutations among affected cases was 58 % versus 16 % in healthy subjects (Kapplinger et al. 2011). Most of the genetic variants detected in the controls were missense, suggesting that many of them were benign. However, the prevalence of ACM mutations dropped to 0.5 % in healthy subjects, compared with 43 % in probands, after assessment for only radical mutations (in/del, splice junction, nonsense). These findings suggest that radical mutations are associated with ACM, whereas missense mutations found in genetic testing for ACM should be considered with caution. Kapplinger et al. (2011) suggested that factors strengthening missense variations pathogenicity might include the conservation of residues within PKP2 and DSG2 and localization to the N-terminal regions of DSP and DSG2. Moreover, compared with controls, pathogenic mutations detected in cases seem to occur more frequently in non-Caucasians, suggesting that ethnicity influences the probability of a missense mutation being truly pathogenic (Kapplinger et al. 2011). The certainty of the pathogenicity of the mutation is crucial for safe and effective cascade screening. Since a family history of sudden death represents an important risk factor, genetic background might prove to be helpful in risk assessment. Once a causative mutation is detected in an ACM patient, family member screening for the presence of the same mutation enables the identification of at-risk asymptomatic subjects. Because many mutations causing ACM are private, careful family assessment demonstrating the co-segregation of the mutation with the disease phenotype is advised.

Molecular pathogenesis of ACM Although genetic testing plays a key role in identifying ACMcausing mutations, much less is known about the pathogenic mechanisms leading to cardiomyocyte death, to fibroadiposis and to the development of the disease phenotype. Together with the disruption of mechanical cell-cell junctions, which is supposed to lead to cardiomyocyte death under mechanical stress and to subsequent fibro-fatty replacement, various signaling pathways have been proposed to be involved in ACM pathogenesis, highlighting the dual function of junctional proteins as both components of intercellular adhesion structures and transcriptional regulators (Table 2). The canonical Wnt/β-catenin/Tcf/Lef pathway is known to regulate adipogenesis, fibrogenesis and apoptosis (Ross et al.

Cell Tissue Res Table 2 Potential mechanisms of ACM. Selected findings from a mouse model and in vitro studies (PG/JUP plakoglobin, TGF transforming growth factor, DSP desmoplakin, PKP2 plakophilin-2, iPSCs induced Targeted protein Model DSP

pluripotent stem cells, IF immunofluorescence, PPAR peroxisome proliferator-activated receptor)

Summary of findings

Reference

PG

DSP+/− mouse; stable transfection of HL-1 Nuclear PG translocation with adipogenic switch cells with short interfering RNA specifically to suppress DSP expression Mouse overexpressing cardiac truncated PG; Suppression of canonical Wnt signaling and induction PG null embryos of pro-adipogenic gene expression Cardiac-restricted Jup knockout mouse Increase of β-catenin signaling in the nucleus

PG

Cardiac-restricted Jup knockout mouse

Increase of TGF-β-mediated signaling

D. Li et al. 2011

DSP/PG/PKP2

Cardiac-restricted heterozygous DSP+/− mouse; cardiac-specific truncated PG mouse; Pkp2 knockdown HL-1 cells Neonatal rat and zebrafish ventricular myocytes expressing the human p.W680GfsX11 mutation in PG; iPSCs from two probands carrying p.Q617X and p.K672RfsX12 mutations in PKP2

Activation of the Hippo pathway, suppression of canonical Wnt signaling and enhanced adipogenesis

Chen et al. 2014

Reduction in INa and IK1 current densities and interruptions in cell boundaries and structural disarray in fishes and in neonatal rat cardiomyocytes; abnormal subcellular distribution of PG, connexin 43, Nav1.5 and SAP97 in neonatal rat cardiomyocytes and iPSCs. In all cases, SB216763 compound reverted ACM pathobiological features Reduction of plakophilin-2 and plakoglobin IF signals; larger ACM iPSCs containing darker lipid droplets Nuclear PG translocation; very low β-catenin expression; increased expression of PPAR-α and hyperactivation of PPAR-γ pathway; lipogenesis, apoptosis and calcium-handling deficits Intracellular lipid droplet accumulation; upregulation of pro-adipogenic PPAR-γ; desmosomal distortion

Asimaki et al. 2014

PG

PG/PKP2

PKP2 PKP2

PKP2

iPSCs from a patient heterozygous for p.L614P mutation in PKP2 iPSCs from two patients carrying homozygous p.G828G or heterozygous p.K672RfsX12 mutations in PKP2 iPSCs from two patients heterozygous for p.A324fs335X or p.T50SfsX110 mutations in PKP2

2000). In a series of studies, Marian’s group was the first to implicate the suppression of the canonical Wnt/β-catenin/Tcf/ Lef pathway in the pathogenesis of ACM. Dsp knockdown in mice and in HL-1 cells causes the translocation of PG into the nucleus where it interferes with β-catenin/TCF transcriptional activity, leading to an adipogenic switch (Garcia-Gras et al. 2006). Subsequently, by genetic fate-mapping experiments, the same group demonstrated that cardiac progenitor cells from the embryonic second heart field are the source of most of the adipocytes observed in the Dsp+/− mouse model (Lombardi et al. 2009). In a more recent study performed in mice overexpressing cardiac wild-type or truncated PG, the nuclear translocation of PG was shown to lead to adipogenesis in c-Kit+cardiac progenitor cells by repressing the canonical Wnt signaling and by inducing the expression of proadipogenic genes (Lombardi et al. 2011). In a conditional cardiac tissue-restricted JUP deletion mouse model for ACM, disruption of junctional integrity causes increased β-catenin stabilization, apparently attributable to activation of AKT, which in turn inhibits glycogen synthase kinase-3β. This finding suggests that β-catenin signaling activation contributes to the cardiac phenotype (J. Li et al. 2011). However, this hypothesis was not confirmed in another cardiomyocyte-restricted Jup knockout mouse (D. Li

Garcia-Gras et al. 2006

Lombardi et al. 2011 J. Li et al. 2011

Ma et al. 2013 Kim et al. 2013

Caspi et al. 2013

et al. 2011). In this model, despite the increase of β-catenin levels at IDs of Jup mutant cardiomyocytes, the Wnt/β-catenin-mediated signaling was not activated, whereas TGFβ signaling was found significantly up-regulated in the early stages of the disease. TGFβ signaling regulates myocyte cell death, including both apoptosis and necrosis, in addition to its paramount influence on cardiac fibrosis and hypertrophy (D. Li et al. 2011). Recently, the activation of another signaling pathway, the Hippo/YAP pathway, was linked to the intercellular adhesion disruption in ACM by means of experiments on ACM human samples, mouse models and Pkp2 knockdown HL-1 myocytes (Chen et al. 2014). Activation of the Hippo kinase cascade results in the phosphorylation and in the cytoplasmic retention of the transcriptional coactivator Yes-associated protein (YAP), whereas the inhibition of Hippo signaling causes YAP entry into the nucleus and its association with the TEAD transcription factor and β-catenin/TCF complexes, determining an increasing in the transcriptional activity of both TEAD and TCF, which are positive regulators of cardiac growth (Heallen et al. 2011; Xin et al. 2011). Recent data suggest that perturbations in ID attributable to mutations in genes encoding ID proteins reduce the localization of protein kinase C-α to these structures, leading to the activation of Neurofibromin 2

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(NF2), the most upstream kinase of the Hippo pathway. Consequently, the Hippo kinases downstream of NF2 are cascade-phoshorylated, which in turn causes YAP phosphorylation and cytoplasmic retention (Chen et al. 2014). Cytoplasmic YAP sequesters β-catenin and PG, resulting in a reduction of both β-catenin/TCF and YAP/TEAD transcriptional activities, followed by increased adipogenesis in ACM hearts. Thus, the impairment of the ID structure is expected not only to affect the mechanical properties of the cardiomyocytes but also to modulate a series of signaling events, such as the crosstalk between the Wnt/β-catenin and Hippo/YAP signaling pathways. Most recently, Asimaki et al. (2014) created a zebrafish ACM model with the cardiomyocyte-specific expression of the human c.2057del2 mutation in the JUP gene. The animals show reductions in INa and IK1 current densities and interruptions in cell-cell boundaries and structural disarray. Highthroughput screening has identified SB216763 as a compound able to prevent heart failure and to reduce mortality in the fish model. The efficacy of SB216763 in the reversion or prevention of ACM pathobiological features has been supported by additional experiments performed in neonatal rat ventricular myocytes expressing the same JUP mutation and in cardiac myocytes derived from induced pluripotent stem cells (iPSCs) from two ACM probands with PKP2 mutations. Treatment with SB216763 restores the subcellular distribution of PG, connexin 43 and Nav1.5 in cardiomyocytes and of SAP97, a protein known to mediate the forward trafficking of Nav1.5 and Kir2.1, in both cell types. These results suggest that defects in ID protein trafficking might play a significant role in cardiomyocyte injury and electrical abnormalities in ACM. A new powerful model system for studying the basic mechanisms of ACM is represented by patient iPSC-derived cardiomyocytes, which overcome limitations arising from species differences in cardiac electrophysiological properties. Up to now, three separate studies have examined the effect of mutations in the PKP2 gene in ACM patient-specific iPSCs: mutant iPSCs are prone to lipid accumulation following treatment with various adipogenic stimuli and display a functional pro-adipogenic state, which is considered to be one of the hallmarks of ACM (Table 2). In iPSCs obtained from a patient heterozygous for the p.L614P missense mutation in PKP2, immunofluorescence assays revealed reduced signals of PKP2 and PG but similar levels of staining for N-cadherin, DSP and connexin-43 (Ma et al. 2013). Furthermore, transmission electron microscopy showed larger ACM iPSCs containing darker lipid droplets compared with controls, thus suggesting an increased adipogenic potential in these cells. In another study, Kim et al. (2013) reprogrammed iPSCs from fibroblasts from two patients carrying either the homozygous c.2482C>T (p.G828G) or the heterozygous c.2013delC (p.K672RfsX12)

mutation in the PKP2 gene. Despite the abnormal nuclear translocation of junction PG and extremely low β-catenin activity and expression, the disease phenotype was not fully recapitulated in standard culture conditions. The induction of an adult-like metabolism by culturing beating mutant embryoid bodies in a lipogenic milieu co-activated master peroxisome proliferator-activated receptor (PPAR)-α-dependent metabolism and the PPAR-γ pathway and recapitulated the classical ACM features consisting in lipogenesis, apoptosis and calcium-handling deficits. These results were confirmed in the most recent study on iPSC-derived cardiomyocytes from two ACM patients carrying two different heterozygous PKP2 frameshift mutations (Caspi et al. 2013). In mutant cells, intracellular lipid droplet accumulation together with the abnormal upregulation of pro-adipogenic PPAR-γ was shown to be correlated with the degree of the desmosomal distortion, suggesting a causal link between desmosomal malfunction and lipid accumulation in ACM. Moreover, the adipogenic stimuli on the mutant cardiomyocytes seemed to be prevented by GSK-3β inhibitor, further suggesting the possible role of the canonical Wnt/βcatenin pathway in ACM pathogenesis. Despite the useful insights supplied by the reported data, in order to determine the true potential of the use of iPSCderived cardiomyocytes with regard to providing a deeper understanding of ACM, further studies are manditory, including those involving the analysis of cells obtained from patients harboring other ID protein mutations.

IDs and ACM Rod-shaped adult cardiomyocytes are joined with one another at their ends in an ID, a specialized intercellular junctional structure that is found only in cardiac tissue, which is critical for enabling coordinated heart function. Originally, an ID was described as being composed of three distinct structures: AJs, desmosomes and gap junctions (GJs). AJs and desmosomes are extremely important for the maintenance of the adhesion and integrity of a tissue such as the myocardium, which is constantly exposed to mechanical stress and they anchor cardiomyocytes via the actin cytoskeleton and intermediate filament system, respectively, at the IDs (Garrod and Chidgey 2008). GJs account for the rapid and coordinated electrical excitation that is a prerequisite for normal rythmic cardiac function (Delmar and Sorgen 2009). Cardiac AJs consist in N-cadherin, a single-pass transmembrane protein responsible for Ca2+-dependent homophilic cellcell adhesion through its five extracellular cadherin domains and binding by its cytoplasmic tail to the catenins or armadillo proteins. β-Catenin or PG (γ-catenin) directly binds to the Cterminal region of N-cadherin in a mutually exclusive manner, whereas the αT- and αE-catenins form a direct or indirect link

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between the cadherin-catenin complex and the F-actin cytoskeleton (Jamora and Fuchs 2002). The molecular structure of the desmosomes is relatively similar to that of AJs (Fig. 1). The cardiac desmosomal cadherins, DSG2 and DSC2, mediate intercellular adhesion through homophylic and heterophylic interactions by their extracellular domains and by associating with desmosomal plaque proteins through their cytoplasmic domain. The armadillo family members PG and PKP2, in turn, bind DSP, which finally links the whole structure with desmin IF. This complex network allows the propagation of the tensile strength from the IF cytoskeleton across the entire tissue and is thus essential for myocardial integrity (Garrod and Chidgey 2008). Until recently, AJs and desmosomes were thought to be distinct junctional complexes at IDs. However, immunoelectron microscopy studies performed by Franke and colleagues (2006) using adult mammalian heart revealed that desmosomal molecules are not confined to desmosomes but are also present in the more abundant junctions with AJ-like morphology (Fig. 1). Because of its hybrid character, this junction has been termed the “area composita” (Franke et al. 2006). Interestingly, the area composita is only found in higher vertebrates and seems to have evolved to withstand the high mechanical load of the four-chamber mammalian heart by anchoring both actin and IF over an extended junctional area of the ID (Pieperhoff and Franke 2007). GJs are intercellular channels providing a low-resistive substrate for the direct intercellular flow of ions and electrical coupling of cardiomyocytes (Delmar and Sorgen 2009). Each GJ channel is formed by the noncovalent interactions of two hemichannels, called connexons, each consisting in six connexin (Cx) molecules that span across the extracellular space, forming a permeable pore. Compared with other tissues, the mammalian ID of the working myocardium contains extremely large Cx43-composed GJ plaques, underlining the importance of this isoform in the efficient propagation of cardiac action potentials throughout the myocardium (Kardami et al. 2003). The other cardiac isoforms, Cx40 and Cx45, are the main constituents of GJs in atrial myocytes and in the conduction system, respectively (Kostin et al. 2003). In addition to mechanical and electrical intercellular complexes, the ID hosts a number of molecules that are not traditionally considered as “junctional” but that form their own molecular and functional complexes thus providing a physical continuum between adjacent cells (Sato et al. 2009). Among these, the most relevant for its function in the heart is the voltage-gated sodium channel complex, which is composed by α (mostly Nav1.5 in heart) and β (β1 to β4) subunits and by the cytoskeletal adaptor protein ankyrin-G. Several co-immunoprecipitation studies have shown that mechanical and electrical junctions and ion channel proteins interact amongst each other (Agullo-Pascual et al. 2013; Rizzo et al. 2012; Sato et al. 2009). The destruction of these

interactions might contribute to abnormal tissue electrophysiology in the concealed phase of ACM, during which arrhythmias might occur in the absence of structural changes. The evidence for the presence of desmosomes and area composita in the cardiac conduction system suggests that electrical abnormalities observed in ACM patients also arise from direct or indirect perturbations of mechanical and electrical junctions in this system (Pieperhoff et al. 2010). Loss and/or mutations of mechanical junction proteins in mouse model hearts have been shown to cause profound defects in the His-Purkinje conduction system, such as right bundle branch block, spontaneous ventricular ectopic beats, ventricular tachycardia and prolonged QRS interval, all typical features of human ACM (Garcia-Gras et al. 2006; Pilichou et al. 2009; Lyon et al. 2014). Based on these findings, the ID emerges as a dynamic structure in which both mechanical and electrical components interact synergically to maintain synchrony in the heart. Since almost half of ACM patients carry one or more mutations in genes encoding mechanical junction proteins expressed in the cardiac ID, ACM should be defined as “a disease of the ID” (Fig. 1).

Concluding remarks and future perspectives ACM is a leading cause of sudden death in the young and in athletes because of ventricular fibrillation. In at least half of the cases, it is caused by single-gene mutations in genes encoding cell-cell junction proteins. The current hypothesis with regard to ACM molecular pathogenesis is that the molecular remodeling of the IDs perturbs signaling pathways regulated at the junctions. Elucidation of the role of junctional-related signaling pathways in the pathogenesis of ACM could help in the development of effective mechanismbased therapies aimed at delaying the onset or progression of this disease. Although enormous progress has been made in the last decade in understanding the clinical features, molecular genetics and pathogenesis of ACM, many questions remain open. The disease gene is undefined in a significant proportion of patients; thus, we need to identify novel genes to clarify the genetic basis of ACM. The majority of the ACMsusceptibility genes have been discovered by using a biologically plausible, candidate-gene approach. Major technological advances in DNA sequencing, allowing the rapid wholegenome or whole-exome interrogation of patient samples for the identification of novel pathogenic mutations, have recently emerged. Moreover, the relationship between genetic, epigenetic and environmental factors and their role in disease expression remains unresolved and requires further studies. The precise function of the nuclear translocation of PG and of the suppression of canonical Wnt signaling is still not

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completely understood. Clarification is urgently required with regard to whether PG is a major mediator of adipogenesis and whether impaired ID assembly could explain the mechanical and electrical dysfunction in ACM. A new appealing tool that might improve the understanding of such pathogenic molecular mechanisms is represented by the differentiation of iPSCs into cardiomyocytes, which have been used to recapitulate ACM features in the context of the genetic background of the patient. This disease model could be important to gain insight into the pathophysiology of the disease and to identify targeted therapies for the individual patient. The newly emerging approaches of next-generation sequencing, epigenetic analysis and patient-specific iPSCs should certainly have a major impact on our understanding of the molecular genetics and pathogenesis of ACM.

References Agullo-Pascual E, Reid DA, Keegan S, Sidhu M, Fenyö D, Rothenberg E, Delmar M (2013) Super-resolution fluorescence microscopy of the cardiac connexome reveals plakophilin-2 inside the connexin43 plaque. Cardiovasc Res 100:231–240 Asimaki A, Syrris P, Wichter T, Matthias P, Saffitz JE, McKenna WJ (2007) A novel dominant mutation in plakoglobin causes arrhythmogenic right ventricular cardiomyopathy. Am J Hum Genet 81: 964–973 Asimaki A, Kapoor S, Plovie E, Karin Arndt A, Adams E, Liu Z, James CA, Judge DP, Calkins H, Churko J, Wu JC, MacRae CA, Kléber AG, Saffitz JE (2014) Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Sci Transl Med 6:240ra74 Basso C, Bauce B, Corrado D, Thiene G (2011) Pathophysiology of arrhythmogenic cardiomyopathy. Nat Rev Cardiol 9:223–233 Bauce B, Frigo G, Marcus FI, Basso C, Rampazzo A, Maddalena F, Corrado D, Winnicki M, Daliento L, Rigato I, Steriotis A, Mazzotti E, Thiene G, Nava A (2008) Comparison of clinical features of arrhythmogenic right ventricular cardiomyopathy in men versus women. Am J Cardiol 102:1252–1257 Bauce B, Nava A, Beffagna G, Basso C, Lorenzon A, Smaniotto G, De Bortoli M, Rigato I, Mazzotti E, Steriotis A, Marra MP, Towbin JA, Thiene G, Danieli GA, Rampazzo A (2010) Multiple mutations in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia. Heart Rhythm 7:22–29 Beffagna G, Occhi G, Nava A, Vitiello L, Ditadi A, Basso C, Bauce B, Carraro G, Thiene G, Towbin JA, Danieli GA, Rampazzo A (2005) Regulatory mutations in transforming growth factor-beta3 gene cause arrhythmogenic right ventricular cardiomyopathy type 1. Cardiovasc Res 65:366–373 Carvajal-Huerta L (1998) Epidermolytic palmoplantar keratoderma with woolly hair and dilated cardiomyopathy. J Am Acad Dermatol 39: 418–421 Caspi O, Huber I, Gepstein A, Arbel G, Maizels L, Boulos M, Gepstein L (2013) Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells. Circ Cardiovasc Genet 6:557–568 Chen SN, Gurha P, Lombardi R, Ruggiero A, Willerson JT, Marian AJ (2014) The hippo pathway is activated and is a causal mechanism

for adipogenesis in arrhythmogenic cardiomyopathy. Circ Res 114: 454–468 Corrado D, Basso C, Thiene G, McKenna WJ, Davies MJ, Fontaliran F, Nava A, Silvestri F, Blomström-Lundqvist C, Wlodarska EK, Fontaine G, Camerini F (1997) Spectrum of clinicopathologic manifestations of arrhythmogenic right ventricular cardiomyopathy/dysplasia: a multicenter study. J Am Coll Cardiol 30:1512–1520 Corrado D, Basso C, Pavei A, Michieli P, Schiavon M, Thiene G (2006) Trends in sudden cardiovascular death in young competitive athletes after implementation of a preparticipation screening program. JAMA 296:1593–1601 Corrado D, Basso C, Pilichou K, Thiene G (2011) Molecular biology and clinical management of arrhythmogenic right ventricular cardiomyopathy/dysplasia. Heart 97:530–539 Delmar M, Sorgen PL (2009) Molecular organization and regulation of the cardiac gap junction channel Connexin43. In: Zipes DP, Jalife J (eds) Cardiac electrophysiology: from cell to bedside. Saunders Elsevier, Philadelphia, pp 85–92 Franke WW, Borrmann CM, Grund C, Pieperhoff S (2006) The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins. Eur J Cell Biol 85:69–82 Franke WW, Dörflinger Y, Kuhn C, Zimbelmann R, WinterSimanowski S, Frey N, Heid H (2014) Protein LUMA is a cytoplasmic plaque constituent of various epithelial adherens junctions and composite junctions of myocardial intercalated disks: a unifying finding for cell biology and cardiology. Cell Tissue Res 357:159–172 Fressart V, Duthoit G, Donal E, Probst V, Deharo JC, Chevalier P, Klug D, Dubourg O, Delacretaz E, Cosnay P, Scanu P, Extramiana F, Keller D, Hidden-Lucet F, Simon F, Bessirard V, Roux-Buisson N, Hebert JL, Azarine A, Casset-Senon D, Rouzet F, Lecarpentier Y, Fontaine G, Coirault C, Frank R, Hainque B, Charron P (2010) Desmosomal gene analysis in arrhythmogenic right ventricular dysplasia/cardiomyopathy: spectrum of mutations and clinical impact in practice. Europace 12:861–868 Garcia-Gras E, Lombardi R, Giocondo MJ, Willerson JT, Schneider MD, Khoury DS, Marian AJ (2006) Suppression of canonical Wnt/betacatenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy. J Clin Invest 116: 2012–2021 Garrod D, Chidgey M (2008) Desmosome structure, composition and function. Biochim Biophys Acta Biomembr 1778:572–587 Gerull B, Heuser A, Wichter T, Paul M, Basson CT, McDermott DA, Lerman BB, Markowitz SM, Ellinor PT, MacRae CA, Peters S, Grossmann KS, Drenckhahn J, Michely B, Sasse-Klaassen S, Birchmeier W, Dietz R, Breithardt G, Schulze-Bahr E, Thierfelder L (2004) Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy. Nat Genet 36:1162–1164 Goossens S, Janssens B, Bonné S, De Rycke R, Braet F, Hengel J van, Roy F van (2007) A unique and specific interaction between alphaTcatenin and plakophilin-2 in the area composita, the mixed-type junctional structure of cardiac intercalated discs. J Cell Sci 120: 2126–2136 Heallen T, Zhang M, Wang J, Bonilla-Claudio M, Klysik E, Johnson RL, Martin JF (2011) Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size. Science 332:458–461 Hengel J van, Calore M, Bauce B, Dazzo E, Mazzotti E, De Bortoli M, Lorenzon A, Li Mura IE, Beffagna G, Rigato I, Vleeschouwers M, Tyberghein K, Hulpiau P, Hamme E van, Zaglia T, Corrado D, Basso C, Thiene G, Daliento L, Nava A, Roy F van, Rampazzo A (2013) Mutations in the area composita protein alpha-T-catenin are associated with arrhythmogenic right ventricular cardiomyopathy. Eur Heart J 34:201–210

Cell Tissue Res Jamora C, Fuchs E (2002) Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol 4:E101–E108 Kaplan SR, Gard JJ, Protonotarios N, Tsatsopoulou A, Spiliopoulou C, Anastasakis A, Squarcioni CP, McKenna WJ, Thiene G, Basso C, Brousse N, Fontaine G, Saffitz JE (2004) Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease). Heart Rhythm 1:3–11 Kapplinger JD, Landstrom AP, Salisbury BA, Callis TE, Pollevick GD, Tester DJ, Cox MG, Bhuiyan Z, Bikker H, Wiesfeld AC, Hauer RN, Tintelen JP van, Jongbloed JD, Calkins H, Judge DP, Wilde AA, Ackerman MJ (2011) Distinguishing arrhythmogenic right ventricular cardiomyopathy/dysplasia-associated mutations from background genetic noise. J Am Coll Cardiol 57: 2317–2327 Kardami E, Banerji S, Doble BW, Dang X, Fandrich RR, Jin Y, Cattini PA (2003) PKC-dependent phosphorylation may regulate the ability of connexin43 to inhibit DNA synthesis.Cell Commun Adhes 10: 293–297 Kim C, Wong J, Wen J, Wang S, Wang C, Spiering S, Kan NG, Forcales S, Puri PL, Leone TC, Marine JE, Calkins H, Kelly DP, Judge DP, Chen H (2013) Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature 494:105–110 Klauke B, Kossmann S, Gaertner A, Brand K, Stork I, Brodehl A, Dieding M, Walhorn V, Anselmetti D, Gerdes D, Bohms B, Schulz U, Zu Knyphausen E, Vorgerd M, Gummert J, Milting H (2010) De novo desmin-mutation N116S is associated with arrhythmogenic right ventricular cardiomyopathy. Hum Mol Genet 19: 4595–4607 Kostin S, Rieger M, Dammer S, Hein S, Richter M, Klövekorn WP, Bauer EP, Schaper J (2003) Gap junction remodeling and altered connexin43 expression in the failing human heart. Mol Cell Biochem 242:135–144 Li D, Liu Y, Maruyama M, Zhu W, Chen H, Zhang W, Reuter S, Lin SF, Haneline LS, Field LJ, Chen PS, Shou W (2011) Restrictive loss of plakoglobin in cardiomyocytes leads to arrhythmogenic cardiomyopathy. Hum Mol Genet 20:4582–4596 Li J, Swope D, Raess N, Cheng L, Muller EJ, Radice GL (2011) Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of beta-catenin signaling. Mol Cell Biol 31:1134–1144 Li Mura IE, Bauce B, Nava A, Fanciulli M, Vazza G, Mazzotti E, Rigato I, De Bortoli M, Beffagna G, Lorenzon A, Calore M, Dazzo E, Nobile C, Mostacciuolo ML, Corrado D, Basso C, Daliento L, Thiene G, Rampazzo A (2013) Identification of a PKP2 gene deletion in a family with arrhythmogenic right ventricular cardiomyopathy. Eur J Hum Genet 21:1226–1231 Lombardi R, Dong J, Rodriguez G, Bell A, Leung TK, Schwartz RJ, Willerson JT, Brugada R, Marian AJ (2009) Genetic fate mapping identifies second heart field progenitor cells as a source of adipocytes in arrhythmogenic right ventricular cardiomyopathy. Circ Res 104:1076–1084 Lombardi R, Graca C-HM da, Bell A, Fromm RR, Willerson JT, Marian AJ (2011) Nuclear plakoglobin is essential for differentiation of cardiac progenitor cells to adipocytes in arrhythmogenic right ventricular cardiomyopathy. Circ Res 109:1342–1353 Lyon RC, Mezzano V, Wright AT, Pfeiffer E, Chuang J, Banares K, Castaneda A, Ouyang K, Cui L, Contu R, Gu Y, Evans SM, Omens JH, Peterson KL, McCulloch AD, Sheikh F (2014) Connexin defects underlie arrhythmogenic right ventricular cardiomyopathy in a novel mouse model. Hum Mol Genet 23:1134–1150 Ma D, Wei H, Lu J, Ho S, Zhang G, Sun X, Oh Y, Tan SH, Ng ML, Shim W, Wong P, Liew R (2013) Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy. Eur Heart J 34: 1122–1133

Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B, Bluemke DA, Calkins H, Corrado D, Cox MG, Daubert JP, Fontaine G, Gear K, Hauer R, Nava A, Picard MH, Protonotarios N, Saffitz JE, Sanborn DM, Steinberg JS, Tandri H, Thiene G, Towbin JA, Tsatsopoulou A, Wichter T, Zareba W (2010) Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the Task Force Criteria. Eur Heart J 31:806–814 McKenna WJ, Thiene G, Nava A, Fontaliran F, Blomström-Lundqvist C, Fontaine G, Camerini F (1994) Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy. Task force of the Working Group Myocardial and Pericardial Disease of the European Society of Cardiology and of the Scientific Council on Cardiomyopathies of the International Society and Federation of Cardiology. Br Heart J 71:215–218 McKoy G, Protonotarios N, Crosby A, Tsatsopoulou A, Anastasakis A, Coonar A, Norman M, Baboonian C, Jeffery S, McKenna WJ (2000) Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease). Lancet 355:2119–2124 Merner ND, Hodgkinson KA, Haywood AF, Connors S, French VM, Drenckhahn JD, Kupprion C, Ramadanova K, Thierfelder L, McKenna W, Gallagher B, Morris-Larkin L, Bassett AS, Parfrey PS, Young TL (2008) Arrhythmogenic right ventricular cardiomyopathy type 5 is a fully penetrant, lethal arrhythmic disorder caused by a missense mutation in the TMEM43 gene. Am J Hum Genet 82: 809–821 Nava A, Thiene G, Canciani B, Scognamiglio R, Daliento L, Buja G, Martini B, Stritoni P, Fasoli G (1988) Familial occurrence of right ventricular dysplasia: a study involving nine families. J Am Coll Cardiol 12:1222–1228 Nava A, Bauce B, Basso C, Muriago M, Rampazzo A, Villanova C, Daliento L, Buja G, Corrado D, Danieli GA, Thiene G (2000) Clinical profile and long-term follow-up of 37 families with arrhythmogenic right ventricular cardiomyopathy. J Am Coll Cardiol 36: 2226–2233 Norgett EE, Hatsell SJ, Carvajal-Huerta L, Cabezas JC, Common J, Purkis PE, Whittock N, Leigh IM, Stevens HP, Kelsell DP (2000) Recessive mutation in desmoplakin disrupts desmoplakinintermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma. Hum Mol Genet 9:2761–2766 Pieperhoff S, Franke WW (2007) The area composita of adhering junctions connecting heart muscle cells of vertebrates. IV. Coalescence and amalgamation of desmosomal and adhaerens junction components—late processes in mammalian heart development. Eur J Cell Biol 86:377–391 Pieperhoff S, Borrmann C, Grund C, Barth M, Rizzo S, Franke WW (2010) The area composita of adhering junctions connecting heart muscle cells of vertebrates. VII. The different types of lateral junctions between the special cardiomyocytes of the conduction system of ovine and bovine hearts. Eur J Cell Biol 89:365–378 Pilichou K, Nava A, Basso C, Beffagna G, Bauce B, Lorenzon A, Frigo G, Vettori A, Valente M, Towbin J, Thiene G, Danieli GA, Rampazzo A (2006) Mutations in desmoglein-2 gene are associated with arrhythmogenic right ventricular cardiomyopathy. Circulation 113:1171–1179 Pilichou K, Remme CA, Basso C, Campian ME, Rizzo S, Barnett P, Scicluna BP, Bauce B, Hoff MJ van den, Bakker JM de, Tan HL, Valente M, Nava A, Wilde AA, Moorman AF, Thiene G, Bezzina CR (2009) Myocyte necrosis underlies progressive myocardial dystrophy in mouse dsg2-related arrhythmogenic right ventricular cardiomyopathy. J Exp Med 206:1787–1802 Protonotarios N, Tsatsopoulou A, Scampardonis G (1988) Arrhythmogenic cardiomyopathy with ectodermal dysplasia. Am Heart J 116:1651–1652 Quarta G, Syrris P, Ashworth M, Jenkins S, Zuborne Alapi K, Morgan J, Muir A, Pantazis A, McKenna WJ, Elliott PM (2012) Mutations in

Cell Tissue Res the Lamin A/C gene mimic arrhythmogenic right ventricular cardiomyopathy. Eur Heart J 33:1128–1136 Rampazzo A (2006) Genetic bases of arrhythmogenic right ventricular cardiomyopathy. Heart Int 2:17 Rampazzo A, Nava A, Malacrida S, Beffagna G, Bauce B, Rossi V, Zimbello R, Simionati B, Basso C, Thiene G, Towbin JA, Danieli GA (2002) Mutation in human desmoplakin domain binding to plakoglobin causes a dominant form of arrhythmogenic right ventricular cardiomyopathy. Am J Hum Genet 71:1200–1206 Rigato I, Bauce B, Rampazzo A, Zorzi A, Pilichou K, Mazzotti E, Migliore F, Marra MP, Lorenzon A, De Bortoli M, Calore M, Nava A, Daliento L, Gregori D, Iliceto S, Thiene G, Basso C, Corrado D (2013) Compound and digenic heterozygosity predicts lifetime arrhythmic outcome and sudden cardiac death in desmosomal gene-related arrhythmogenic right ventricular cardiomyopathy. Circ Cardiovasc Genet 6:533–542 Rizzo S, Lodder EM, Verkerk AO, Wolswinkel R, Beekman L, Pilichou K, Basso C, Remme CA, Thiene G, Bezzina CR (2012) Intercalated disc abnormalities, reduced Na+current density, and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes. Cardiovasc Res 95:409–418 Roberts JD, Herkert JC, Rutberg J, Nikkel SM, Wiesfeld AC, Dooijes D, Gow RM, Tintelen JP van, Gollob MH (2013) Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy. Clin Genet 83:452–456 Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, MacDougald OA (2000) Inhibition of adipogenesis by Wnt signaling. Science 289:950–953 Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patino GA, Taffet SM, Isom LL, Delmar M (2009) Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res 105:523–526 Sato T, Nishio H, Suzuki K (2011) Sudden death during exercise in a juvenile with arrhythmogenic right ventricular cardiomyopathy and desmoglein-2 gene substitution: a case report. Leg Med (Tokyo) 3: 298–300 Sen-Chowdhry S, Syrris P, Pantazis A, Quarta G, McKenna WJ, Chambers JC (2010) Mutational heterogeneity, modifier genes, and environmental influences contribute to phenotypic diversity of arrhythmogenic cardiomyopathy. Circ Cardiovasc Genet 3:323–330

Syrris P, Ward D, Evans A, Asimaki A, Gandjbakhch E, Sen-Chowdhry S, McKenna WJ (2006) Arrhythmogenic right ventricular dysplasia/ cardiomyopathy associated with mutations in the desmosomal gene desmocollin-2. Am J Hum Genet 79:978–984 Taylor M, Graw S, Sinagra G, Barnes C, Slavov D, Brun F, Pinamonti B, Salcedo EE, Sauer W, Pyxaras S, Anderson B, Simon B, Bogomolovas J, Labeit S, Granzier H, Mestroni L (2011) Genetic variation in titin in arrhythmogenic right ventricular cardiomyopathy-overlap syndromes. Circulation 124: 876–885 Thiene G, Nava A, Corrado D, Rossi L, Pennelli N (1988) Right ventricular cardiomyopathy and sudden death in young people. N Engl J Med 318:129–133 Tiso N, Stephan DA, Nava A, Bagattin A, Devaney JM, Stanchi F, Larderet G, Brahmbhatt B, Brown K, Bauce B, Muriago M, Basso C, Thiene G, Danieli GA, Rampazzo A (2001) Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum Mol Genet 10:189–194 Xin M, Kim Y, Sutherland LB, Qi X, McAnally J, Schwartz RJ, Richardson JA, Bassel-Duby R, Olson EN (2011) Regulation of insulin-like growth factor signaling by Yap governs cardiomyocyte proliferation and embryonic heart size. Sci Signal 4:ra70 Xu T, Yang Z, Vatta M, Rampazzo A, Beffagna G, Pilichou K, Scherer SE, Saffitz J, Kravitz J, Zareba W, Danieli GA, Lorenzon A, Nava A, Bauce B, Thiene G, Basso C, Calkins H, Gear K, Marcus F, Towbin JA (2010) Multidisciplinary study of right ventricular dysplasia investigators. Compound and digenic heterozygosity contributes to arrhythmogenic right ventricular cardiomyopathy. J Am Coll Cardiol 55:587–597 Zwaag PA van der, Rijsingen IA van, Asimaki A, Jongbloed JD, Veldhuisen DJ van, Wiesfeld AC, Cox MG, Lochem LT van, Boer RA de, Hofstra RM, Christiaans I, Spaendonck-Zwarts KY van, Lekanne dit Deprez RH, Judge DP, Calkins H, Suurmeijer AJ, Hauer RN, Saffitz JE, Wilde AA, Berg MP van den, Tintelen JP van (2012) Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy: evidence supporting the concept of arrhythmogenic cardiomyopathy. Eur J Heart Fail 14:1199–1207