Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype with a yet-to-be-defined molecular mechanism. We established atrial myocyte cell lines expressing siRNA against desmoplakin (DP), responsible for human ARVC. We show suppression of DP expression leads to nuclear localization of the desmosomal protein plakoglobin and a 2-fold reduction in canonical Wnt/b-catenin signaling through Tcf/Lef1 transcription factors. The ensuing phenotype is increased expression of adipogenic and fibrogenic genes and accumulation of fat droplets. We further show that cardiac-restricted deletion of Dsp, encoding DP, impairs cardiac morphogenesis and leads to high embryonic lethality in the homozygous state. Heterozygous DP-deficient mice exhibited excess adipocytes and fibrosis in the myocardium, increased myocyte apoptosis, cardiac dysfunction, and ventricular arrhythmias, thus recapitulating the phenotype of human ARVC. We believe our results provide for a novel molecular mechanism for the pathogenesis of ARVC and establish cardiac-restricted DP-deficient mice as a model for human ARVC. These findings could provide for the opportunity to identify new diagnostic markers and therapeutic targets in patients with ARVC.

Rationale Mutations in the intercalated disc (ID) proteins, such as plakophilin 2 (PKP2) cause arrhythmogenic cardiomyopathy (AC). AC is characterized by the replacement of cardiac myocytes by fibro-adipocytes, cardiac dysfunction, arrhythmias and sudden death. Objective To delineate the molecular pathogenesis of AC. Methods and Results Localization and levels of selected ID proteins including signaling molecules were markedly reduced in the human hearts with AC. Altered protein constituents of IDs was associated with activation of the upstream Hippo molecules in the human hearts, Nkx2.5-Cre:DspW/F and Myh6:Jup mouse models of AC, and in the plakophilin 2 (PKP2) knock down HL-1 myocytes (HL-1PKP2:shRNA). Level of active PKC-α, which requires PKP2 for activity, was reduced. In contrast, neurofibromin (NF2 or Merlin), a molecule upstream to the Hippo pathway, which is inactivated by PKC-α, was activated. Consequently, the downstream Hippo molecules MST1/2, LATS1/2 and YAP; the latter is the effector of the pathway, were phosphorylated. Co-immunoprecipitation detected binding of pYAP, pβ-catenin, and JUP, the latter translocated from the junction. RNA sequencing, transcript qPCR and reporter assays showed suppressed activity of TEAD and TCF7L2, transcription factors of the Hippo and the canonical Wnt signaling, respectively. In contrast, adipogenesis was enhanced. Simultaneous KD of Lats1/2, molecules upstream to YAP, rescued inactivation of YAP and β-catenin and adipogenesis in the HL-1PKP2:shRNA myocytes. Conclusions Molecular remodeling of the IDs leads to pathogenic activation of the Hippo pathway, suppression of the canonical Wnt signaling and enhanced adipogenesis in AC. The findings offer novel mechanisms for the pathogenesis of AC.

Rationale Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disease of desmosome proteins characterized by fibroadipogenesis in the myocardium. We have implicated signaling properties of junction protein plakoglobin (PG) in the pathogenesis of ARVC. Objective To delineate the pathogenic role of PG in adipogenesis in ARVC. Methods and Results We generated mice overexpressing PG, either a wildtype (PGWT) or a truncated (PGTR), known to cause ARVC, in the heart; and PG null (PG−/−) embryos. PGWT and PGTR mice exhibited fibro-adiposis, cardiac dysfunction, and premature death. Subcellular protein fractionation and immunofluorescence showed nuclear localization of PGWT and PGTR and reduced membrane localization of PGTR. Coimmunoprecipitation showed reduced binding of PGTR but not PGWT to desmosome proteins DSP and DSG2. Transgene PGWT and PGTR were expressed in c-Kit+:Sca1+ cardiac progenitor cells (CPCs) isolated from the hearts of PGWT and PGTR by fluorescence activated cell sorting. CPCs isolated from the transgenic hearts showed enhanced adipogenesis, increased levels of adipogenic factors KLF15, C/EBP-α and noncanonical Wnt5b, and reduced level of CTGF, an inhibitor of adipogenesis. Treatment with BIO activated the canonical Wnt signaling, reversed the proadipogenic transcriptional switch and prevented adipogenesis in a dose-dependent manner. Moreover, c-Kit+ CPCs, isolated from PG−/− embryos, were resistant to adipogenesis, expressed high mRNA levels of CTGF and other canonical Wnt signaling targets. Conclusions Nuclear PG provokes adipogenesis in c-Kit+ CPCs by repressing the canonical Wnt signaling and inducing a proadipogenic gene expression. The findings suggest that adipocytes in ARVC, at least in part, originate from c-Kit+ CPCs.

Abstract-The phenotypic hallmark of arrhythmogenic right ventricular cardiomyopathy, a genetic disease of desmosomal proteins, is fibroadipocytic replacement of the right ventricle. Cellular origin of excess adipocytes, the responsible mechanism(s) and the basis for predominant involvement of the right ventricle are unknown. We generated 3 sets of lineage tracer mice regulated by cardiac lineage promoters ␣-myosin heavy chain (␣MyHC), Nkx2.5, or Mef2C. We conditionally expressed the reporter enhanced yellow fluorescent protein while concomitantly deleting the desmosomal protein desmoplakin in cardiac myocyte lineages using the Cre-LoxP technique. Lineage tracer mice showed excess fibroadiposis and increased numbers of adipocytes in the hearts. Few adipocytes in the hearts of ␣MyHC-regulated lineage tracer mice, but the majority of adipocytes in the hearts of Nkx2.5-and Mef2C-regulated lineage tracer mice, expressed enhanced yellow fluorescent protein. In addition, rare cells coexpressed adipogenic transcription factors and the second heart field markers Isl1 and Mef2C in the lineage tracer mouse hearts and in human myocardium from patients with arrhythmogenic right ventricular cardiomyopathy. To delineate the responsible mechanism, we generated transgenic mice expressing desmosomal protein plakoglobin in myocyte lineages. Transgene plakoglobin translocated to nucleus, detected by immunoblotting and immunofluorescence staining and coimmunoprecipitated with Tcf7l2, a canonical Wnt signaling transcription factor. Expression levels of canonical Wnt/Tcf7l2 targets bone morphogenetic protein 7 and Wnt5b, which promote adipogenesis, were increased and expression level of connective tissue growth factor, an inhibitor of adipogenesis, was decreased. We conclude adipocytes in arrhythmogenic right ventricular cardiomyopathy originate from the second heart field cardiac progenitors, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear plakoglobin. Key Words: adipocytes Ⅲ progenitor cells Ⅲ Wnt signaling Ⅲ desmosomes Ⅲ heart failure A rrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disease characterized by the unique phenotype of fibroadipocytic replacement of cardiac myocytes, predominantly in the right ventricle. [1][2][3] Clinical manifestations of ARVC include ventricular arrhythmias, typically originating from the right ventricle; sudden cardiac death, which often is its first manifestation; and right ventricular aneurysmal dilatation and failure. 1,4 The left ventricle is also commonly involved in the advanced stages. 1,5 ARVC is typically an autosomal dominant disease. Recessive forms in conjunction with palmoplantar keratoderma and woolly hair (Naxos disease) or predominant involvement of the left ventricle (Carvajal syndrome) are referred to as "cardiocutaneous syndromes." 6,7 Recently, mutations in 5 genes for ARVC, namely, DSP, JUP, PKP2, DSC2, and DSG2, encoding desmosomal proteins desmoplakin (Dsp), plakoglobin (PG), plakophilin 2, desmocollin 2, and ...