Purpose. Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is emerging as a relatively common cause of sudden death in teenagers and athletes. ARVC is considered a disease of cell adhesion because mutations in desmosomal genes have been involved in the pathogenesis of ARVC in a significant proportion of patients. The aim of the present study is to elucidate the molecular pathogenesis of ARVC by generating DSG2 transgenic animal models. Methods. Human full−length wild−type and mutant cDNA sequences for DSG2 were cloned into a vector containing alpha−MyHC promoter. Transgenic lines showing a comparable expression level of transgene have been generated for G100R, N266S, K294E and Q558X mutations and for wild-type DSG2. Phenotypic characterisation was performed by electrophysiological and histological analyses. In order to investigate on the molecular pathogenesis, electron miscroscopic, immunohystochemical and Western blot examination was performed as well. Results. Histological examination of 6-month-old mouse hearts evidentiated cardiomyocytes loss and fibro-fatty tissue replacement, resembling the phenotype of ARVC patients and confirming the pathogenic effects of DSG2 mutations in an animal model. Immunofluorescent staining showed correct localization of DSG2 G100R, N266S, and K294E into the desmosomal complexes, while in mice carrying the DSG2-Q558X mutation the protein was retained in the cytoplasm. Electron microscopic analyses showed a reduced number of desmosomes at the intercalated discs, myofibrillar degeneration and damaged mitochondria. Beta-catenin overexpression has been detected by Western blotting analysis and immunofluorescence in hearts of transgenic mice carrying the mutated proteins. DSG2 transgenic mice have been inbreeded with beta-catenin-activated transgene driving expression of nuclear beta-galactosidase reporter (BAT-gal) transgenic mice, in order to confirm beta-catenin overexpression. Analyses of BAT-gal expression in embrionic and neonatal transgenic hearts revealed a higher beta-catenin transcriptional activity compared to wild-type hearts. Conclusions. DSG2 transgenic mice represents an invaluable model to investigate molecular pathogenesis of ARVC. Further studies will be needed to assess if beta-catenin overexpression could lead to beta-catenin signalling defects.

Is beta-catenin signalling involved in the molecular pathogenesis of arrhythmogenic right ventricular cardiomyopathy? Study on DSG2 transgenic mice

BEFFAGNA, GIORGIA;LORENZON, ALESSANDRA;GRUMATI, PAOLO;BONALDO, PAOLO;BRAGHETTA, PAOLA;RAMPAZZO, ALESSANDRA
2010

Abstract

Purpose. Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is emerging as a relatively common cause of sudden death in teenagers and athletes. ARVC is considered a disease of cell adhesion because mutations in desmosomal genes have been involved in the pathogenesis of ARVC in a significant proportion of patients. The aim of the present study is to elucidate the molecular pathogenesis of ARVC by generating DSG2 transgenic animal models. Methods. Human full−length wild−type and mutant cDNA sequences for DSG2 were cloned into a vector containing alpha−MyHC promoter. Transgenic lines showing a comparable expression level of transgene have been generated for G100R, N266S, K294E and Q558X mutations and for wild-type DSG2. Phenotypic characterisation was performed by electrophysiological and histological analyses. In order to investigate on the molecular pathogenesis, electron miscroscopic, immunohystochemical and Western blot examination was performed as well. Results. Histological examination of 6-month-old mouse hearts evidentiated cardiomyocytes loss and fibro-fatty tissue replacement, resembling the phenotype of ARVC patients and confirming the pathogenic effects of DSG2 mutations in an animal model. Immunofluorescent staining showed correct localization of DSG2 G100R, N266S, and K294E into the desmosomal complexes, while in mice carrying the DSG2-Q558X mutation the protein was retained in the cytoplasm. Electron microscopic analyses showed a reduced number of desmosomes at the intercalated discs, myofibrillar degeneration and damaged mitochondria. Beta-catenin overexpression has been detected by Western blotting analysis and immunofluorescence in hearts of transgenic mice carrying the mutated proteins. DSG2 transgenic mice have been inbreeded with beta-catenin-activated transgene driving expression of nuclear beta-galactosidase reporter (BAT-gal) transgenic mice, in order to confirm beta-catenin overexpression. Analyses of BAT-gal expression in embrionic and neonatal transgenic hearts revealed a higher beta-catenin transcriptional activity compared to wild-type hearts. Conclusions. DSG2 transgenic mice represents an invaluable model to investigate molecular pathogenesis of ARVC. Further studies will be needed to assess if beta-catenin overexpression could lead to beta-catenin signalling defects.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2531866
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